| Endometriosis(EMs)is a common gyneacological disease.Endometriosis has been estimated to affect 10%-15%of reproductive aged women.The influence of endometriosis on follicular development is an important cause of infertility.50%of endometriosis patients with infertility.The follicular development of endometriosis and pregnancy rate and live birth rate after laparoscopic surgery can be effectively improved by using traditional Chinese medicine of activating blood circulation,regulating Gan,tonifying Shen sequential therapy by Professor Zhao Ruihua.Previous experimental studies showed that traditional Chinese medicine can improve the number of IVF-ET ovulation and fertilization in endometriosis model rats,improve pregnancy rate and live birth rate of IVF-ET.This study begins with the effect to follicular development in endometriosis,focuses on apoptosis and autophagy of follicular development.Vivo and vitro experiments were carried out to clarify the mechanism of Huoxue Xiaoyi Decoction to improve follicular development.Objective(1)To explore the effect of Huoxue Xiaoyi Decoction on apoptosis and autophagy of ovarian granulosa cells in endometriosis model rats,and to clarify the possible mechanism of Huoxue Xiaoyi Decoction through ROS-JNK signaling pathway.(2)To explore the effect and mechanism of drug-containing serum of Huoxue Xiaoyi Decoction on the apoptosis of rat ovarian granulosa cells in oxidative stress induced by H2O2.MethodsIn vivo experiment:Thirty 8-week-old female SD rats were divided into four groups:blank group,sham operation group,model group and Huoxue Xiaoyi Decoction group.Huoxue Xiaoyi Decoction group were given Huoxue Xiaoyi Decoction,while blank group,sham operation group and model group were given distilled water for 15 days.After intragastric administration,blood samples from abdominal aorta of rats were collected to detect oxidative and antioxidative indexes such as ROS,T-SOD,CAT.The morphology of follicles were observed by HE staining and every stage of follicles were calculated.The location of granulosa cells and Bax,Bcl-2,caspase-3 were stained by immunohistochemistry staining.The apoptosis of granulosa cells were stained by TUNEL staining and the rate of apoptosis were calculated.Autophagy and apoptosis related proteins including p-JNK,Bax,Bcl-2,caspase-3,Beclin-1,LC3? were detected by Western blot.Apoptosis related genes including Bax,Bcl-2,caspase-3 mRNA were detected by qRT-PCR.Ultrastructure of granulosa cells were observed by transmission electron microscope and autophagy cytoplasmic area ratio were calculated.In vitro experiment:Fifty 21-25 day female SD rats were selected.The ovarian granulosa cells were collected by the combination of mechanical separation and pancreatin digestion.The expression of rabbit anti-FSHR receptor polyclonal antibody was observed by immunofluorescence staining,and the purity of ovarian granulosa cells was tested.The cell growth state was observed and the growth curve was drawn.Combined with the literature and cell growth state,50 ?m,100 ?m,200 ?m,500 ?m,1mm H2O2 were used to intervene the ovarian granulosa cells.CCK-8 method was used to select the appropriate concentration in order to establish the rat ovarian granulosa cells model of oxidative stress.5%,10%,20%drug-containing serum of Huoxue Xiaoyi Decoction were used to intervene.CCK-8 method was used to screen the concentration and intervention time.200nm,400nm,800nm SP600125,a JNK signaling pathway inhibitor,were used to intervene with ovarian granulosa cells,and CCK-8 method is used to select the appropriate concentration.The ovarian granulosa cells in the logarithmic growth period were randomly divided into the blank group,the model oxidative stress group intervened by H2O2(model group),drug-containing serum of Huoxue Xiaoyi Decoction group(TCM group),and the JNK signaling pathway inhibitor group.After intervention,ROS was tested by confocal laser scanning microscope and mean optical density were calculated for each group;TUNEL was tested by confocal laser scanning microscope and the rate of apoptosis were calculated for each group,apoptosis-related protein including p-JNK,Bax,caspase-3 were tested by Western blot and ultrastructure of granulosa cells were detected by transmission electron microscope.ResultsIn vivo experiment:(1)Compared with the blank group and the sham operation group,the serum ROS in the model group increased,T-SOD and CAT decreased,the difference was statistically significant(P<0.05).Compared with the model group,the serum ROS in the Huoxue Xiaoyi Decoction group decreased,T-SOD and CAT increased,the difference was statistically significant(P<0.05).(2)HE staining:Compared with the blank group and the sham operation group,the number of secondary follicles in the model group decreased(P<0.05).Compared with the model group,the number of secondary follicles in the Huoxue Xiaoyi Decoction group increased(P<0.05).(3)Immunohistochemistry staining:The ovarian granulosa cells in the model group were scattered,and those in the blank group,the sham operation group and the Huoxue Xiaoyi Decoction group were orderly arranged.Compared with the blank group and the sham operation group,the expression of Bax,caspase-3 in the model group increased,and the expression of Bcl-2 decreased(P<0.01).Compared with the model group,the expression of Bax,caspase-3 in the Huoxue Xiaoyi Decoction group decreased and the expression of Bcl-2 increased(P<0.01).(4)TUNEL staining:Compared with the blank group and the sham operation group,the apoptosis rate of ovarian granulosa cells in the model group increased(P<0.001).Compared with the model group,the apoptosis rate in the Huoxue Xiaoyi Decoction group decreased(P<0.001).(5)Western blot:Compared with the blank group and the sham operation group,the expression of p-JNK,Bax and caspase-3 protein in the model group increased,while the expression of Bcl-2,Beclin-1 and LC3? protein decreased(P<0.05).Compared with the model group,the expression of p-JNK,Bax,caspase-3 protein in the Huoxue Xiaoyi Decoction group decreased,the expression of Bcl-2,Beclin-1,LC3? protein increased(P<0.05).(6)qRT-PCR:Compared with the blank group and the sham operation group,the expression of Bax mRNA and caspase-3 mRNA in the model group increased,the expression of Bcl-2 mRNA decreased(P<0.01).Compared with the model group,the expression of Bax mRNA and caspase-3 mRNA in the Huoxue Xiaoyi Decoction group decreased,and the expression of Bcl-2 mRNA increased(P<0.05).(7)Transmission Electron Microscope:The ultrastructural damage of the ovarian granulosa cell in the model group were obvious,and the ultrastructure of the blank group,the sham operation group and the Huoxue Xiaoyi Decoction group were normal;There were no significant difference in the autophagy cytoplasmic area ratio of the ovarian granulosa in the blank group,the sham operation group,the model group and the Huoxue Xiaoyi Decoction group(P>0.05).In vitro experiment:(1)The purity of ovarian granulosa cells collected by mechanical separation and pancreatin digestion was 98%,which met the requirements of cell experiment.The inoculation density of ovarian granulosa cells should be 5 × 104 cells/well(100 ?L).Oxidative stress of ovarian granulosa cell model can be established by using 200 ?m H2O2;The optimum concentration of drug-containing serum of Huoxue Xiaoyi Decoction group was 10%,the time of intervention was 24h.The concentration of SP600125 was 800 nm.(2)Intracellular ROS:Compared with the blank group,the mean optical density of ROS in the model group,the TCM group and the JNK signaling pathway inhibitor group increased(P<0.05).Compared with the model group and the JNK signaling pathway inhibitor group,the mean optical density of ROS in the TCM group decreased(P<0.05)(3)TUNEL staining:Compared with the blank group,the apoptotic rate of the model group,the TCM group and the JNK signaling pathway inhibitor group increased(P<0.05).Compared with the model group,the apoptotic rate in the TCM group and the JNK signaling pathway inhibitor group decreased(P<0.05).(4)Western blot:The expression of p-JNK,Bax and caspase-3 protein in the model group were higher than that in the blank group,the TCM group and the JNK signaling pathway inhibitor group(5)Transmission Electron Microscope:The ultrastructure of ovarian granulosa cells in model group induced by H2O2 was significantly damaged,and the ultrastructural damage of ovarian granulosa cells in the TCM group was smaller than that in the model group.Conclusion(1)Huoxue Xiaoyi Decoction may improve the oxidative stress state,decrease the apoptosis of ovarian granulosa cells,increase the autophagy of ovarian granulosa cells and improve the development of follicles in endometriosis rats through ROS-JNK signaling pathway.(2)Drug-containing serum of Huoxue Xiaoyi Decoction may reduce the apoptosis of ovarian granulosa cells in oxidative stress induced by H2O2 by inhibiting ROS-JNK signaling pathway. |
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