Effects Of Nordihydroguaiaretic Acid On The Endometriosis In Rats

Objective: Endometriosis is a common gynecological disease among reproductive aged women, which seriously impact the patients' physical and mental health. Its pathogenesis is still not clear, and freshman angiogenesis become a new kind of etiology doctrine. Subsequently anti-angiogenic treatment research on endometriosis become a new hotspot. Nordihy- droguaiaretic acid(NDGA) is a natural drug ingredients extracted from evergreen shrubs. Now it also can be synthesized artificially. NDGA has inhibitory effect on lipoxygenases, specificly on 5-lipoxygenase and 12-lipoxygenase. It has been proved to have various biological effects, such as anti-inflammatory, anti-oxidation, lowering blood sugar, antitumor, allergies. The NDGA used in this study is a natural drug ingredients extracted from Larrea tridentata evergreen shrubs. The study is to investigate the effect of NDGA on the expresion of VEGF, KDR and 5-LOX in the eutopic endometrium and ectopic endometrium of endometriosis rat model,so as to explore the feasibility of the treatment for endometriosis, opening up new remedial approaches.Methods:1 Establish endometriosis model of ratsWe used 45 healthy sexual maturity female SD rats, which were not mating, weighting between 180g and 220g. All the rats were given diethylstilbestrol by intragastric administration five days before the operations to make them in estrogen period in unity. We anaesthetized the rats by the way of intraperitoneal injection chloral hydrate with the concentration of 43g/L on dose of 300mg/kg. Cutted open an about 2cm long incision along the medioventral line 1cm upon the pubis. Separated out the right womb. Cutted an about 1cm long uterine segment on the nearly fallopian tube end. Cutted the uterine segment in uterine pieces about in 5mm×5mm. And fixed them on the abdominal wall by surgery line. Dripped 0.2ml gentamycin sulfate injection in to enterocoelia, sutured layered. Intramuscular injection 0.1ml gentamycin sulfate after surgery for five days. From the tenth day give diethylstilbestrol by intragastric administration for five days. Opened abdominal to observe the ectopic endometrium growth situation and measure the volume.2 Treatment to endometriosis model of ratsSeparated the 39 successfully modeled rats in to five groups for therapeutic investigation: model control group, 30mg/kg dose of NDGA group, 60mg/kg dose of NDGA group, 90mg/kg dose of NDGA group and mifepristone influences group. Subcutaneous injection 3% NDGA tertianly to the three NDGA dose groups for 20 days. So as to model control group with subcutaneous injection PBS liquid. Intragastric administration mifepristone on dose of 2mg/kg for 28 days. Collected celiac rinses and 3ml abdominal aortic blood, centrifuged respectively. Preserved supernatant fluid in -70℃refrigerator, used ABC-ELISA method to detect 5-LOX level. Saved the eutopic endometrium and ectopic endometrium in 10% formalin to fix specimens, using immunohistochemistry PV two-steps method to detect the expression of VEGF, KDR and 5-LOX.All data are analyzed by the statistical software SPSS13.0, P < 0.05 was deemde to have statistical significance. Materials meet normality and Homoscedasticity analized by Compare Means One-Way ANOVA. Others analized by Nonparametric test.Result:1 Ectopic lesions growth situationThe survival rate of transplant endometrium was 78% 4 weeks after operation.The average volume is 129.639±17.627mm~3. Ectopic endometrium inhibition rate in control group, low dose group, moderate dose group, high dose group and mifepristone influences group were respectively -17.90±2.45%, 14.49±1.04%, 40.26±2.73%, 59.05±1.82%, 57.16±2.57% after treatments. Volumes of control group increased significantly, and there was no significant reduction of low dose group compared to volumes before treatments. The inhibition rate of NDGA increased along with the ascending dose. There was statistically significant differences between each other in the three groups. Inhibiting rate of mifepristone influences group was significantly higher than NDGA low dose group and the dose group(P<0.05), but no significant differences compared to NDGA high dose group(P>0.05).2 Serum and celiac liquid 5-LOX levelSerum and celiac liquid 5-LOX level of the control group was significantly higher compared to the three NDGA groups. Serum and celiac liquid 5-LOX levels dropped more obviously along with the increase of NDGA doses. There was statistically significant differences between each other in the three NDGA groups(P<0.05). The 5-LOX level of mifepristone influences group was far above other treatment groups with statistically significant differences(P<0.05). The celiac liquid 5-LOX levels is significantly higher than serum 5-LOX levels in control group and NDGA low dose group(P<0.05). There was no significant differences between serum and celiac liquid 5-LOX levels of NDGA high dose group and mifepristone influences group and moderate dose group(P>0.05).3 Immunohistochemical results3.1 VEGF protein mainly expressed in cytoplasm of glandular epithelium cells in eutopic endometrium and ectopic endometrium. The VEGF protein expression in NDGA low dose group, moderate doses group, high dose groups, mifepristone influences group declined in turn, and all were significantly lower than control group (P < 0.05). The VEGF expression intensity of mifepristone influences group had no statistically significant differences compared to NDGA moderate and high dose groups(P>0.05).3.2 KDR protein mainly expressed in cytoplasm of glandular epithelium cells, endometrial interstitial, part of vascular endothelial cells' cytoplasm in eutopic endometrium and ectopic endometrium. The KDR protein expression in NDGA low dose group, moderate doses group, high dose groups, mifepristone influences group declined in turn, and all were significantly lower than control group (P < 0.05). The KDR expression in NDGA groups declined along with NDGA dose with statistically significant differences. The KDR expression in mifepristone influences group is observably higher than in NDGA moderate group(P < 0.05), but lower than NDGA high dose group with no statistically significant differences.3.3 5-LOX protein mainly expressed in cytoplasm and nucleiof glandular epithelium cells in eutopic endometrium and ectopic endometrium. The 5-LOX protein expression in control group, NDGA low dose group, moderate doses group and high dose groups declined in turn with statistically significant differences(P < 0.05). 5-LOX protein oerexpressed in mifepristone influences group. There were statistically significant differences compared to other groups(P < 0.05).Conclusion:1 Establish autologous womb transplants endometriosis model of rats is feasible.2 Moderate dose(60 mg/kg) group and high dose (90 mg/kg) of group NDGA has inhibiting effect on endometriosis model of rats. They can observably inhibit the expression of VEGF, KDR and 5-LOX in the eutopic endometrium and ectopic endometrium, and lowered the serum and celiac liquid 5-LOX levels. Low dose of NDGA(30 mg/kg) has no effectively prohibitive effect on endometriosis. The high dose of NDGA(30 mg/kg) is de most effective group.3 NDGA can inhibit endometriosis dose dependently. The inhibiting effect on the expression of VEGF and KDR by the way of inhibiting the expression of 5-LOX and its activation, so to inhibit angiogenesis of ectopic endometrium.4 The oerexpression of 5-LOX in mifepristone influences group maybe due to its inhibition to the Cyclooxygenase-2(COX-2) and aromatase cytochrome P450(P450arom).