cAMP response element binding (CREB) in the brain induced by CR upregulates SIRT1 gene transcription, resulting in neuroprotective responses that oppose brain aging

[Uhohinc]

Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1
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N Fujitsuka^1,2, A Asakawa¹, A Morinaga¹, M S Amitani¹, H Amitani¹, G Katsuura¹, Y Sawada³, Y Sudo³, Y Uezono³, E Mochiki⁴, I Sakata⁵, T Sakai⁵, K Hanazaki⁶, T Yada⁷, K Yakabi⁸, E Sakuma⁹, T Ueki⁹, A Niijima¹⁰, K Nakagawa¹¹, N Okubo¹¹, H Takeda^11,12, M Asaka¹³ and A Inui¹

1. ¹Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
2. ²Tsumura Research Laboratories, Tsumura, Ibaraki, Japan
3. ³Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo, Japan
4. ⁴Department of Digestive Tract and General Surgery, Saitama Medical Center, Saitama Medical University, Saitama, Japan
5. ⁵Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama, Japan
6. ⁶Department of Surgery, Kochi Medical School, Kochi, Japan
7. ⁷Department of Physiology, Jichi Medical University School of Medicine, Tochigi, Japan
8. ⁸Department of Gastroenterology and Hepatology, Saitama Medical Center, Saitama Medical University, Saitama, Japan
9. ⁹Department of Integrative Anatomy, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
10. ¹⁰Department of Physiology, Niigata University School of Medicine, Niigata, Japan
11. ¹¹Pathophysiology and Therapeutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan
12. ¹²Hokkaido University Hospital Gastroenterological Medicine, Sapporo, Japan
13. ¹³Cancer Preventive Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan

Correspondence: Professor A Inui, Department of Psychosomatic Internal Medicine, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan. E-mail: in...@m.kufm.kagoshima-u.ac.jp

Received 15 May 2015; Revised 1 December 2015; Accepted 15 December 2015
Advance online publication 2 February 2016

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Abstract

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP–CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

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Introduction

The increased lifespan of world population illustrates the success of modern medicine; however, the risk of developing many diseases increases exponentially with old age.¹ Pharmacological therapies that delay or prevent aging-related diseases, such as dementia, cancer, diabetes mellitus, osteoporosis and vascular disease, are highly desired. Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms.² The beneficial effects of CR are considered to be mediated by several signaling pathways, including sirtuin (SIRT), insulin-like growth factor-1 (IGF-1)/insulin, adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin. Hypothalamic SIRT1 is required for normal response to CR³ and regulates energy balance, which is implicated in aging/longevity.^{4, 5} However, key regulated hormone activated by CR or precise mechanisms involved remain uncertain.

Ghrelin is an acylated hormone that is secreted mainly from stomach in response to fasting and CR.⁶ Ghrelin receptors (growth hormone secretagogue receptor type 1a; GHS-R1a) are expressed in several organs, including the brain and heart.⁷ Ghrelin has much broader physiological functions as an orexigen, including stimulation of growth hormone (GH) secretion,⁸ gastrointestinal motility⁹ and cardiac contraction,¹⁰ inhibition of energy metabolism⁶ and insulin secretion^{11, 12} and inhibition of inflammation¹³ and apoptosis. Orexigenic effect of ghrelin is mediated by activated AMPK in neuropeptide Y neurons in the hypothalamic arcuate nucleus.^{14, 15, 16} AMPK enhances SIRT1 activity by increasing cellular nicotinamide adenine dinucleotide (NAD⁺) levels and regulates energy expenditure.¹⁷ Therefore, we hypothesized that in CR ghrelin has a role in protecting aging-related diseases through a mediation of SIRT1.

The traditional Japanese Kampo medicine rikkunshito is able to potentiate ghrelin signaling by stimulating ghrelin secretion via serotonin 2b/2c receptor antagonism and by enhancing GHS-R activity.^{18, 19, 20, 21, 22} Of the 43 major chemical compounds contained in rikkunshito, atractylodin shows a marked increase in ghrelin/GHS-R-binding activity and ghrelin-induced cytosolic calcium ion (Ca²⁺) concentration in GHS-R-expressing cells through an allosteric mechanism. Atractylodin is a major ingredient detected in the plasma of rikkunshito-treated healthy volunteers.²³

In this study, we examined the impact of ghrelin signaling on the survival of three mouse strains with different genetic and biochemical backgrounds. These animal models mimic accelerated or normal human aging. These models are useful in studying the common protective mechanisms regulating many age-related diseases. We show here that the elevated plasma acyl ghrelin concentration was observed in homozygous klotho mutant (kl/kl; klotho-deficient) and senescence-accelerated mouse prone/8 (SAMP8) mice. The treatment with ghrelin antagonist (D-Lys3)-GHRP-6 hastened death, whereas the ghrelin signaling potentiators rikkunshito and atractylodin, but not ghrelin, prolonged survival in klotho-deficient mice. Rikkunshito extended the survival of another pathological model of both SAMP8 and aged ICR mice, a model of normal aging. These results suggest the importance of potentiating endogenous ghrelin signaling or attenuation of ghrelin resistance in the extension of lifespan in animal models of human aging. Several age-related diseases were also ameliorated in all rodent models, including improved cardiac involvement in both klotho-deficient and SAMP8 mice and improved learning in aged ICR mice. The effects on survival were mostly independent of the orexigenic activity and interactions with insulin and IGF-1 signaling. As a protective mechanism against the aging process, ghrelin signaling was considered to be associated with increased SIRT1 activity and decreased microglial activation in the brain.

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Materials and methods

Klotho-deficient, SAMP8 and ICR mice as models of aging and GHS-R knockout mice were used. All experimental procedures were performed according to the ‘Guidelines for the Care and Use of Laboratory Animals’ approved by each Laboratory Animal Committee. The detailed description of the materials and methods is provided in Supplementary Information.

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Results

Role of ghrelin in aging process of klotho-deficient mice

A defect in klotho gene expression in mice leads to systemic age-dependent degeneration and a reduced lifespan. Multiple degenerations occur after 4 weeks of age, and premature death occurs at approximately 2 months of age.²⁴ We examined the potential role of ghrelin signaling in klotho-deficient mice. We found that 5-week-old klotho-deficient mice showed an increase in acyl ghrelin, desacyl ghrelin, GH and corticosterone under fed or fasted conditions, while a decrease in acyl ghrelin/desacyl ghrelin (A/D) ratio suggestive of cachectic state, IGF-1, insulin and glucose were observed in klotho-deficient mice (Supplementary Figure S1a). The hypothalamic gene expression of neuropeptide Y and agouti-related peptide increased and proopiomelanocortin expression decreased in fasted klotho-deficient mice (Supplementary Figure S1b). These hormonal changes were consistent with those observed in cachexia.^{18, 25}

The plasma GH concentration, but not IGF-1, increased immediately after ghrelin administration (100 μg kg⁻¹, intraperitoneally) in both klotho-deficient and age-matched wild-type mice (Supplementary Figure S2a). Klotho-deficient mice showed decreased food intake and body weight loss compared with wild-type mice, but ghrelin-induced increase in food intake or body weight was not observed in contrast to wild-type mice (Supplementary Figure S2b). These findings suggest that klotho-deficient mice have a marked resistance to the appetite-stimulating effect of ghrelin. Daily ghrelin administration (30 or 100 μg kg⁻¹, intraperitoneally twice a day) failed to prolong survival, but ghrelin antagonist (D-Lys3)-GHRP-6 (10 μmol kg⁻¹, intraperitoneally) decreased the median survival without changing body weight in klotho-deficient mice (Figures 1a and b and Supplementary Figure S2c). These findings indicate that endogenous ghrelin signaling is important in preventing premature death, but ghrelin resistance appears to cancel ghrelin’s effect, similar to cancer cachexia.^{18, 25}

Figure 1.

Role of ghrelin in aging in klotho-deficient mice. (a and b) Daily ghrelin (a) administration failed to prolong survival, but the growth hormone secretagogue receptor (GHS-R) antagonist (D-Lys3)-GHRP-6 (b) decreased median survival. *P<0.05. (c–f) Daily administration of rikkunshito (RKT) (c) and atractylodin (d) prolonged survival in klotho-deficient mice. (e) There was no effect of RKT on the weight loss of klotho-deficient mice. (f) Myocardial calcification, but not pericarditis, was observed in klotho-deficient mice. It was significantly decreased by RKT (1000 mg kg⁻¹, p.o.) treatment. *P<0.05, **P<0.01. (g–i) There was no significant effect of RKT on food intake (g) for 24 h when klotho-deficient mice resided in individual houses. Four-day treatment with RKT (1000 mg kg⁻¹, p.o.) did not affect plasma acyl ghrelin concentrations (h) but increased sirtuin1 (SIRT1) activity in the hypothalamus and SIRT1 protein expression in the heart (i) in klotho-deficient mice under the fed condition, which is used here because fasting is a severe stress leading to death in this model. *P<0.05, **P<0.01 (n=8–10). (j and k) The inflammatory activation of microglia in the brains of Klotho-deficient mice (Klo) was considerably reduced in the existence of RKT. (k) The number of aberrantly activated microglia with amoeboid morphology (arrow heads) decreased. **P<0.01 (n=14). cont; wild-type mice.

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Daily rikkunshito (1000 mg kg⁻¹, p.o.) and atractylodin (1 mg kg⁻¹, p.o.) administration significantly increased median survival (Figures 1c and d), without change in body weight or aging score in klotho-deficient mice (Figure 1e and Supplementary Figures S3a and b). A significant decrease in myocardial calcification was observed in rikkunshito-treated mice (Figure 1f and Supplementary Table S1). Rikkunshito (1000 mg kg⁻¹, p.o.) did not affect 24-h food intake (Figure 1g) and failed to stimulate ghrelin secretion (Figure 1h) on day 4 in klotho-deficient mice. The other hormonal parameters, except insulin, were not changed by rikkunshito treatment (Supplementary Figure S3d). However, rikkunshito increased SIRT1 activity in the hypothalamus of klotho-deficient mice. The SIRT1 activity in the heart was difficult to determine owing to its extremely low levels, but a significant increase in heart SIRT1 protein by rikkunshito was observed in klotho-deficient mice (Figure 1i).

Klotho-deficient mice showed increased hypothalamic gene expression of interleukin-6 and tumor necrosis factor-α, which were not affected by rikkunshito and atractylodin administration (Supplementary Figure S4a). A microarray analysis of hypothalamic gene expression in klotho-deficient mice demonstrated that rikkunshito and atractylodin markedly improved the expression of age-related genes affecting inflammation, apoptosis, DNA repair and migration, with a modest change in proopiomelanocortin, IGF-1 and arginine vasopressin expression (Supplementary Figures S4b and c). Behavioral analyses could not be performed in klotho-deficient mice owing to their fragility.

Antiaging effect of ghrelin signal potentiator rikkunshito in SAMP8 mice

Senescence-accelerated mice have been inbred selectively based on age-associated pathological phenotypes. SAMP8 mice show age-related deficits and a shorter lifespan.²⁶ The median survival of SAMP8 mice was shorter than senescence-accelerated mouse resistant/1 (SAMR1) mice; however, it was significantly increased by the daily rikkunshito (1%) administration (Figure 2a), with no change in the aging score (Supplementary Figure S5a). Decreases in food intake and body weight were observed in both SAMP8 and SAMR1 mice during aging process (Figure 2b). Food intake, body weight and food efficiency as expressed changes per 5 weeks were decreased in SAMP8 mice compared with SAMR1 mice. Rikkunshito improved food intake (Figure 2c and Supplementary Figure S5b). SAMP8 mice exhibited decreased locomotor activity, especially during the nighttime, which was recovered after rikkunshito administration (Figure 2d). There were no differences in anxiety-like behavior in an open-field test or memory disturbance in a step-through passive-avoidance test after rikkunshito treatment (Supplementary Figures S5c and d). Several pathological changes, such as pericarditis, atrophy of myocardial and muscle (sarcopenia) fiber (Figures 2e and f) and leukemia development (Supplementary Table S2), were significantly inhibited by the treatment.

Figure 2.

Antiaging effect of the ghrelin signaling potentiator rikkunshito in senescence-accelerated mouse prone/8 (SAMP8) mice. (a–f) Rikkunshito (RKT) improved the short lifespan (a) of SAMP8 (P8) mice. Decreases in food intake and body weight (b) were observed in both SAMP8 and SAMR1 (R1) mice during aging process, and RKT improved the rates of change in food intake (c) in SAMP8 mice. Thirty-nine-week-old SAMP8 mice exhibited decreased locomotor activity (d), which was recovered after rikkunshito administration. Pathological changes, such as pericarditis and atrophy of myocardial (e) and muscle fibers (f), at the end of the survival study were inhibited in RKT (1%)-treated mice. The mean dose of RKT was 600 mg kg⁻¹ (0.5%) and 1240 mg kg⁻¹ (1%) per day during the survival study. *P<0.05, **P<0.01 (n=17–20). (g) A 19-week treatment with RKT (1%) did not affect plasma acyl ghrelin concentrations in SAMP8 mice. **P<0.01 (n=15–19). (h) RKT (1000 mg kg⁻¹, p.o. for 4 days) increased sirtuin1 (SIRT1) activity in the hypothalamus, but not SIRT1 protein expression in the heart, in 18-week-old SAMP8 mice. *P<0.05 (n=9–10). (i and j) RKT (1%) significantly ameliorated microglial pathological activation in the brains of SAMP8 (P8) mice, in which the number of Iba-1-positive amoeboid microglia (arrow heads) decreased. DAPI (4,6-diamidino-2-phenylindole) staining demonstrates the nucleus. The scale bar indicates 100 μm. **P<0.01 (n=14–16). R1; SAMR1 mice.

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Significant increases in the plasma concentrations of acyl ghrelin, desacyl ghrelin and GH were observed in 42-week-old SAMP8 mice compared with age-matched SAMR1 mice (Figure 2g and Supplementary Figure S6a). Increases in the hypothalamic gene expression of interleukin-1β, tumor necrosis factor-α and ionized calcium-binding adaptor molecule 1 (Iba-1), a microglia marker, were observed in SAMP8 mice (Supplementary Figure S6b). Rikkunshito treatment (1% for 19 weeks) increased plasma IGF-1 concentration but did not affect cytokines and Iba-1 expression (Supplementary Figure S6).

Gastric mucosal atrophy was observed in SAMP8 mice. The decrease in gastric pit thickness and the numbers of ghrelin-positive cells and the increase in activated macrophage were recovered by rikkunshito (Supplementary Figure S7). The treatment with rikkunshito increased SIRT 1 activity in the hypothalamus but had no effect on SIRT1 protein expression in the heart of SAMP8 mice (Figure 2h).

Antiaging effect of rikkunshito in ICR and GHS-R knockout mice

As a model of normal aging, 16- to 18-month-old ICR mice were used. They were assessed and grouped using aging scores and body weight reflecting growth curve because of a difference in the age of the animals available. Rikkunshito (0.5% and 1%) prolonged the median survival (Figure 3a), with no effect on food intake, body weight or aging scores in ICR mice (Figure 3b and Supplementary Figure S8a). This animal model exhibited the focal atrophy of myocardial fiber but not cardiac calcification and pericarditis, which was inhibited by rikkunshito (1%) treatment (Figure 3c and Supplementary Table S3). Rikkunshito facilitated the memory consolidation of passive avoidance learning in ICR mice 2 months after treatment (Figure 3d). There were no differences in anxiety-like behavior in the open-field and elevated plus-maze tests (Supplementary Figures S8b and c).

Figure 3.

Antiaging effect of the ghrelin signaling potentiator rikkunshito in ICR and growth hormone secretagogue receptor (GHS-R) knockout mice. (a–d) Rikkunshito (RKT) prolonged the median survival (a) in ICR mice. Twenty-four-hour food intake and body weight (b) were not affected by RKT treatment. Pericarditis (c) was rarely observed, while focal atrophy of myocardial fiber was inhibited by RKT treatment in ICR mice after death. Two-month treatment with RKT facilitated the memory consolidation of passive avoidance learning (d) in ICR mice. The mean dose of RKT was 420 mg kg⁻¹ (0.5%) and 850 mg kg⁻¹ (1%) per day in this study. *P<0.05, **P<0.01 (n=19-23). (e and f). No significant change of plasma concentrations of acyl ghrelin (e) and SIRT1 (f) were observed between 4-month-old (young) ICR mice and 26-month-old (aged) ICR mice. Eight-month treatment with RKT (1%) did not affect plasma acyl ghrelin concentrations and sirtuin1 (SIRT1) protein expression in the heart but increased SIRT1 activity in the hypothalamus in aged ICR mice. *P<0.05 (n=6–8). (g and h) RKT significantly decreased the number of amoeboid microglia (arrow head) in the brains of aged ICR mice, while inflammatory activation of microglia maintained in the brains of control ICR mice. *P<0.05 (n=8). (i) Hypothalamic SIRT1 activity decreased in RKT-treated GHS-R knockout mice. *P<0.05 (n=5–7).

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No significant change of plasma concentrations of acyl ghrelin was observed in 26-month-old aged ICR mice compared with 4-month-old young ICR mice. Eight-month treatment with rikkunshito did not affect plasma acyl ghrelin concentration and SIRT1 protein expression in the heart but increased SIRT 1 activity in the hypothalamus of aged ICR mice (Figures 3e and f).

Twelve-week-old GHS-R knockout, heterozygous and wild-type C57BL/6 mice were treated with rikkunshito (1%) containing chow or control chow for 4 weeks. Hypothalamic SIRT1 activity decreased in rikkunshito-treated GHS-R knockout mice (Figure 3i).

Activation of SIRT1 pathway by ghrelin signaling

We examined the cellular effects of ghrelin signaling on SIRT1 activity in GHS-R1a-expressing human embryonic kidney (HEK) 293 (293-GHS-R) cells. Ghrelin and rikkunshito increased SIRT1 activity in 293-GHS-R cells (Figures 4a and b), the effect of which was enhanced by treatment with both ghrelin and rikkunshito (Figure 4c).

Figure 4.

Activation of sirtuin1 (SIRT1) by ghrelin and rikkunshito (RKT) in growth hormone secretagogue receptor type 1a (GHS-R1a)-expressing HEK293 (293-GHS-R) cells. (a–c) Ghrelin (a) and RKT (b) increased SIRT1 activity in 293-GHS-R cells, which was enhanced by treatment with both ghrelin and RKT (c). *P<0.05, **P<0.01 (n=6). (d and e) Activation of SIRT1 by RKT was observed in 293-GHS-R cells (d) and mock cells (293-Mock) (e), which was inhibited by treatment with GHS-R inverse agonist (SP-A) in 293-GHS-R cells but not in mock cells. *P<0.05, **P<0.01 (n=6). (f) RKT potentiated ghrelin-induced intracellular Ca²⁺ flux in 293-GHS-R cells. *P<0.05 (n=4). (g–i) Ghrelin (g) increased cyclic adenosine monophosphate (cAMP) in 293-GHS-R cells, the effect of which was enhanced by treatment with RKT (h). RKT (i) increased cAMP in 293-GHS-R cells, which was inhibited by treatment with SP-A. *P<0.05, **P<0.01 (n=6).

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GHS-R was found as a G-protein coupled receptor (GPCR) that initiates multiple intracellular signaling cascades.²⁷ GPCRs coupled to Gαq/11 activate phospholipase C, which opens the endoplasmic inositol trisphosphate-gated Ca²⁺ channel and activates protein kinase C through production of diacylglycerol. On the other hand, GPCRs coupled to Gαs activate adenylyl cyclase, produce cyclic adenosine monophosphate (cAMP) and activate protein kinase A (PKA) and other downstream effectors. In this study, ghrelin elicited an increase in intracellular Ca²⁺ concentration in 293-GHS-R cells (Figure 4f). Rikkunshito had no effect on the intracellular Ca²⁺ in 293-GHS-R cells and mock-transfected (293-Mock) cells. However, it potentiated the ghrelin-induced Ca²⁺ flux in 293-GHS-R cells (Figure 4f), as we previously showed in COS cells and hypothalamic neurons^{18, 19} by rikkunshito and atractylodin. The impedance-based cell assay showed that rikkunshito potentiated the subsequent change in intracellular signaling induced by ghrelin, but not by vehicle, in 293-GHS-R cells (Supplementary Figure S9a). Gastric vagal afferent activity decreased in rats treated with ghrelin or rikkunshito. Similar effect was observed following the administration of its constituents atractylodes lancea rhizome, poria sclerotium and citrus unshiu peel (Supplementary Figure S10a).

Ghrelin increased intracellular cAMP in 293-GHS-R cells, which was also enhanced by treatment with rikkunshito (Figures 4g and h). It was reported that cAMP response element binding (CREB) in the brain induced by CR upregulates SIRT1 gene transcription, resulting in neuroprotective responses that oppose brain aging.²⁸ In the present study, ghrelin increased cAMP response element (CRE) reporter activity and phosphorylated CREB in 293-GHS-R cells and the effect was augmented by rikkunshito (Figure 5a and Supplementary Figure S11). These results suggest that ghrelin-induced SIRT1 activity is mediated by the cAMP–CREB pathway.

Figure 5.

cAMP response element (CRE) reporter activity. (a and b) Rikkunshito (RKT) increased CRE reporter activity, which was enhanced by treatment with ghrelin and inhibited by treatment with growth hormone secretagogue receptor (GHS-R) inverse agonist (SP-A) in GHS-R1a-expressing HEK293 (293-GHS-R) cells (a) but not in mock cells (b). **P<0.01 (n=3). (c–f) RKT-induced CRE reporter activity was suppressed by protein kinase A inhibitor H89 but not by mitogen-activated protein kinase kinase inhibitor U0126 and enhanced by phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) in 293-GHS-R cells (c and e) and mock cells (d and f). **P<0.01 (n=3). (g) In human osteosarcoma (U2OS) cells, RKT increased CRE reporter activity, which was not influenced by treatment with ghrelin or SP-A (n=3).

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Rikkunshito increased cAMP (Figure 4i), CRE reporter activity (Figure 5a) and SIRT1 activity (Figure 4d) in 293-GHS-R cells without ghrelin treatment. These effects were inhibited by the treatment with GHS-R inverse agonist (D-Arg1, D-Phe5, D-Trp7,9, Leu11)-substance P (SP-A) (Figures 4d and i and 5a), suggesting the mediation by GHS-R that shows ligand-independent constitutive signaling.²⁹ Rikkunshito-induced CRE reporter activity was suppressed by the PKA inhibitor H89 but not by the mitogen-activated protein kinase kinase inhibitor U0126 and enhanced by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (Figures 5c and e). Additionally, these effects of rikkunshito were observed in 293-Mock cells and human osteosarcoma (U2OS) cells (Figures 4e and 5b, d, f and g), suggesting a partial involvement of GHS-R-independent signaling related to cAMP/PKA pathway.

Next, the effect of ghrelin on SIRT1 was examined in human umbilical vein endothelial cells (HUVECs), which express both ghrelin and GHS-R1a.³⁰ SIRT1 (ref. 31) and AMPK³² protect against age-associated vascular endothelial dysfunction. We found that rikkunshito elicited increases in SIRT1 activity and protein expression in HUVECs, as in ghrelin and atractylodin (Supplementary Figures S12a and b). These effects of rikkunshito and atractylodin were inhibited by treatment with (D-Lys3)-GHRP-6 or SP-A (Supplementary Figures S12a and c).

Calcium/calmodulin-dependent protein kinase kinase-β (CamKKβ) acts upstream of AMPK in mammalian cells.³³ AMPK enhances SIRT1 activity by increasing cellular NAD⁺ levels, resulting in deacetylation and modulation of the activity of downstream SIRT1 targets.¹⁷ Activation of exchange protein directly activated by cAMP (Epac1), a cAMP effector protein, increases intracellular Ca²⁺ levels and activates the CamKKβ–AMPK pathway.³⁴ Rikkunshito elevated phosphorylated AMPK levels, which was inhibited by the treatment with (D-Lys3)-GHRP-6 (Supplementary Figure S12d), suggesting the involvement of GHS-R signaling. Additionally, the SIRT1 protein expression levels in HUVECs were increased by the AMPK activator 5-amino-4-imidazolecarboxamide-1-beta-d-ribofuranoside and decreased by the AMPK inhibitor Compound C (Supplementary Figure S12e), suggesting that AMPK induces SIRT1 protein expression.

Ghrelin in combination with rikkunshito inhibited the hydrogen peroxide-induced apoptosis of 293-GHS-R cells, a model of the oxidative cell death (Supplementary Figure S9b).

The inhibitory effect of SIRT1 pathway activation on microglia-mediated inflammation

Previous studies demonstrated that the activation of SIRT1 pathway ameliorated microglia-mediated inflammation through its inhibitory effect on receptor activator of nuclear factor-κB.³⁵ To examine the possible involvement of SIRT1 in amelioration of microglial inflammation, rikkunshito was orally administered to klotho-deficient mice, an animal model of progeria, and microglial pathological activation in the dissected brains was morphologically analyzed. Rikkunshito significantly reduced the number of pathologically activated microglia with amoeboid morphology, which was predominantly observed in the brains of non-administered klotho-deficient mice (Figures 1j and k). In the brains of treated mice, the morphology of microglia was reversed to that of ramified resting microglia, which was featured with fine processes and few cytoplasm. Rikkunshito also significantly decreased the number of amoeboid microglia in the brains of both SAMP8 (Figures 2i and j) and aged ICR mice (Figures 3g and h).

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Discussion

We found that ghrelin antagonist (D-Lys3)-GHRP-6 hastened death, whereas the ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases and prolonged survival in three mouse models of accelerated or normal human aging. These findings suggest that the elevated endogenous ghrelin signaling has an important role in preventing aging-related premature death.

Plasma acyl ghrelin concentrations were increased in both klotho-deficient and SAMP8 mice. It is most likely due to the anorexia and decreased body weight, because weight loss is a potent stimulus to ghrelin secretion in humans and animals.^{36, 37} Increased hypothalamic inflammatory cytokines observed in these mice may underlie the resistance to appetite-stimulating effects of ghrelin, the situation similar to cachexia associated with cancer and other diseases.^{18, 25} In SAMP8 mice only, rikkunshito improved in part the adaptive feeding and body weight response, suggesting that the effects on survival were mostly independent of the orexigenic activity.

Ghrelin, GHS and rikkunshito were reported to improve the GH–IGF-1 axis decline in aged animals and humans.^{38, 39, 40, 41} In this study of pathological aging, ghrelin increased GH but not IGF-1 in klotho-deficient mice and the GH–IGF-1 axis appeared not to downregulate in SAMP8 mice. Despite the studies indicating the importance of adequate levels of circulating GH and IGF-1 for healthy aging, the role of these anabolic hormones in the genesis of aging phenotypes and in the extension of lifespan remains highly controversial.⁴²

Neurodegenerative disorders are one of the most potent risk factors for any age-related diseases. CR delays brain senescence and prevents neurodegeneration.^{28, 43, 44} In particular, hypothalamus has a programmatic role in the development of whole-body aging via immune-neuroendocrine integration.⁴⁵ Enhanced ghrelin signaling improved the levels of neuronal genes involved in inflammation, apoptosis, DNA repair and migration in the hypothalamus of klotho-deficient mice. These findings are consistent with previous reports in which the beneficial effect of CR on age-related hypothalamic gene changes⁴⁶ and the neuroprotective effect of ghrelin⁴⁷ were observed. Cancer and dementia are the most debilitating conditions associated with aging. Rikkunshito facilitated memory consolidation of passive avoidance learning in aged ICR mice. The improved ghrelin signaling appears to protect against several age-associated neurodegenerative changes, including the pathology of brain and heart, as in CR.

Cardiovascular disease is one of the leading causes of death and disability in the aged society. Klotho gene polymorphisms in humans are associated with an altered risk for coronary artery disease.⁴⁸ Klotho attenuates cellular apoptosis and senescence in vascular cells.⁴⁹ CR attenuates oxidative stress and improves endothelial function in the aorta of aged F344 rats.⁵⁰ Recently, ghrelin have been demonstrated to have beneficial effects in the cardiovascular system through a combination of direct and indirect actions.¹⁰ Ghrelin improves cardiac function and survival in heart failure using a mouse model of inherited dilated cardiomyopathy.⁵¹ Ghrelin inhibits sympathetic nervous system activity and stimulates GH secretion, which may help treat cardiovascular diseases and cardiac cachexia.^{10, 52} Histological analysis in this study revealed that pericarditis and calcification and focal atrophy of cardiac muscle developed in SAMP8 or aged ICR mice were improved by the treatment with rikkunshito. Calcification was also significantly attenuated by rikkunshito in klotho-deficient mice, suggesting a reason for the better prognosis. Ghrelin was reported to attenuate myocardial calcification induced by nicotine and vitamin D3 via an increase and decrease in osteopontin and endothelin-1 expression, respectively, in the myocardium.⁵³ The mechanism through which leukemia, the main cause of death in SAMP8 mice, was inhibited by treatment with rikkunshito remains clarified in relation to ghrelin.

Changes in muscle mass during aging influence both lifespan and healthspan. The present study demonstrated improved atrophy of myocardial and muscle fiber by treatment with rikkunshito. These findings suggest ghrelin signaling improves age-related sarcopenia and could contribute to prolonged survival in mouse models. Rikkunshito administration recovered the decreased locomotor activity in SAMP8 mice. This result may also be mediated by improvement of muscle atrophy. Ghrelin and rikkunshito decreased the sympathetic nerve activity to brown adipose tissues. This finding suggests the reduced basal energy expenditure.

Ghrelin and rikkunshito increased SIRT1 activity and protein expression in 293-GHS-R cells and HUVECs through the GHS-R–cAMP–CREB pathway. GHS-Rs are expressed in several organs, including the hypothalamus and heart. Rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression in the heart in all three mouse models of aging. However, hypothalamic SIRT1 activity significantly decreased in rikkunshito-treated GHS-R knockout mice. These results suggest that SIRT1 activity in aged mice is mediated by ghrelin signaling.

Present findings that administration of rikkunshito to SAMP8, klotho-deficient and physiologically senescent mice resulted in the amelioration of microglial pathological activation are consistent with the previous report in which SIRT1 diminished the activation of nuclear factor-κB in microglia and the toxicity of amyloid β.³⁵ Recent studies demonstrated that gut and intestine influenced the microglial behavior in the brain and determines the prognosis of some neurological disorders, such as multiple sclerosis; however, the pathophysiology has not been appreciated well.^{45, 54} Our findings indicate that ghrelin signaling activates SIRT1, and treatment for ghrelin resistance may exert a protective effect against brain and other organ/tissue pathologies during the process of aging through the SIRT1 pathway. These outcomes may be independent of the orexigenic activity of ghrelin^{3, 55} (Supplementary Figure S13).

CR has been established to extend the lifespan and improve the health in several species. A long-term CR study in rhesus monkeys demonstrated that CR lowered the incidence of aging-related deaths and delayed the onset of age-associated pathologies, such as diabetes, cancer, cardiovascular disease and brain atrophy,^{56, 57} although these results are still somewhat controversial. Many studies have shown that CR results in reduced oxidative stress, which is considered to be a key mechanism underlying the aging process.⁵⁸ These CR effects are mediated by several signaling pathways, including SIRT, IGF-1/insulin, AMPK and mammalian target of rapamycin. These pathways may interact and may all have important roles in mediating different aspects of the response.⁵⁹ Rapamycin has been shown to extend both the mean and maximum lifespan, and the development of newer, safer antiaging therapies based on this analog are expected.⁶⁰

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Conclusion

The present study demonstrates that the key regulated hormones and mechanisms activated by CR should include ghrelin–GHS-R signaling pathways. The potentiation of ghrelin signaling may be an additional approach for the improvement of both healthspan and lifespan in modern aging societies.

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Conflict of interest

AI has received grant support from Tsumura. The other authors declare no conflict of interest.

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References

1. Heemels MT. Ageing. Nature 2010; 464: 503. | Article | PubMed |
2. Maxmen A. Calorie restriction falters in the long run. Nature 2012; 488: 569. | Article | PubMed |
3. Baur JA, Ungvari Z, Minor RK, Le Couteur DG, de Cabo R. Are sirtuins viable targets for improving healthspan and lifespan? Nat Rev Drug Discov 2012; 11: 443–461. | Article | PubMed | CAS |
4. Toorie AM, Nillni EA. Minireview: central Sirt1 regulates energy balance via the melanocortin system and alternate pathways. Mol Endocrinol 2014; 28: 1423–1434. | Article | PubMed |
5. Satoh A, Imai S. Hypothalamic Sirt1 in aging. Aging (Albany NY) 2014; 6: 1–2. | PubMed |
6. Inui A. Ghrelin: an orexigenic and somatotrophic signal from the stomach. Nat Rev Neurosci 2001; 2: 551–560. | Article | PubMed | ISI | CAS |
7. Gnanapavan S, Kola B, Bustin SA, Morris DG, McGee P, Fairclough P et al. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002; 87: 2988. | Article | PubMed | ISI | CAS |
8. Kojima M, Hosoda H, Date Y, Nakazato

[Uhohinc]

Scientists discover a big benefit to eating cinnamon

By Arden Dier

Published July 14, 2016

Newser

Facebook66 Twitter0 livefyre2 Email Print

Mmm cinnamon bun. (AP Photo/Matthew Mead)

Scientists say they've discovered "one of the safest and the easiest approaches to convert poor learners to good learners." And all you have to do is eat cinnamon.

Researchers at Rush University Medical Center say that feeding cinnamon to mice with a poor learning ability turned them into a bunch of brainiacs by transforming the part of the brain that controls memory.

Previous research has found poor learners have less of a protein vital to memory and learning, known as CREB, and more of a protein known as GABRA5 in the hippocampus.

However, poor-learning mice showed increased CREB and decreased GABRA5 after a month of daily cinnamon doses, study author Kalipada Pahan explains in a release. Essentially, the body converts cinnamon into sodium benzoate, which promotes healthy neurons, reports the Epoch Times.

The mice were then able to navigate a maze in half the time it took them before, even though the exit moved with each test. The ability was similar to that of so-called good-learning mice.

Mice who were given cinnamon but were already good learners, however, didn't exhibit any change. "We have successfully used cinnamon to reverse biochemical, cellular, and anatomical changes that occur in the brains of mice with poor learning," says Pahan, adding "if these results are replicated in poor learning students, it would be a remarkable advance." Interestingly, Pahan notes cinnamon is superior to straight doses of sodium benzoate because the chemical is slowly released from cinnamon but is "quickly excreted out through the urine" when taken on its own.

(This doesn't mean you should take the cinnamon challenge.)

This article originally appeared on Newser: Cinnamon Might Make Us Better Learners

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EdLowery 5ptsFeatured
5 days ago

But which cinnamon are we talking about here? Saigon or Ceylon? They taste about the same, but have significantly different chemical properties.

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witness2hope 5ptsFeatured
5 days ago

@EdLowery if you click the link to the release, it says they use Ceylon

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[MrPoonz1]

I'm buying cinnamon tonight after work, though I'm sure I'll get sick of it in a hurry... Expecting memory recall power close to that of a savant😈

[Uhohinc]

I checked on the  GABRA5 really not much on the internet to lead to anywhere. The research indicated they used Chinese and also Ceylon cinnamon. But nothing about how the lab animals or inference how a human eating cinnamon can get it thru the digestive enzymes, the gastrointestinal, and into the blood and past the blood brain barrier to reach the neurons in the hippocampus. I assume it does make it there. But then how is the cAMP binding response (CREB) reached as it is in the cells nuclei.

At least there has been enough abstracts to give methodology of pathways of how melanocyte stimulating hormone does this, or Scenesse will to some extent. To note, I do not know how much natural human msh/ or Scenesse or the various sub types of commercially available melanocyte stimulating hormone can also reach thru the blood brain barrier. Of course some of the natural human msh is known to be created in the left anterior lobe of the pituitary, therefore already in almost center of the brain itself.  If need be, I am certain Clinuvel could formulate an msh more specific to reach more past the bbb and more specific to other melanocortin receptors other than mostly number one.

On Tuesday, July 19, 2016 at 7:25:25 PM UTC-7, MrPoonz1 wrote:

[Uhohinc]

Agrp neurons mediate Sirt1's action on the melanocortin system and energy balance: roles for Sirt1 in neuronal firing and synaptic plasticity.
Author
Marcelo O Dietrich, Catiele Antunes, Gan Geliang, Zhong-Wu Liu, Erzsebet Borok, Yongzhan Nie, Allison W Xu, Diogo O Souza, Qian Gao, Sabrina Diano, Xiao-Bing Gao, Tamas L Horvath
Journal
JOURNAL OF NEUROSCIENCE SCI(E) SCOPUS

view options
11815p ~ 11840p ISSN 0270-6474 E-ISSN 1529-2401
Publisher
Medline
Cited Count
71
Partners
MEDLINE®/PubMed®
Paid
MEDLINE®/PubMed®
Category
Medicine > Neuroscience
Keywords
Agouti-Related Protein, biosynthesis, physiology, Animals, Carbazoles, administration & dosage, Drug Combinations, Eating, drug effects, Energy Metabolism, Female, Male, Melanocortins, metabolism, Melanocyte-Stimulating Hormones, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Mitochondria, Neuronal Plasticity, Neurons, Oxidation-Reduction, Sirtuin 1, antagonists & inhibitors, deficiency, Synapses, Synaptic Potentials
References
Cited Articles
References 51
1.
Sirt1 contributes critically to the redox-dependent fate of neural progenitors.
Timour Prozorovski et al. Nature Cell Biology
Biology cited 119 times
2.
Fasting-dependent glucose and lipid metabolic response through hepatic sirtuin 1.
Joseph T Rodgers et al. Proceedings of the National Academy of Sciences of the United States of America
General Sciences cited 162 times
3.
UCP2 mediates ghrelin's action on NPY/AgRP neurons by lowering free radicals.
Zane B Andrews et al. Nature
General Sciences cited 157 times
4.
Brain SIRT1: anatomical distribution and regulation by energy availability.
Giorgio Ramadori et al. JOURNAL OF NEUROSCIENCE
Neuroscience cited 79 times
5.
Exercise-induced synaptogenesis in the hippocampus is dependent on UCP2-regulated mitochondrial adaptation.
(2008) Marcelo O Dietrich et al. JOURNAL OF NEUROSCIENCE
Neuroscience cited 24 times
6.
A fasting inducible switch modulates gluconeogenesis via activator/coactivator exchange.
(2008) Yi Liu et al. Nature
General Sciences cited 166 times
7.
STAT3 inhibition of gluconeogenesis is downregulated by SirT1.
Yongzhan Nie et al. Nature Cell Biology
Biology cited 68 times
8.
Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation.
Aparna Purushotham et al. Cell Metabolism
Biotechnology cited 227 times
9.
Loss of GABAergic signaling by AgRP neurons to the parabrachial nucleus leads to starvation.
Qi Wu et al. CELL
General Sciences cited 100 times
10.
De novo neurogenesis in adult hypothalamus as a compensatory mechanism to regulate energy balance.
Andrew A Pierce et al. JOURNAL OF NEUROSCIENCE
Neuroscience cited 65 times
previous page 1 2 3 4 5 6 next page
Articles referencing this article 71
1.
Obesity is associated with hypothalamic injury in rodents and humans.
Joshua P Thaler et al. JOURNAL OF CLINICAL INVESTIGATION
General Medicine cited 294 times
2.
Circadian rhythms, sleep, and metabolism.
Wenyu Huang et al. JOURNAL OF CLINICAL INVESTIGATION
General Medicine cited 129 times
3.
Sirtuin 1 and sirtuin 3: physiological modulators of metabolism.
Ruben Nogueiras et al. PHYSIOLOGICAL REVIEWS
Physiology cited 89 times
4.
Sirtuin 1 in lipid metabolism and obesity.
Thaddeus T Schug et al. ANNALS OF MEDICINE
General Medicine cited 76 times
5.
The central Sirtuin 1/p53 pathway is essential for the orexigenic action of ghrelin.
Douglas A Velásquez et al. DIABETES
Internal Medicine cited 44 times
6.
Mitochondrial dynamics controlled by mitofusins regulate Agrp neuronal activity and diet-induced obesity.
Marcelo O Dietrich et al. CELL
General Sciences cited 36 times
7.
Protective effects and mechanisms of sirtuins in the nervous system.
Feng Zhang et al. Progress in Neurobiology
Neuroscience cited 30 times
8.
AMPK: regulating energy balance at the cellular and whole body levels.
D Grahame Hardie et al. Physiology
Physiology cited 30 times
9.
SIRT1 in neurodevelopment and brain senescence.
A Zara Herskovits et al. Neuron
Neuroscience cited 29 times
10.
Mammalian sirtuins and energy metabolism.
Xiaoling Li et al. International Journal of Biological Sciences
Biology cited 29 times
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[Uhohinc]

NDP-MSH binding melanocortin-1 receptor ameliorates neuroinflammation and BBB disruption through CREB/Nr4a1/NF-κB pathway after intracerebral hemorrhage in mice

• Xuan Wu, 
• Siming Fu, 
• Yun Liu, 
• Hansheng Luo, 
• Feng Li, 
• Yiying Wang, 
• Meng Gao, 
• Yuan Cheng & 
• Zongyi Xie 

Journal of Neuroinflammation volume 16, Article number: 192 (2019) Cite this article

• 3830 Accesses

• 44 Citations

• Metricsdetails

Abstract

Background

Neuroinflammation and blood-brain barrier (BBB) disruption are two vital mechanisms of secondary brain injury following intracerebral hemorrhage (ICH). Recently, melanocortin-1 receptor (Mc1r) activation by Nle4-D-Phe7-α-MSH (NDP-MSH) was shown to play a neuroprotective role in an experimental autoimmune encephalomyelitis (EAE) mouse model. This study aimed to investigate whether NDP-MSH could alleviate neuroinflammation and BBB disruption after experimental ICH, as well as the potential mechanisms of its neuroprotective roles.

Methods

Two hundred and eighteen male C57BL/6 mice were subjected to autologous blood-injection ICH model. NDP-MSH, an agonist of Mc1r, was administered intraperitoneally injected at 1 h after ICH insult. To further explore the related protective mechanisms, Mc1r small interfering RNA (Mc1r siRNA) and nuclear receptor subfamily 4 group A member 1 (Nr4a1) siRNA were administered via intracerebroventricular (i.c.v) injection before ICH induction. Neurological test, BBB permeability, brain water content, immunofluorescence staining, and Western blot analysis were implemented.

Results

The Expression of Mc1r was significantly increased after ICH. Mc1r was mainly expressed in microglia, astrocytes, and endothelial cells following ICH. Treatment with NDP-MSH remarkably improved neurological function and reduced BBB disruption, brain water content, and the number of microglia in the peri-hematoma tissue after ICH. Meanwhile, the administration of NDP-MSH significantly reduced the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9 and increased the expression of p-CREB, Nr4a1, ZO-1, occludin, and Lama5. Inversely, the knockdown of Mc1r or Nr4a1 abolished the neuroprotective effects of NDP-MSH.

Conclusions

Taken together, NDP-MSH binding Mc1r attenuated neuroinflammation and BBB disruption and improved neurological deficits, at least in part through CREB/Nr4a1/NF-κB pathway after ICH.

Background

Intracerebral hemorrhage (ICH) is a severe cerebral vascular disease with high morbidity and mortality, and its incidence is increasing annually [1]. Mounting evidence has demonstrated that neuroinflammation and blood-brain barrier (BBB) disruption are two critical mechanisms of ICH-induced brain injury, which are closely associated with poor prognosis [2]. Therefore, a therapeutic strategy targeting neuroinflammation and BBB disruption would be beneficial for attenuating brain injury following ICH.

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a member of the melanocortin family, a group of peptides derived from pro-opiomelanocortin (POMC) [3]. α-MSH exerts well-established roles in the regulation of skin pigmentation and energy homeostasis, as well as inflammatory reaction [4, 5]. The biological function of α-MSH is mediated by five melanocortin receptors (termed Mc1r to Mc5r) [6]. Melanocortin-1 receptor (Mc1r), a G protein-coupled receptor, is best known as a mediator of the synthesis of melanin pigments, and it is also implicated in inflammation which is regulated by NF-κB signaling pathway [7,8,9]. α-MSH is released from cells in the central nervous system; however, the chemical property of α-MSH is unstable, transformed into the protease-stable Nle4-D-Phe7-α-MSH (NDP-MSH), which has a specific higher affinity to Mc1r [8, 10, 11]. Treatment with NDP-MSH was proven to reduce inflammation and vasospasm after subarachnoid hemorrhage [12]. Likewise, the administration of NDP-MSH ameliorated blood-brain barrier (BBB) disruption by activating Mc1r in a model of experimental autoimmune encephalomyelitis (EAE) [13]. Despite the well-recognized roles of NDP-MSH and Mc1r on inflammation, the effects of NDP-MSH and Mc1r on neuroinflammation and BBB integrity after ICH have not been elucidated.

Nuclear receptor subfamily 4 group A member 1 (Nr4a1), a member of Nur nuclear receptor family of transcriptional factors, is involved in neuroinflammation as a regulator of microglia activation in EAE in mice [14]. A previous study indicated that Nr4a1 was induced and functions immediately downstream of Mc1r signaling in melanocytic cells [15]. Furthermore, Mykicki et al. showed that NDP-MSH binding to Mc1r initiated the phosphorylation of cAMP response element-binding protein (CREB), and activated Nr4a1, subsequently exerted long-lasting neuroprotective roles in mice with EAE [13]. It was reported that Nr4a orphan receptors could regulate NF-κB signaling in microglial and myeloid cells [16, 17]. Moreover, mounting evidence revealed that Nr4a1 negatively modulated the transcriptional activity of NF-κB and inhibited inflammatory gene expression [18,19,20,21].

In the present study, we hypothesized that Mc1r activation by NDP-MSH could attenuate neuroinflammation and preserve BBB integrity after experimental ICH, and the protective mechanism is mediated through CREB/Nr4a1/NF-κB pathway.

Methods

Animals

All experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University. The study complied with the National Institutes of Health guide for the care and use of Laboratory Animals and the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines. A total of 218 C57BL/6 mice (male, weight about 25 g) were purchased from and bred at the Animal Center of Chongqing Medical University. All mice were housed in a light- and temperature-controlled room with free access to food and water.

Experimental design

Four separate experiments were designed as follows (Fig. 1). A total of 218 mice were used (Additional file 1: Table S1).

Fig. 1

Experimental design and animal groups. ICH, intracerebral hemorrhage; Mc1r, melanocortin-1 receptor; Nr4a1, nuclear receptor subfamily 4 group A member 1; IF staining, immunofluorescence staining; WB, Western blot; Scr siRNA, scrambled siRNA

Full size image

Experiment 1

The time course of endogenous Mc1r in the peri-hematoma tissue was measured by Western blot. The cellular localization of Mc1r was detected by double immunofluorescence staining at 24 h after ICH.

Experiment 2

To evaluate the effects of NDP-MSH on neuroinflammation and BBB integrity, three doses of NDP-MSH (1.5, 5, 15 μg/mouse, Anaspec, USA) dissolved in phosphate-buffered saline (PBS) were administered intraperitoneally at 1 h after ICH insult. Mice were randomly divided into five groups: sham, ICH + vehicle (PBS), ICH + NDP-MSH (1.5 μg/mouse), ICH + NDP-MSH (5 μg/mouse), and ICH + NDP-MSH (15 μg/mouse). Neurological test and brain water content were examined at 24 and 72 h after ICH. Evans blue (EB) extravasation was evaluated at 24 h after ICH.

Experiment 3

To assess the effect of in vivo knockdown of Mc1r on neuroinflammation and BBB permeability, Mc1r small interfering RNA (siRNA) was administered by intracerebroventricular (i.c.v) injection at 48 h before ICH induction, and then followed with NDP-MSH (5 μg/mouse) treatment at 1 h after ICH. Neurological test, brain water content, EB extravasation, immunofluorescence staining, and Western blot were carried out at 24 h post-ICH. Mice were randomly divided into five groups: sham, ICH + vehicle (PBS), ICH + NDP-MSH (5 μg/mouse), ICH + NDP-MSH (5 μg/mouse) + scrambled siRNA (Scr siRNA), and ICH + NDP-MSH (5 μg/mouse) + Mc1r siRNA. In addition, to verify the knockdown efficiency of Mc1r siRNA, the expression of Mc1r in the right hemisphere was analyzed by Western blot. Mice were randomly assigned to four groups: Naive+Scr siRNA, Naive+Mc1r siRNA, ICH + Scr siRNA, and ICH + Mc1r siRNA.

Experiment 4

To investigate the underlying mechanisms of NDP-MSH-mediated neuroprotective effects, Nr4a1 siRNA was administered by i.c.v injection at 48 h before ICH induction, and then followed with NDP-MSH (5 μg/mouse) treatment at 1 h after ICH. Neurological test, brain water content, and Western blot were implemented at 24 h following ICH. Mice were randomly allotted into five groups: sham, ICH + vehicle, ICH + NDP-MSH (5 μg/mouse), ICH + NDP-MSH (5 μg/mouse) + Scr siRNA, and ICH + DNP-MSH (5 μg/mouse) + Nr4a1 siRNA. Moreover, to validate the knockdown efficiency of Nr4a1 siRNA, the expression of Nr4a1 was measured by Western blot. Mice were randomly assigned to four groups: Naive + Scr siRNA, Naive + Nr4a1 siRNA, ICH + Scr siRNA, and ICH + Nr4a1 siRNA.

ICH mouse model induction

The ICH model was induced by autologous blood injection as previously described [22]. Briefly, the mice were anesthetized and fixed prone in a stereotaxic frame. Drill a small hole about 1 mm in diameter at 2 mm to the right of the bregma. Then 30 μl autologous arterial blood without anticoagulation was drawn from the central artery of the tail and delivered into the basal ganglion (stereotaxic coordinates: 0.2 mm anterior, 2.3 mm right lateral to the bregma, and 3.5 mm ventral to the skull). Firstly, 5 μl of blood was injected at 0.7 mm above the target position. Five minutes later, the remaining 25 μl blood was delivered at 3.5 mm depth. The needle was left for 10 min more after injection and withdrawn slowly at a rate of 1 mm/min. Bone wax was then applied to cover the drilled hole. The sham-operated animals were delivered an equal volume of sterile saline at the same position.

Intracerebroventricular injection

Intracerebroventricular injection was performed as previously described [23]. Briefly, mice were anesthetized and placed in a stereotactic head frame in the prone position. A longitudinal incision was made along the midline and a burr hole was drilled to the right of the bregma (1.0 mm lateral of the bregma). Following the manufacturer’s instructions, Mc1r siRNA (Thermo Fisher Scientific, USA, MSS275666, GCG AUU CUG UAU GCC CAC AUG UUC A, UGA ACA UGU GGG CAU ACA GAA UCG C), Nr4a1 siRNA (Thermo Fisher Scientific, USA, MSS205160, GAA GAU GCC GGU GAC GUG CAA CAA U, AUU GUU GCA CGU CAC CGG CAU CUU C), or scramble siRNA was dissolved in sterile RNase-free water. Mc1r siRNA mixture or scramble siRNA (100 pmol/2 μl) was delivered into the ipsilateral ventricle at the depth of 2.5 mm. The needle was left for an additional 5 min after injection to avert possible leakage and was slowly withdrawn at a rate of 1 mm/min. The burr hole was sealed with bone wax, and the incision was closed with sutures. Mice were placed in an individual recovery cage.

Neurobehavioral function test

Neurobehavioral functions were evaluated using the modified Garcia test and corner turn test at 24 or 72 h following ICH by a blinded investigator as previously described [24]. In the modified Garcia test, seven items including spontaneous activity, axial sensation, vibrissae touch, limb symmetry, lateral turning, forelimb walking, and climbing were tested. In the corner turn test, mice were allowed to approach a 30° corner. The mice exited the corner with either a right turn or left turn. Ten trials were performed, with at least a 30-s break between the trials. The percentage of a right turn to 10 trials was then calculated.

BBB permeability

To evaluate BBB permeability, Evans blue (Aladdin, China) was injected intraperitoneally (100 μl of 4% solution in saline) as previously described with a slight modification [25]. After 3 h circulation, mice were transcardially perfused with cold phosphate-buffered saline (0.1 M, PBS, pH 7.4) under deep anesthesia. Afterwards, the brain was removed and divided into left and right hemispheres and stored at − 80 °C immediately. The right part of the brain was homogenized in 1100 μl PBS, sonicated, and centrifuged (12,000 g, 4 °C, 30 min). The supernatant was collected and added an equal amount of trichloroacetic acid (TCA) to incubate overnight by 4 °C. After centrifugation (12,000 g, 4 °C, 30 min), Evans blue stain was measured by spectrophotometer (Thermo Fisher Scientific, USA) at 610 nm.

Brain water content

Brain water content was measured at 24 h and 72 h after ICH by an investigator blind to group information as previously described [26]. In short, mice were sacrificed under deep anesthesia. The brain was immediately removed and cut into 4 mm coronal slice. The brain slice was separated into five parts: ipsilateral and contralateral basal ganglia, ipsilateral and contralateral cortex, and cerebellum. The cerebellum was retained as an internal control. Each part was immediately weighed on an electronic analytical balance (FA2204B, Techcomp, USA) to determine the wet weight (WW) and then dried at 100 °C for 72 h to determine the dry weight (DW). Brain water content (percentage) was calculated as [(WW − DW)/WW] × 100%.

Immunofluorescence staining

Double fluorescence staining was performed as described previously [27]. The mice were deeply anesthetized and were transcardially perfused with 20 ml ice-cold PBS followed by 20 ml of 4% paraformaldehyde at 24 h post-ICH. The whole brain was collected and then fixed in 4% paraformaldehyde for another 24 h. Afterwards, the brain was fixed in 20% sucrose solution until the tissue sink to the bottom followed by 30% sucrose solution for another 24 h. After being frozen at − 25 °C, the brain was cut into 10-μm-thick coronal sections using a cryostat (CM1860; Leica Microsystems, Germany). To conduct double immunohistochemistry staining, the brain sections were incubated with primary antibody of anti-ionized calcium-binding adaptor molecule 1 (Iba-1, 1:100, Abcam, ab153696), anti-glial fibrillary acidic protein (GFAP, 1:200, CST, 3670, AB_561049), anti-von Willebrand factor (vWF, 1:50, Santa Cruz, sc-365712, AB_10842026), anti-NeuN (1:100, Abcam, ab104224, AB_10711040), and anti-Mc1r (1:50, Genetex, GTX108190) overnight at 4 °C. After being incubated with the appropriate secondary antibody (1:200, Bioss) at 37 °C for 1 h, the sections were visualized and photographed with a fluorescence microscope (U-HGLGPS, OLYMPUS, Japan). Microphotographs were analyzed with cellSens Standard software. The numbers of Iba-1-positive cells were identified and counted in three different fields in peri-hematoma area from five random coronal sections per brain, and data were expressed as cells/field.

Western blotting

After mice were perfused with ice-cold PBS (0.1 M, pH 7.4) at 24 h post-operation, the peri-hematoma tissues were collected and stored in − 80 °C freezer until use. Western blotting was performed as previously described [28]. After sample preparation, equal amounts of protein were loaded onto an SDS-PAGE gel. After being electrophoresed and transferred to a PVDF membrane, the membrane was blocked 2 h at 37 °C followed by incubated with the primary antibody overnight at 4 °C. The primary antibodies were anti-Mc1r (1:1000, Abcam, ab180776), anti-Nr4a1 (1:500, Abcam, ab13851, AB_300679), anti-phospho-CREB (1:1000, cell signaling, 9198, Ser133, AB_2561044), anti-CREB (1:1000, cell signaling, 9197, AB_331277), anti-phospho-NF-κB p65 (1:1000, cell signaling, 3033, AB_331284), anti-NF-κB p65 (1:1000, cell signaling, 8242, AB_10859369), anti-IL-1β (1:1000, cell signaling, 31202), anti-TNF-α (1:1000, cell signaling, 11948, AB_2687962), anti-MMP-9 (1:500, Abcam, ab38898, AB_776512), anti-occludin (1:50000, abcam, ab167161, AB_2756463), anti-ZO-1 (1:1000, affinity, AF5145), anti-Lama5 (1:1000, abcam, ab184330), and anti-β-actin (1:5000, proteintech, 60008-1-Ig). The secondary antibodies (ZSGB-BIO) were incubated for 1 h at 37 °C. Immunoblots were then probed with an ECL Plus chemiluminescence reagent kit (4A Biotech) and visualized with the image system (Bio-Rad, Universal Hood III). All data were analyzed using the software ImageJ.

Statistics analysis

All data were expressed as mean and standard deviation (mean ± SD). All analyses were performed using SigmaPlot 11.0 and GraphPad Prism 6 (GraphPad software, San Diego, CA, USA). Firstly, Shapiro-Wilk normality test was implemented in determining data normality. For the data that conformed to normal distribution, one-way ANOVA analysis followed by Tukey’s post hoc test was used for multiple-group comparisons. For the data that failed the normality test, Kruskal-Wallis one-way ANOVA on ranks, followed by Tukey’s multiple comparison post hoc analysis was performed. Statistical differences between two groups were analyzed using Student’s unpaired, two-tailed t test. P value of less than 0.05 was defined statistically significant (Additional file 2).

Results

Mortality and exclusion

The total mortality of ICH mice was 9.34% (17/182) in this study. None of the sham group mice died. There was no significant difference in mortality rate among the experimental groups. Six mice were ruled out from this study due to no hemorrhage (Additional file 1: Table S1).

Expression of Mc1r after ICH

As shown in Fig. 2a, the Mc1r expression in the peri-hematoma tissue was significantly increased at 24 h and reached its peak at 72 h after ICH, when compared to the sham group. Double immunofluorescence staining showed that Mc1r was mainly expressed in the microglia, astrocytes, and endothelial cells in the peri-hematoma tissue at 24 h after ICH (Fig. 2c).

Fig. 2

Expression of Mc1r after intracerebral hemorrhage (ICH). a Representative Western blot band and quantitative analyses of Mc1r time-dependent expression from the peri-hematoma tissue after ICH. #P < 0.05 vs sham. n = 6 per group. b Representative brain sample with schematic illustration presenting the four regions in peri-hematoma area (indicated by black boxes). c Representative images of double immunofluorescence staining showed that Mc1r was colocalized with microglia (Iba-1), endothelium (vWF), astrocyte (GFAP), and neuron (NeuN) and at 24 h after ICH. n = 3 per group. Scale bar = 50 μm

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Administration of NDP-MSH improved neurological deficits and reduced brain edema and BBB permeability after ICH

The neurological deficits and brain edema were evidently worse at 24 and 72 h post-ICH in the ICH + vehicle and ICH + NDP-MSH (1.5 μg/mouse) groups, when compared with sham group. However, the administration of NDP-MSH (5 μg/mouse) and NDP-MSH (15 μg/mouse) significantly improved the neurological deficits (Fig. 3a, b) and reduced brain edema in ipsilateral basal ganglion (Fig. 3c). Based on these results, the optimal dose of NDP-MSH was 5 μg/mouse, which was used for the rest of the experiments. BBB permeability was assessed by EB extravasation in the right cerebral hemispheres. EB extravasation in the ICH + vehicle group was significantly increased at 24 h after ICH, whereas NDP-MSH treatment (5 μg/mouse) prominently decreased EB dye leakage compared with the ICH + vehicle group (Fig. 3d).

Fig. 3

The neuroprotective effects of NDP-MSH on neurological functions, brain water content, and blood-brain barrier permeability after intracerebral hemorrhage (ICH). Treatment with NDP-MSH significantly improved neurological deficits (a and b) and reduced brain water content (c) at 24 and 72 h, as well as decreased EB extravasation at 24 h after ICH (d). n = 6 for each group. Brain sections were divided into five parts: ipsilateral basal ganglia (ipsi-BG), contralateral basal ganglia (contra-BG), ipsilateral cortex (ipsi-CX), contralateral cortex (contra-CX), and cerebellum. #P < 0.05 vs sham; *P < 0.05 vs vehicle and NDP-MSH (1.5 μg)

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Mc1r in vivo knockdown aggravated neurological deficits, brain edema, and BBB disruption after ICH

To further investigate, the protective role of NDP-MSH and Mc1r siRNA was administered by i.c.v injection to knockdown the expression of endogenous Mc1r. Western blot showed that the Mc1r expression was inhibited by Mc1r siRNA at 72 h after injection (Fig. 4a). The knockdown of Mc1r abolished the protective effect of NDP-MSH on neurological functions (Fig. 4b, c), brain edema (Fig. 4d), and BBB integrity (Fig. 4e) at 24 h post-ICH.

Fig. 4

The effect of Mc1r siRNA on neurological functions, brain water content and BBB integrity at 24 h after ICH. a The expression of Mc1r was obviously reduced in the right hemisphere by Mc1r siRNA at 24 h post-ICH. &P < 0.05 vs Scr siRNA. b–e Knockdown of Mc1r using Mc1r siRNA aggravated neurological deficits and increased brain edema and BBB permeability at 24 h following ICH. n = 6 per group. #P < 0.05 vs sham; *P < 0.05 vs ICH + vehicle; @P < 0.05 vs ICH + NDP-MSH, and ICH + NDP-MSH + Scr siRNA. Scr siRNA, scrambled siRNA

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Effect of NDP-MSH treatment and knockdown of Mc1r on the expression of downstream molecules after ICH

Treatment with NDP-MSH increased phospho-CREB (p-CREB) expression in the peri-hematoma tissue at 24 h post-ICH, which increased the expression of downstream molecules including Nr4a1, ZO-1, occludin, and laminin-α5 (Lama5) and inhibited the expression of downstream inflammation-related proteins and MMP-9 (Fig. 5a-j), compared with ICH + vehicle group. In contrast, the knockdown of Mc1r using specific siRNA got opposite changes on the expression of downstream signaling molecules (Fig. 5a–j), compared with the ICH + NDP-MSH group.

Fig. 5

The effects of NDP-MSH treatment and knockdown of Mc1r on expression of downstream molecules at 24 h after ICH. a Representative Western blot bands of the downstream signaling pathway protein. b-j Densitometric quantification suggested that the administration of NDP-MSH, a agonist of Mc1r, prominently upregulated the levels of p-CREB, Nr4a1, ZO-1, occludin, and laminin-α5 (Lama5) at 24 h post-ICH . In addition, treatment with NDP-MSH significantly decreased p-NF-κB p65, IL-1β, TNF-α, and MMP-9 at the same time. In contrast, the knockdown of Mc1r led to a decrease of p-CREB, Nr4a1, ZO-1, occludin, and Lama5 and an increase of p-NF-κB p65, IL-1β, TNF-α, and MMP-9. #P < 0.05 vs sham; *P < 0.05 vs ICH + vehicle; @P < 0.05 vs ICH + NDP-MSH, and ICH + NDP-MSH + Scr siRNA. Scr siRNA, scrambled siRNA

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Treatment with NDP-MSH decreased microglial counts after ICH

We investigated whether the anti-inflammatory function of NDP-MSH was associated with the decrease in the numbers of microglia in peri-hematoma tissue. As presented in Fig. 6, the numbers of Iba-1-positive cells were dramatically increased in ICH + vehicle group at 24 h post-ICH. The administration of NDP-MSH significantly reduced the number of Iba-1-positive cells, whereas the knockdown of Mc1r abolished this effect.

Fig. 6

Microglial counts in the peri-hematoma area at 24 h following ICH. Representative microphotographs and quantification of Iba-1-stained microglia showed that NDP-MSH treatment reduced the number of Iba-1-positive cells, while this effect was reversed by Mc1r siRNA. #P < 0.05 vs sham; *P < 0.05 vs ICH + vehicle; @P < 0.05 vs ICH + NDP-MSH. n = 3 per group, scale bar = 50 μm

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Knockdown of Nr4a1 abolished the neuroprotective effects of NDP-MSH after ICH

To further determine whether the neuroprotective effects of NDP-MSH were regulated by Nr4a1, Nr4a1 siRNA was administered by i.c.v injection at 48 h before ICH induction and treated with NDP-MSH at 24 h post-ICH. Nr4a1 siRNA significantly decreased Nr4a1 expression at 72 h after injection (Fig. 7a). The knockdown of Nr4a1 exacerbated neurological impairments (Fig. 7b, c) and increased brain water content (Fig. 7d) at 24 h after ICH. Furthermore, the knockdown of Nr4a1 significantly increased the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9 with a decrease of ZO-1, occludin, and Lama5 in the peri-hematoma tissue (Fig. 7e–l).

Fig. 7

Knockdown of Nr4a1 reversed the neuroprotection of NDP-MSH following ICH. a The expression of Nr4a1 was significantly decreased in the right hemisphere by Nr4a1 siRNA at 24 h post-ICH. &P < 0.05 vs Scr siRNA. b–d Knockdown of Nr4a1 aggravated neurological deficits and increased brain edema at 24 h following ICH. e–l Knockdown of Nr4a1 reversed the neuroprotection of NDP-MSH-induced change in protein levels of p-NF-κB p65, IL-1β, TNF-α, MMP-9, ZO-1, occludin, and Lama5 after ICH. #P < 0.05 vs sham; *P < 0.05 vs ICH + vehicle; @P < 0.05 vs ICH + NDP-MSH, and ICH + NDP-MSH + Scr siRNA. Scr siRNA, scrambled siRNA

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Discussion

The novel findings in the present study were as follows: (1) Mc1r was significantly increased in the peri-hematoma tissue after ICH; (2) the administration of NDP-MSH attenuated brain edema and BBB disruption and improved neurological deficits following ICH; (3) treatment with NDP-MSH inhibited the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9, as well as increased the expression of p-CREB, Nr4a1, ZO-1, occludin, and Lama5, thereby ameliorated brain injury post-ICH; (4) knockdown of Mc1r and Nr4a1 by specific siRNA aggravated neurological deficits, BBB damage, and inflammatory response after ICH; (5) CREB/Nr4a1/NF-κB signaling pathway was the potential mechanism of neuroprotection of NDP-MSH. Taken together, our findings indicated that NDP-MSH, by binding to Mc1r, attenuated neruoinflammation and BBB disruption after ICH, which is at least in part mediated by CREB/Nr4a1/NF-κB signaling pathway.

An ongoing body of researches demonstrated that inflammatory reaction and BBB disruption are critical factors to induce secondary brain injury following ICH [29, 30]. Following ICH, blood components rapidly enter the cerebral parenchyma and cause an inflammatory response. Furthermore, intensive inflammatory cascades aggravate BBB disruption, contribute to blood components infiltration into the brain in turn, and subsequently trap in a vicious circle to exacerbate brain injury after ICH.

Numerous studies have revealed that α-MSH analog NDP-MSH could inhibit inflammation and preserve BBB integrity [12, 13, 31]. In rat microglial cells, NDP-MSH exerted its anti-inflammatory effect by promoting a M2-like phenotype in microglia [31]. Following subarachnoid hemorrhage, treatment with NDP-MSH reduced vasospasm and inflammation through the decrease in the phosphorylation of extracellular-signal-regulated kinases (ERK1/2) [12]. Furthermore, NDP-MSH preserved BBB integrity and ameliorated neuroinflammation by preventing immune cell infiltration into the brain in mice with EAE through Mc1r/CREB/Nr4a1 signaling pathway [13]. Consistent with previous findings, our results revealed that treatment with NDP-MSH contributed to the upregulation of p-CREB, Nr4a1, ZO-1, occludin, Lama5, and downregulation of MMP-9 and inflammation-related molecules, and thus, attenuated neuroinflammation and BBB breakdown after ICH.

NDP-MSH exerts an anti-inflammatory effect by binding to different melanocortin receptors (Mc1r to Mc5r) [10, 13, 32, 33]. However, it has been proven that NDP-MSH has a specific higher affinity for Mc1r than other receptors [8, 10, 11]. Mc1r is widely distributed among various cell types, including macrophage, neutrophils, endothelial cells, and astrocytes [10]. In the present study, we observed that Mc1r was mainly expressed in the microglia, astrocytes, and endothelial cells after ICH. Moreover, the knockdown of Mc1r with Mc1r siRNA significantly abolished neuroprotective effects of NDP-MSH by increasing the expression of the inflammation-related molecules and MMP-9 and by decreasing the expression of ZO-1, occludin, and Lama5. Therefore, it is reasonable to speculate that Mc1r activation mediates NDP-MSH-induced neuroprotective effects after ICH. However, the finding was different from the previous observations, which showed that activating Mc4r with NDP-MSH or RO27-3225 could alleviate inflammatory reaction in the animal model of testicular ischemia and ICH [34, 35]. We supposed that such discrepancy may be due to the difference in animal models and tissue types.

Nr4a1 has been shown to inhibit inflammatory response by regulating the transcriptional activity of NF-κB [14, 18,19,20]. Nr4a1 also regulated microvessel permeability by increasing endothelial nitric-oxide synthase expression and by destabilizing endothelial junctions [36]. The NF-κB signaling pathway is well-known to be involved in mediating inflammatory response and BBB integrity after stroke [28, 37]. In the current study, the knockdown of Nr4a1 increased the expression of p-NF-κB p65, IL-1β, TNF-α, and MMP-9; decreased the expression of ZO-1, occludin, and Lama5; and resulted in neuroinflammation and BBB disruption. Therefore, knockdown of Nr4a1 reversed the neuroprotective roles of NDP-MSH.

There are some limitations in our study. NDP-MSH had been reported to possess multiple beneficial properties in a central nervous system disease, including anti-inflammation, anti-apoptosis, and anti-oxidation [13, 31, 34, 38, 39]. In this study, we only investigated the neuroprotective functions of NDP-MSH on neuroinflammation and BBB integrity after ICH. Thus, we cannot rule out the possibility that NDP-MSH-mediated anti-apoptosis and anti-oxidation may be involved in the neuroprotective effects after ICH. Further studies are needed to explore other functions of NDP-MSH after ICH and its underlying mechanisms. Second, we did not investigate the NDP-MSH-induced long-term neurological benefits following ICH. In addition, we only used male mice in this study. Thus, we cannot infer the effect of NDP-MSH on female mice after ICH.

Conclusion

NDP-MSH binding Mc1r could alleviate neuroinflammation and BBB disruption and improve neurological impairments after ICH in mice. The neuroprotective role of NDP-SMH was mediated at least via CREB/Nr4a1/NF-κB signaling pathway (Fig. 8). Therefore, NDP-MSH might serve as a potential therapeutic agent against neuroinflammation for ICH patients.

Fig. 8

The schematic diagram of potential molecular mechanisms of neuroprotective effects of NDP-MSH through CREB/Nr4a1/NF-κB pathway after ICH

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Availability of data and materials

The data used in the present study are available from the corresponding author on reasonable request.

Abbreviations

BBB:

Blood-brain barrier

CREB:

cAMP response element-binding protein

GFAP:

Glial fibrillary acidic protein

Iba-1:

Ionized calcium-binding adaptor molecule 1

ICH:

Intracerebral hemorrhage

Mc1r:

Melanocortin-1 receptor

NDP-MSH:

Nle4-D-Phe7-α-MSH

Nr4a1:

Nuclear receptor subfamily 4 group A member 1

vWF:

von Willebrand factor

α-MSH:

α-Melanocyte-stimulating hormone

References

1. Hemphill JC 3rd, Adeoye OM, Alexander DN, Alexandrov AW, Amin-Hanjani S, Cushman M, George MG, LeRoux PD, Mayer SA, Qureshi AI, et al. Clinical performance measures for adults hospitalized with intracerebral hemorrhage: performance measures for healthcare professionals from the American Heart Association/American Stroke Association. Stroke. 2018;49:e243–61.

   Article Google Scholar 

2. Xi T, Jin F, Zhu Y, Wang J, Tang L, Wang Y, Liebeskind DS, He Z. MicroRNA-126-3p attenuates blood-brain barrier disruption, cerebral edema and neuronal injury following intracerebral hemorrhage by regulating PIK3R2 and Akt. Biochem Biophys Res Commun. 2017;494(1–2):144–51.

   Article CAS Google Scholar 

3. Brzoska T, Luger TA, Maaser C, Abels C, Bohm M. Alpha-melanocyte-stimulating hormone and related tripeptides: biochemistry, antiinflammatory and protective effects in vitro and in vivo, and future perspectives for the treatment of immune-mediated inflammatory diseases. Endocr Rev. 2008;29:581–602.

   Article CAS Google Scholar 

4. Anderson EJ, Cakir I, Carrington SJ, Cone RD, Ghamari-Langroudi M, Gillyard T, Gimenez LE, Litt MJ. 60 YEARS OF POMC: regulation of feeding and energy homeostasis by alpha-MSH. J Mol Endocrinol. 2016;56:T157–74.

   Article CAS Google Scholar 

5. Singh M, Mukhopadhyay K. Alpha-melanocyte stimulating hormone: an emerging anti-inflammatory antimicrobial peptide. Biomed Res Int. 2014;2014:874610.

   PubMed PubMed Central Google Scholar 

6. Catania A. Neuroprotective actions of melanocortins: a therapeutic opportunity. Trends Neurosci. 2008;31:353–60.

   Article CAS Google Scholar 

7. Holloway PM, Durrenberger PF, Trutschl M, Cvek U, Cooper D, Orr AW, Perretti M, Getting SJ, Gavins FNE. Both MC1 and MC3 receptors provide protection from cerebral ischemia-reperfusion-induced neutrophil recruitment. Arterioscler Thromb Vasc Biol. 2015;35:1936–44.

   Article CAS Google Scholar 

8. Holloway PM, Smith HK, Renshaw D, Flower RJ, Getting SJ, Gavins FNE. Targeting the melanocortin receptor system for anti-stroke therapy. Trends Pharmacol Sci. 2011;32:90–8.

   Article CAS Google Scholar 

9. Herraiz C, Garcia-Borron JC, Jimenez-Cervantes C, Olivares C. MC1R signaling. Intracellular partners and pathophysiological implications. Biochim Biophys Acta. 2017;1863:2448–61.

   Article CAS Google Scholar 

10. Catania A, Gatti S, Colombo G, Lipton JM. Targeting melanocortin receptors as a novel strategy to control inflammation. Pharmacol Rev. 2004;56:1–29.

    Article CAS Google Scholar 

11. Getting SJ. Targeting melanocortin receptors as potential novel therapeutics. Pharmacol Ther. 2006;111:1–15.

    Article CAS Google Scholar 

12. Gatti S, Lonati C, Acerbi F, Sordi A, Leonardi P, Carlin A, Gaini SM, Catania A. Protective action of NDP-MSH in experimental subarachnoid hemorrhage. Exp Neurol. 2012;234:230–8.

    Article CAS Google Scholar 

13. Mykicki N, Herrmann AM, Schwab N, Deenen R, Sparwasser T, Limmer A, Wachsmuth L, Klotz L, Köhrer K, Faber C, et al. Melanocortin-1 receptor activation is neuroprotective in mouse models of neuroinflammatory disease. Sci Transl Med. 2016;8:146.

    Article Google Scholar 

14. Rothe T, Ipseiz N, Faas M, Lang S, Perez-Branguli F, Metzger D, Ichinose H, Winner B, Schett G, Kronke G. The nuclear receptor Nr4a1 acts as a microglia rheostat and serves as a therapeutic target in autoimmune-driven central nervous system inflammation. J Immunol. 2017;198:3878–85.

    Article CAS Google Scholar 

15. Smith AG, Luk N, Newton RA, Roberts DW, Sturm RA, Muscat GE. Melanocortin-1 receptor signaling markedly induces the expression of the NR4A nuclear receptor subgroup in melanocytic cells. J Biol Chem. 2008;283:12564–70.

    Article CAS Google Scholar 

16. De Miranda BR, Popichak KA, Hammond SL, Jorgensen BA, Phillips AT, Safe S, Tjalkens RB. The Nurr1 activator 1,1-bis(3′-indolyl)-1-(p-chlorophenyl)methane blocks inflammatory gene expression in BV-2 microglial cells by inhibiting nuclear factor kappaB. Mol Pharmacol. 2015;87:1021–34.

    Article Google Scholar 

17. McEvoy C, de Gaetano M, Giffney HE, Bahar B, Cummins EP, Brennan EP, Barry M, Belton O, Godson CG, Murphy EP, et al. NR4A receptors differentially regulate NF-kappaB signaling in myeloid cells. Front Immunol. 2017;8:7.

    Article Google Scholar 

18. Hamers AA, van Dam L, Teixeira Duarte JM, Vos M, Marinkovic G, van Tiel CM, Meijer SL, van Stalborch AM, Huveneers S, Te Velde AA, et al. Deficiency of nuclear receptor Nur77 aggravates mouse experimental colitis by increased NFkappaB activity in macrophages. PLoS One. 2015;10:e0133598.

    Article Google Scholar 

19. Popichak KA, Hammond SL, Moreno JA, Afzali MF, Backos DS, Slayden RD, Safe S, Tjalkens RB. Compensatory expression of Nur77 and Nurr1 regulates NF-kappaB-dependent inflammatory signaling in astrocytes. Mol Pharmacol. 2018;94:1174–86.

    Article CAS Google Scholar 

20. Liu TY, Yang XY, Zheng LT, Wang GH, Zhen XC. Activation of Nur77 in microglia attenuates proinflammatory mediators production and protects dopaminergic neurons from inflammation-induced cell death. J Neurochem. 2017;140:589–604.

    Article CAS Google Scholar 

21. You B, Jiang YY, Chen S, Yan G, Sun J. The orphan nuclear receptor Nur77 suppresses endothelial cell activation through induction of IkappaBalpha expression. Circ Res. 2009;104:742–9.

    Article CAS Google Scholar 

22. Rynkowski MA, Kim GH, Komotar RJ, Otten ML, Ducruet AF, Zacharia BE, Kellner CP, Hahn DK, Merkow MB, Garrett MC, et al. A mouse model of intracerebral hemorrhage using autologous blood infusion. Nat Protoc. 2008;3:122–8.

    Article CAS Google Scholar 

23. Xie Z, Huang L, Enkhjargal B, Reis C, Wan W, Tang J, Cheng Y, Zhang JH. Recombinant Netrin-1 binding UNC5B receptor attenuates neuroinflammation and brain injury via PPARgamma/NFkappaB signaling pathway after subarachnoid hemorrhage in rats. Brain Behav Immun. 2018;69:190–202.

    Article CAS Google Scholar 

24. Krafft PR, McBride DW, Lekic T, Rolland WB, Mansell CE, Ma Q, Tang J, Zhang JH. Correlation between subacute sensorimotor deficits and brain edema in two mouse models of intracerebral hemorrhage. Behav Brain Res. 2014;264:151–60.

    Article Google Scholar 

25. Xie Z, Enkhjargal B, Reis C, Huang L, Wan W, Tang J, Cheng Y, Zhang JH. Netrin-1 preserves blood-brain barrier integrity through deleted in colorectal cancer/focal adhesion kinase/RhoA signaling pathway following subarachnoid hemorrhage in rats. J Am Heart Assoc. 2017;19:6.

    Google Scholar 

26. Tong LS, Shao AW, Ou YB, Guo ZN, Manaenko A, Dixon BJ, Tang J, Lou M, Zhang JH. Recombinant Gas6 augments Axl and facilitates immune restoration in an intracerebral hemorrhage mouse model. J Cereb Blood Flow Metab. 2017;37:1971–81.

    Article CAS Google Scholar 

27. Xie Z, Huang L, Enkhjargal B, Reis C, Wan W, Tang J, Cheng Y, Zhang JH. Intranasal administration of recombinant Netrin-1 attenuates neuronal apoptosis by activating DCC/APPL-1/AKT signaling pathway after subarachnoid hemorrhage in rats. Neuropharmacology. 2017;119:123–33.

    Article CAS Google Scholar 

28. Zhu Q, Enkhjargal B, Huang L, Zhang T, Sun C, Xie Z, Wu P, Mo J, Tang J, Xie Z, et al. Aggf1 attenuates neuroinflammation and BBB disruption via PI3K/Akt/NF-kappaB pathway after subarachnoid hemorrhage in rats. J Neuroinflammation. 2018;15:178.

    Article Google Scholar 

29. Zhou Y, Wang Y, Wang J, Anne Stetler R, Yang QW. Inflammation in intracerebral hemorrhage: from mechanisms to clinical translation. Prog Neurobiol. 2014;115:25–44.

    Article CAS Google Scholar 

30. Keep RF, Andjelkovic AV, Xiang J, Stamatovic SM, Antonetti DA, Hua Y, Xi G. Brain endothelial cell junctions after cerebral hemorrhage: changes, mechanisms and therapeutic targets. J Cereb Blood Flow Metab. 2018;38:1255–75.

    Article CAS Google Scholar 

31. Carniglia L, Ramirez D, Durand D, Saba J, Caruso C, Lasaga M. [Nle4, D-Phe7]-alpha-MSH inhibits toll-like receptor (TLR)2- and TLR4-induced microglial activation and promotes a M2-Like phenotype. PLoS One. 2016;11:e0158564.

    Article Google Scholar 

32. Saba J, Carniglia L, Ramirez D, Turati J, Imsen M, Durand D, Lasaga M, Caruso C. Melanocortin 4 receptor activation protects striatal neurons and glial cells from 3-nitropropionic acid toxicity. Mol Cell Neurosci. 2019;94:41–51.

    Article CAS Google Scholar 

33. Catania A, Lonati C, Sordi A, Leonardi P, Carlin A, Gatti S. The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning. Peptides. 2010;31:116–22.

    Article CAS Google Scholar 

34. Minutoli L, Bitto A, Squadrito F, Irrera N, Rinaldi M, Nicotina PA, Arena S, Magno C, Marini H, Spaccapelo L, et al. Melanocortin 4 receptor activation protects against testicular ischemia-reperfusion injury by triggering the cholinergic antiinflammatory pathway. Endocrinology. 2011;152:3852–61.

    Article CAS Google Scholar 

35. Chen S, Zhao L, Sherchan P, Ding Y, Yu J, Nowrangi D, Tang J, Xia Y, Zhang JH. Activation of melanocortin receptor 4 with RO27-3225 attenuates neuroinflammation through AMPK/JNK/p38 MAPK pathway after intracerebral hemorrhage in mice. J Neuroinflammation. 2018;15:106.

    Article Google Scholar 

36. Zhao D, Qin L, Bourbon PM, James L, Dvorak HF, Zeng H. Orphan nuclear transcription factor TR3/Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing endothelial junctions. Proc Natl Acad Sci U S A. 2011;108:12066–71.

    Article CAS Google Scholar 

37. Liu Y, Tang G, Li Y, Wang Y, Chen X, Gu X, Zhang Z, Wang Y, Yang GY. Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion. J Neuroinflammation. 2014;11:177.

    Article Google Scholar 

38. Ramirez D, Saba J, Turati J, Carniglia L, Imsen M, Mohn C, Scimonelli T, Durand D, Caruso C, Lasaga M. NDP-MSH reduces oxidative damage induced by palmitic acid in primary astrocytes. J Neuroendocrinol. 2019;31:e12673.

    Article Google Scholar 

39. Ramirez D, Saba J, Carniglia L, Durand D, Lasaga M, Caruso C. Melanocortin 4 receptor activates ERK-cFos pathway to increase brain-derived neurotrophic factor expression in rat astrocytes and hypothalamus. Mol Cell Endocrinol. 2015;411:28–37.

    Article CAS Google Scholar 

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Acknowledgements

The authors thank Prof. Mei Yang from the Department of Human Anatomy, Chongqing Medical University, for providing experimental assistance.

Funding

This work was supported by the National Natural Science Foundation of China (81771961) and Kuanren Talents Program of the Second Affiliated Hospital of Chongqing Medical University (201959).

Author information

Authors and Affiliations

1. Department of Neurosurgery, The Second Affiliated Hospital, Chongqing Medical University, 76th Linjiang Road, Yuzhong District, Chongqing, 400010, China

   Xuan Wu, Siming Fu, Yun Liu, Hansheng Luo, Feng Li, Yuan Cheng & Zongyi Xie

2. Department of Human Anatomy, Chongqing Medical University, Chongqing, 400016, China

   Yiying Wang & Meng Gao

Contributions

XW and ZX designed the research. XW, SF, YW, and MG performed the research. YL, HL, FL, and YC analyzed the data. XW and ZX wrote the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Zongyi Xie.

Ethics declarations

Ethics approval and consent to participate

All experimental protocols for this study were approved by the Animal Ethics Committee of Chongqing Medical University in accordance with the National Institutes of Health guidelines for the care and use of experimental animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Supplementary information

MOESM1 of NDP-MSH binding melanocortin-1 receptor ameliorates neuroinflammation and BBB disruption through CREB/Nr4a1/NF-ÎşB pathway after intracerebral hemorrhage in mice

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Table S1. Summary of experimental groups and mortality rate in the study

Experimental

G

roups

Neurological test

Brain

water content

Evans

blue

IF

staining

WB

Exclusion

Mortality

(%)

Subtotal

Experiment 1

Sham

6

0

0

6

ICH (6 h, 12 h, 24 h, 72 h, 7d)

3

6×5

2

5 (12.82%)

40

Experiment 2

sham

6

6

12

ICH+PBS

6

6

1

2 (13.33%)

15

ICH+NDP-MSH (1.5

μ

g)

6

6

0

2 (14.29%)

14

ICH+NDP-MSH (5

μ

g)

6

6

0

0

12

ICH+NDP-MSH (15

μ

g)

6

6

1

1 (7.14%)

14

Experiment 3

sham

3

6

0

0

9

ICH+PBS

3

6

0

1 (10%)

10

ICH+NDP-MSH (5

μ

g)

3

6

0

0

9

ICH

+NDP-MSH (5

μ

g)

+Scr siRNA

6

6

6

0

18

ICH+Mc1r siRNA

+NDP-MSH (5

μ

g)

6

6

3

6

1

3 (12%)

25

Na

i

ve

+Scr siRNA

3

0

0

3

Na

i

ve

+Mc1r siRNA

3

0

0

3

ICH+Scr siRNA

3

0

0

3

ICH+Mc1r siRNA

3

0

0

3

Experiment 4

ICH+Nr4a1 siRNA

+NDP-MSH (5

μ

g)

6

6

1

2 (13.33%)

15

Na

i

ve

+Nr4a1 siRNA

3

0

0

3

ICH+Nr4a1 siRNA

3

0

1 (25%)

4

Total

48

42

15

90

6

17 (9.34%)

218

ICH, intracerebral hemorrhage. PBS,

phosphate-buffered saline

. Mc1r, m

elanocortin-1 receptor

. Nr4a1,

n

uclear receptor subfamily 4 group A member 1

. siRNA,

small interfering

RNA.

1 / 2

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Additional File 1

Additional file 1: Table S1. Summary of experimental groups and mortality rate in the study.

Additional file 1: Table S1.

Summary of experimental groups and mortality rate in the study.

Additional file 2: Table S2.

The t statistic and degrees of freedom of results.

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