Diphereline 0.1

[Hasan Fogg]

Endometriosisan estrogen-dependent inflammatory disease affecting 10-25% of women, is associated with significant reductions in fertility and is one of the most common benign gynecological diseases. Retrograde menstruation with subsequent adhesion formation, invasion, and neovascularization are believed to give rise to the occurrence of endometriosis lesions. The most common locations for endometrial implants are the ovaries, fossa ovarica, uterosacral ligaments, and posterior cul-de-sac (1).

Although different medications are used to control endometriosis, their adverse effects following long-term use and recurrence of disease after discontinuation of therapy limit their applications. Additionally, these medications are not useful for endometriosis-associated infertility (2). Regarding the fact that no ideal medical treatment is available to control endometriosis, introducing new medical agents with minimal side effects and improved effectiveness for infertility treatment, are required.

Gonadotropin releasing hormone agonists (GnRHa) such as diphereline, as standard medications for the treatment of endometriosis, are able to induce inactivation and degeneration of endometrial implants via suppression of hypothalamic-pituitary-gonadal axis and ovarian estrogen production (3). GnRHa not only induces amenorrhea, but also may cause hot flush, depression, headache, hair loss, musculoskeletal stiffness, vaginal dryness and bone loss (4).

It is known that in women with endometriosis, the growth of endometrial cells within the peritoneal cavity is induced by inflammatory mechanisms (5); so, anti-inflammatory drugs are suggested to control endometriosis growth. Cyclooxygenase enzymes (COXs), known as prostaglandin-endoperoxide synthase, are responsible for formation of inflammatory mediators such as prostaglandins. COX-1 is expressed in almost all cells for maintenance of cell. COX-2 is produced at sites of inflammation, angiogenesis, and estrogenic cellular processes. Pharmacological inhibition of COX-2 was able to reduce the survival and growth of endometrial tissues at ectopic sites (6). NSAIDs (non-steroidal anti-inflammatory drugs) such as celecoxib, inhibit cyclooxygenase isoforms and induce gastrointestinal side effects (7).

Using herbal medicine has always played a significant role in Iranian culture and civilization and some of these herbs have been recommended for treatment of infertility- related diseases. Licorice (Glycyrrhiza glabra), is one of the most widely used herbal drugs in Iranian traditional medicine. Licorice root contains triterpene, saponins, flavonoids, isoflavonoids, hydroxycoumarins, steroids and volatile oil. Licoricidin, is a potent compound isolated from licorice root (8). Studies showed that licoricidin is a selective COX-2 inhibitor and inhibits phospholipase A2 activity that is a critical enzyme involved in numerous inflammatory processes (9, 10). Licorice root with its anti- inflammatory/anti-platelet, antiviral, antifungal and mineralocorticoid functions has been used for the treatment of gastric ulcers, cough and bronchitis since the ancient times. Licorice is not recommended to be used for more than 6 weeks. Complications such as hypokalemia, hypernatremia, edema, hypertension and cardiac complaints are associated with long-term time use of licorice (8).

We hypothesized that licorice or celecoxib might be good candidates for treatment of endometriosis as an inflammatory condition. In the present study, we compared the effects of licorice, celecoxib and diphereline on the growth of endometrial implants in rats.

In this experimental study, 48 mature female Sprague- Dawley rats (almost 8 weeks old, weighting 220 20 g) were purchased from the Center of Comparative and Experimental Medicine at Shiraz University of Medical Sciences (SUMS), Shiraz, Iran. The animals were kept on 12 hours light: 12 hours dark cycles at a controlled temperature with free access to water and food. The animal experiments were performed according to the principles of the care and use of laboratory animals established by the National Institutes of Health, Bethesda, MD, USA, and approved by the Institutional Animal Ethics Committee at SUMS (No. 92-01-01-6869). These animal experiments were performed in the animal house of Shiraz University of Medical Sciences. To select the rats with normal estrous cycle, daily vaginal smears were taken and evaluated by a light microscope. Rats with three normal estrous cycles were used in the next steps.

Licorice roots were purchased from herbal stores in Shiraz, Iran). Glycyrrhiza glabra was preserved in herbarium after authentication by a botanist (Voucher No. PM 684). L. Licorice roots were air-dried, powdered and an alcoholic extract was produced by using ethanol (80%) and percolation method. Then, solvent was completely removed by drying under reduced pressure in a rotary evaporator. The extract was stored at 4C until use.

Endometriosis was induced surgically using the method described by Vernon and Wilson with little modifications (11) (Fig .1). It should be mentioned that as the growth of endometriosis is estrogen-dependent, if induction of endometriosis is performed in an ovariectomized animal, estrogen supplementation is mandatory (11, 12). However similar to the previous researches, since in our study adult intact rats were used, we did not use exogenous estrogen for induction of endometriosis (11-13).

At this stage, 44 female rats were divided into 4 groups (11 rats in each group). The control group was treated by 0.5 ml of saline 0.9%/day, the second group by licorice root extract (3000 mg/kg/day) and the third group took celecoxib (Damloran Razak Pharmaceutical Co., Iran, 50 mg/kg, twice a day, dissolved in 0.5 ml of saline 0.9%) for the next 6 weeks. All the treatments of these three groups were administered by oral gavage. The fourth group received a single IM injection of diphereline S.R. 11.25 mg (3 mg/kg, Ipsen, France). Six weeks after the treatments the rats were sacrificed and endometrial implants were evaluated as shown in Figure 2.

Endometrial implants in different times and groups. A. The first laparotomy: auto-transplant of endometrial implants on the peritoneum, B. The second look surgery: shows the adhesion bands and endometrial implants six weeks after induction of endometriosis, C. The third surgery: necropsy of a rat in the control group showing growth of implanted lesions of endometriosis, and D. The third surgery: necropsy of a rat in diphereline group showing regression of the implants.

All of the endometrial implants were fixed in formalin, placed in paraffin, cut into 5 m sections, stained with hematoxylin-eosin and evaluated by the same pathologist. Photographs were taken by a digital camera (Sony, Japan). To classify the persistence of epithelial cells in grafts, the scoring system applied by Keenan et al. (14) was used with score 0 showing no epithelial layer, and scores 1, 2 and 3 show poorly, moderately and well-preserved epithelial layers, respectively. The percentage of hemosiderin-laden macrophages (HLMs) was also measured in all of the sections. The surgeon, pathologist, and the lab technicians were blinded to the groupings, medications, and specimens.

For statistical analysis, the software SPSS 15 (SPSS Inc., Chicago, USA) was employed. To compare the mean area and the mean volume, ANOVA followed by Tukey HSD test was performed. To compare the histopathologic scoring, Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction were applied. A P

Two rats died during laparotomy due to hemorrhage and in two other rats the implants did not grow. The remaining 44 rats were divided into 4 groups and treated. In licorice group, the mean area and volume values of endometrial implants were significantly lower than those of the control group (P=0.042 and P

Specimens of the treated groups stained by hematoxylin-eosin. A. Well preserved epithelial layer of endometrial implants in the control group (grade 3) (scale bar: 50 m), B. Poorly preserved epithelial layer of endometrial implants in the licorice group (grade 1) (scale bar: 50 m), C. Moderately preserved epithelial layer of endometrial implants in licorice group (grade 2) (scale bar: 100 m), and D. Poorly preserved epithelial layer of endometrial implants in diphereline group (grade 1) (scale bar: 100 m). Arrows demonstrate epithelial layer of endometrial implants.

The percentage of HLMs in endometrial implants of rats in celecoxib, licorice and diphereline group was significantly lower than that of the control group (P=0.004, P=0.000 and P=0.000, respectively). Also, the percentage of HLMs was significantly lower in licorice and diphereline group compared to celecoxib group (P

We compared the effects of licorice, celecoxib, and diphereline in a rat model of endometriosis induced by auto-transplantation of endometrium on the peritoneal surface as a well-established method (11). Licorice decreased the growth of endometrial implants; celecoxib had no significant effect and diphereline had the highest potency in suppression of the endometrial growth. According to our knowledge, this is the first study on the effect of licorice on the endometrial implants.

Previous studies showed that glycyrrhetinic acid as a constituent of licorice extract, inhibits thrombin-induced platelet aggregation (9, 15) and has steroid-like anti-inflammatory effects similar to glucocorticoids (16, 17). Park and colleagues showed that administration of hexane/ ethanol extract of Glycyrrhiza uralensis to mice decreases cell proliferation, inhibits the expression of angiogenic and inflammatory proteins and induces cell cycle arrest or apoptosis (18). Also, they observed that licoricidin reduces macrophages number and tumor growth in the tumor microenvironment. In another study, it was shown that licoricidin inhibits the metastatic and invasive capacity of malignant prostate cancer cells in vitro (19). La et al. (20) reported that licoricidin suppresses the production of inflammatory cytokines. The anti-inflammatory property of licoricidin is due, in part, to the inhibition of phospholipase A2 activity, resulting in inhibition of cyclooxygenase activity and prostaglandin formation (9, 16, 17). Licoricidin also inhibits an isomer of platelet-activating factor and acetyltransferase resulting in an anti-inflammatory activity (21).

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