The Protective Effect Of Erxian Decoction And Its Core Medicine On Curculio-Xianlingpi Against Cisplatin-induced Ovarian Oxidative Damage

Objective:Premature Ovarian Failure refers to ovarian function declines in advance before the age of 40,POF keeps company with estrogen levels decline,follicle-stimulating hormone and luteinizing hormone levels rise.POF brings out menstrual disease,amenorrhea,infertility and so on.The pathogeny of Premature Ovarian Failure is complex,including genetic factors,immune factors and psychological factors,and so on.The incidence of Premature Ovarian Failure is 1%³%.POF is seriously affect the patient's physical and mental health,the low estrogen level can lead to osteoporosis,Premature Ovarian Failure is becoming a hot spot of the modern medicine research.Erxian soup is a kind of Chinese herbal compound for relieving premature ovarian failure.Erxian soup contains two core medicine Xianmao and Yinyanghuo,Erxian soup can effectively cure Premature Ovarian Failure.This experiment from the point of oxidative stress and the angiogenesis as the entry point to explore the curative mechanism of how Erxian soup and Xianmao-Yinyanghuo treat POF,with nuclear transcription factor Nrf 2 and endothelial factor VEGF as the research target,to research the Nrf 2-ARE signal transduction pathway and vascular endothelial factor VEGF and VEGFR2 signal transduction pathways,to investigate the curative effect and the mechanism of Erxian soup and its core medicine Xianmao-Yinyanghuo treating premature ovarian failure.Methods&ResultsSeventy female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were divided into 7 groups by random numble table method:the normal group,the model group,the Er-xian soup group,the positive control group,the Xianmao group,the Yinyanghuo group,and the couplet medicines group.All the rats except the normal group were injected with cisplatin through abdominal cavity to establish the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the herbs treated group were fed with herbs every day.These rats were killed after 15 days.The serum biochemical indexes includeing sex hormones?P and E2,FSH,LH?and oxidation and anti-oxidation index?MDA,SOD and CAT?were detected by radioimmunoassay,The histopathological changes of ovarian tissue were observed by HE staining.The protein levels of Nrf 2?HO-1?NQO1?VEGF and VEGFR2 were detected by Western Blot,The content and location of Nrf 2?HO-1?NQO1?VEGF and VEGFR2 were detected by Immunohistochemical.The cell apoptosis of each group were detected by by Tunel.Experiment 1:Method:Forty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were randomly divided into 4groups:the normal group,the model group,the positive control group and the Erxian soup group.All the rats except the normal group were injected with cisplatin to copy the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the Erxian soup group was fed with Erxian soup everyday.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The serum biochemical indexes includeing sex hormones?FSH,LH and E₂?and oxidation and anti-oxidation index?MDA,SOD and CAT?were detected by radioimmunoassay,The histopathological changes of ovarian tissue were observed by HE staining.Result:Compared with the normal group,the levels of follicle-stimulating hormone?FSH?and luteinizing hormone?LH?in model group were significantly increased?P<0.01?,estrogen?E2?levels decreased significantly?P<0.01?.Compared with model group,the Erxian soup can obviously reduce the levels of FSH and LH?P<0.01 or P<0.05?,increase estrogen?E2?level?P<0.01?,at the same time can also reduce rats serum malondialdehyde?MDA?level?P<0.01?,improve the content of superoxide dismutase?SOD?and catalase?CAT??P<0.01?.The morphological change in Er-xian soup treated group had decreased under the light microscope,Er-xian soup can improve ovarian follicle state.Experiment 2:Method:Forty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were randomly divided into 4groups:the normal group,the model group,the positive control group and the Erxian soup group.All the rats except the normal group were injected with cisplatin to copy the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the Erxian soup group was fed with Erxian soup everyday.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The protein levels of Nrf 2?HO-1 and NQO1 were detected by Western Blot,The content and location of Nrf2?HO-1 and NQO1 were detected by Immunohistochemical.The cell apoptosis of each group were detected by by Tunel.Result:WB and IHC-P results indicate:compared with normal group,model group could significantly reduced the expression of Nrf 2,HO-1 and NQO1 in ovarian tissue?P<0.01?.Compared with model group,positive group and Er-xian soup group could significantly increase the contens of Nrf 2,HO-1 and NQO1?P<0.01?.Tunel results suggest:the model group apoptosis number increased significantly than the normal group,the number of cells apoptosis dramatically reduced in the Er-xian soup group.Experiment 3:Method:Forty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were randomly divided into 4groups:the normal group,the model group,the positive control group and the Erxian soup group.All the rats except the normal group were injected with cisplatin to copy the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the Erxian soup group was fed with Erxian soup everyday.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The protein levels of VEGF and VEGFR2 were detected by Western Blot,The content and location of VEGF and VEGFR2 were detected by Immunohistochemical.Result:WB and IHC-P results indicate:compared with normal group,model group could significantly reduced the expression of VEGF and VEGFR2 in ovarian tissue?P<0.01?.Compared with model group,positive group and Er-xian soup group could significantly increase the contens of VEGF and VEGFR2?P<0.01?.Er-xian soup could promote the expression of VEGF and VEGFR2 in ovarian tissue.Experiment 4:Method:Sixty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were divided into 6 groups by random numble table method:the normal group,the model group,the positive control group,the Xianmao group,the Yinyanghuo group,and the couplet medicines group.All the rats except the normal group were injected with cisplatin through abdominal cavity to establish the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the herbs treated group were fed with herbs every day.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The serum biochemical indexes includeing sex hormones?FSH,LH and E₂?and oxidation and anti-oxidation index?MDA,SOD and CAT?were detected by radioimmunoassay,The histopathological changes of ovarian tissue were observed by HE staining.Result:Compared with model group,the herbs treated groups could obviously reduce the follicle-stimulating hormone?FSH?and luteinizing hormone?LH?levels,increased estrogen?E2?and Progesterne?P?levels.The results of correlation coefficient q calculated according to Jin's formula showed that two medicines had synergy effect,the therapeutic effect was superior to the single use.The morphological change in herbs treated group had decreased under the microscope,the herds could improve ovarian follicle state.Experiment 5:Method:Sixty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were divided into 6 groups by random numble table method:the normal group,the model group,the positive control group,the Xianmao group,the Yinyanghuo group,and the couplet medicines group.All the rats except the normal group were injected with cisplatin through abdominal cavity to establish the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the herbs treated group were fed with herbs every day.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The protein levels of Nrf 2?HO-1 and NQO1 were detected by Western Blot,The content and location of Nrf 2?HO-1 and NQO1 were detected by Immunohistochemical.The cell apoptosis of each group were detected by by Tunel.Result:WB and IHC-P results indicate:compared with normal group,model group could significantly reduced the expression of Nrf 2,HO-1 and NQO1 in ovarian tissue?P<0.01?.Compared with model group,positive group and couplet medicines group could significantly increase the contens of Nrf 2,HO-1 and NQO1?P<0.01?.compared with couplet medicines group,Xianmao group and Yinyanghuo group could significantly reduced the expression of Nrf 2,HO-1 and NQO1 in ovarian tissue?P<0.01or P<0.05?.Tunel results suggest:the model group apoptosis number increased significantly than the normal group,the number of cells apoptosis decreased in Xianmao group and Yinyanghuo group,the number of cells apoptosis dramatically reduced in the couplet medicines group.Experiment 6:Method:Sixty female SD rats were Monitored the estrus cycle and then chose the rats who had the normal estrus cycle into the experiment,the rats were divided into 6 groups by random numble table method:the normal group,the model group,the positive control group,the Xianmao group,the Yinyanghuo group,and the couplet medicines group.All the rats except the normal group were injected with cisplatin through abdominal cavity to establish the model of premature ovarian failure.The normal group and the model group were fed with double distilled water,the positive control group was fed with estradiol,and the herbs treated group were fed with herbs every day.Monitoring the estrus of each rat everyday at the same time.These rats were killed after 15 days.The protein levels of VEGF and VEGFR2 were detected by Western Blot,The content and location of VEGF and VEGFR2 were detected by Immunohistochemical.Result:WB and IHC-P results indicate:compared with normal group,model group could significantly reduced the expression of VEGF and VEGFR2 in ovarian tissue?P<0.01?.Compared with model group,positive group and couplet medicines group could significantly increase the contens of VEGF and VEGFR2?P<0.01?,Xianmao group and Yinyanghuo group could significantly reduced the expression of VEGF and VEGFR2?P<0.05?.Compared with couplet medicines group,Xianmao group and Yinyanghuo group could significantly reduced the expression of VEGF and VEGFR2 in ovarian tissue?P<0.01or P<0.05?.Conclusion1.Erxian soup can effectively improve the rat ovarian follicle state in the organization,and restore the structure and function of the ovarian tissue,it can relieve ovary injury and improve ovarian function obviously by regulating oxidative stress in POF rats.2.Erxian soup and its core medicine Xianmao-Yinyanghuo can increase the level of Nrf2 then by activating Nrf 2-ARE signaling pathways to promote the expression of downstream protein.Er-xian soup and its core medicine Xianmao-Yinyanghuo treat premature ovarian failure by improving the oxidative stress status.3.Erxian soup and its core medicine Xianmao-Yinyanghuo can increase the level of VEGF then by activating VEGF-VEGFR2 signaling pathways to promote the expression of downstream protein,they treat premature ovarian failure by improving the angiogenesis.4.The effect of couplet medicines group is significantly better than Xianmao group and Yinyanghuo group,the results indicate that Xianmao and Yinyanghuo used tongther can show synergy effect.