| Background:Endometriosis is a chronic gynecologic disease that affects approximately 10%-20% of reproductive-age women. It is defined as the presence of endometrial tissue at extrauterine locations, most commonly on the ovaries and uterosacral ligament (USL). The pathogenesis of endometriosis is still unclear, however, it frequently results in chronic pelvic pain, severe dysmenorrhoea, and subfertility, and eventually severely affects the quality of life of the women.There are three types of endometriosis: superficial endometriosis, ovarian endometrioma and deeply infiltrating endometriosis (DIE). DIE is a specific entity, histologically defined in arbitrary manner when endometriotic lesions extend more than 5 mm underneath the peritoneum. DIE implants are found in specific locations, primarily in the posterior area, such as, USL, posterior vaginal wall and the anterior rectal wall. DIE is responsible for painful symptoms (dysmenorrhea, deep dyspareunia and nonmenstrual pain) whose severity is strongly correlated with the depth of the DIE lesions.The treatment options of endometriosis at present are limited to hormonal therapies and/or surgical ablation of the lesions. It is suggested that surgery followed by medical treatment is the first choice of treatment for endometriosis with middle to severe chronic pelvic pain. However, this option of therapy only improves the outcome but not affects the chance of conception, and is also characterized by high recurrence rates, significant side-effects and limited duration of administration. DIE is a severe form of pelvic endometriosis in which pharmacological treatment is relatively ineffective. Laparoscopic surgical treatment is effective, but has the potential risks of bowel perforation and colostomy formation, and doesn't improve the recurrence rates.Accumulated evidence suggests that apoptosis helps to maintain cellular homeostasis during the menstrual cycle by eliminating senescent cells from the functional layer of the uterine endometrium during the late secretory and menstrual phase of the cycle. It has been reported that there are some fundamental differences between eutopic endometrium from women with endometriosis and normal endometrium of women without endometriosis and the susceptibility of endometrial tissue to spontaneous apoptosis is significantly lower in women with endometriosis than that in women without endometriosis. The differences could contribute to the survival of regurgitating endometrial cells into the peritoneal cavity and the development of endometriosis. We suggest that decreased susceptibility of endometrial tissue to apoptosis may contribute to the etiology or pathogenesis of endometriosis, and may also provide a treatment for endometriosis.Gossypol is a polyphenolic compound isolated from cotton seeds, and has been successfully used as a male contraceptive drug for many years. Several proposed clinical applications of gossypol have been reported including antiviral, antitumor effects and gynecological disease, but it was restricted for the severe hypopotassaemia when oral application. And the mechanism was still limited. Recent observations have suggested the antiproliferative and antimetastatic activities of gossypol in several tumor cells including leukemia cells, colon carcinoma cells, glioma cells and prostate carcinoma cells. The local application of gossypol acetate for endometriosis may be an appealing way.To develop animal models for endometriosis is a way of research into the pathophysiology of this disease. Up to date, these experimental endometriosis models include rodents, mice, chicken chorioallantoic membrane (CAM). In this study, we established an endometriosis animal model in rats. Gossypol acetate-loaded vaginal bar was made and placed into rat vagina to detect the releasing state in vitro and in vivo, to observe the change of ectopic cyst after medication and the possible mechanism.Material and Method:Female adult SD rats weighing (230±20)g were supplied by the Animal Laboratory Center of Zhejiang Chinses Medical University (n=56). Operational self-transplantation was applied to establish the EMs model, which sutured both endometrium and myometrium in the cul-de-sac. Operation was at estrus or non-estrus. Opening the abdominal cavity 2/3/4 weeks after the model was made to invest the adhesion, cyst coulor and volume, vascularization, and compare the modeling rate of different stage.Establishing the EMs model at estrus(n=21), opened the abdominal cavity after modeling three weeks and detected the cyst volume. The rats were divided randomly into two groups: (1) the experimental group: medication with gossypol acetate-loaded vaginal bar (4.08mg/kg, n=10); (2) the control group: medication with silica gel-loaded vaginal bar (4.08mg/kg, n=11). Opening the abdominal cavity the third time to observe the adhesion and measure the cyst volume again, the ectopic cyst and normal endometrium of the rats were taken out under aseptic conditions, which were then put into 4% formaldehyde. Regular methods of paraffin section at a thickness of 4μm were taken. Hematoxylin-Eosin Staining to observe the gland, and stroma; immunohistochemical staining with CK and vimentin to identify the gland cell and and stromal cell respectively. Immunohistochemical staining with bcl-2 and bax, and analysis with Image-pro plus 6.0 analysis software to compare the difference between the experiment group and the control group.Preparation of the gossypol acetate-loaded vaginal bar: Silicone gel and its vulcanizing agent were mixed at the ratio of 0.9:1.0(w:w). 10% quantity of gossypol acetate was added and the final mixture was fixed into a rod of 0.2 cm diameter polyvinyl chloride. The fixed rod was vulcanized for 2 h at 80℃.To test the release rate of gossypol acetate from the vagina in vitro, 1 mg of Gossypol acetate-loaded vaginal bar was cut and placed into 100 ml of dissolution medium containing 0.5% Tween-80, and stirred at 60 rpm at 37℃. After 0, 4, 12, 24, 48, 96h, 6, 8,10,12, 14,16, 20, 24, 30, 35,40,45, 50days, all of dissolution medium was removed and assayed for gossypol acetate after filtration through a 0.45μm membrane. The gossypol acetate concentration was detected using the high-performance liquid chromatography (HPLC) method described below. The HPLC conditions for gossypol acetate assay were as follows. Mobile phase: methanol: water 90:10; chromatographic column: Dikma C18-ODS(250 mm×4.6 mm, 5 um); flow rate: 1 ml min-1; detection wavelength: 238 nm; column temperature: 40℃; injection volume: 20μL.To test the release rate of gossypol acetate from the vagina in vivo, medication with the EMs model with gossypol acetate-loaded vaginal bar (4.08mg/kg) (n=18), taking the blood samples at d0, d1, d2, d3, d4, d5, d10,d11, d12 after the medication respectively. The blood was collected in tubes with heparin sodium, the plasma separated by centrifugation, and the sample stored at -20℃. The gossypol acetate concentration in the plasma was assayed using the HPLC method. The HPLC conditions for gossypol acetate assay were as follows. Mobile phase: 0.2%HAC: Acetonitrile=25:75; chromatographic column: Agilent Eclipse-XDB(150×4.6mm, 5μm); flow rate: 1.2 ml min-1; detection wavelength:235 nm; column temperature: 25℃.Result1.The total achievement ratio of modeling(3 weeks) was 82.35%(42/51), modeling rate on estrus phase was 82.93%(34/41), modeling rate on non-estrus phase was 80.0%(8/10). There was no significant difference in the cyst voume of modeling between 3 weeks and 4 weeks.2.The releasing characteristics of gossypol aceate bar in vitro can be described by two-phase dynamic model. The accumulative releasing percentage of 24h was 25%, the release then followed zero-order kinetics, It released about 0.3% everyday after 15th day.3.The blood drug level of gossypol acetate reached the peak at 24h, without obvious fluctuation then.4.The volume inhibition rate of cyst in the experiment group after medication for 7d was higher than the control group, but no statistically significant difference was observed (p=0.247). There was no statistically significant difference between the experiment group and the control group about adhesion (p=0.790).5.Bcl-2, bax are localized to lunimal epithelial cells and glandular epithelial cells in the rat's uterus and cyst. In the experiment group, Bcl-2 expressed lower (p=0.012) and bax expressed higher (p=0.004) in cyst, and bcl-2 expressed lower (p=0.015) and bax expressed higher (p=0.007) in rat's endometrium.Conclusions1.There was no significant difference between modeling rate on estrus phase and non-estrus phase. The modeling phase doesn't affect the achievement ratio.2.The releasing characteristics of gossypol aceate bar in vitro could be described by two-phase dynamic model. The 24h swift releasing maked it enough drug concentration in local and produced therapy effect, the latter releasing was to maintain the drug concentration and long-term care.3.The blood drug level of gossypol acetate was low, it indicated that the vaginal medication had lower system side effects.4.Medication for 7d in the experiment group inhibited the cyst volume, gossypol acetate-loaded vaginal bar may form high drug level in local and inhibited the focus at the cul-de-sac directly. Gossypol acetate didn't improve the adhesion.5.Gossypol acetate may promoted the apoptosis of DIE. |
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