Clinical Study Of Premature Ovarian Insufficiency Improved By Bu Shen Huo Xue Tang And Mechanism Study Of Keap1/Nrf2/ARE Signaling Pathway Regulated By Bu Shen Huo Xue Tang In Model Mice

Background:In recent years,the incidence of premature ovarian insufficiency(POI)is increasing and shows a trend of younger.POI seriously affects female reproductive health.The pathogenesis of POI is complex and unclear.The early diagnosis is not perfect,and the treatment effect is not satisfactory.Therefore,to explore the cause and mechanism of POI,search for early diagnosis criteria and find effective treatment methods are still the major and difficult issues facing the current medical field.The traditional Chinese medicine(TCM)therapy has the overall regulation function of multi-target,multi-component,and multi-pathway.The tonifying kidney and promoting blood circulation is a common gynecological treatment method for POI with kidney deficiency and blood stasis.Based on the relevant theories of traditional Chinese and western medicine,clinical curative effcet observation and experimental research results,this subject explained the clinical effect and molecular mechanism of Bu Shen Huo Xue Tang(BSHXT)in the prevention and treatment of POI,and provided a scientific basis for TCM therapy in POI.Objective:This study aimed to evaluate the prevention and treatment effect of BSHXT on POI with kidney deficiency and blood stasis through clinical study,and to investigate the underlying molecular mechanism of BSHXT via the Nrf2/Keap1 signaling pathway in the treatment of POI through experimental study.Methods:1.Clinical researchSixty POI patients were randomly divided into a BSHXT group of 20 patients(taken BSHXT orally),a fenmatol group of 20 patients(taken fenmatol 1/10 orally),and a BSHXT and fenmatol group of 20 patients(given the combined treatment of BSHXT and fenmatone 1/10 orally).All patients were treated with corresponding drugs for 3 menstrual cycles.Observed the improvement of TCM syndrome score and Kupperman score.The estradiol(E2),follicle-stimulating hormone(FSH),superoxide dismutase(SOD)and malondialdehyde(MDA)in serum were measured.Ovarian volume(OV),antral follicle number(AFC)and ovarian stromal blood flow resistance index(RI)were recorded by ultrasound Doppler.2.Experimental research2.1 The chemical components of BSHXT water extract was analyzed by the UPLC-Q-TOF-MS/MS technology.2.2 Twenty female C57BL/6J mice with normal estrus cycle were screened.The mice were randomly divided into blank control group,model group,positive drug(estradiol valerate tablets)group,and BSHXT group.Except for 5 mice in control group,the other 15 POI model mice were injected with zona pellucida glycoprotein 3(ZP3)polypeptide subcutaneously.One week later,the model mice were immunized by injecting ZP3 again to establish an autoimmune POI animal model.After one week of the last immunization,the control group and the model group mice were given with 0.2ml saline per day by gavage.Positive group mice were given 0.13 mg/kg estradiol valerate.BSXHT group mice were given 12.64g/kg BSHXT water extract.The treatment lasted for 28 days.In the last 10 days,the vaginal exfoliated cells were collected and stained to observe the changes in the estrous cycle of mice.Collected samples the day after the end of administration.Body weight and organ indices were recorded.The levels of ZP antibodies,FSH,anti-Mullerian hormone(AMH),E2,luteinizing hormone(LH),anti-ZP antibody(AzpAb)levels,SOD and MDA were measured.The pathological morphology of the ovaries was observed.The estrous cycle of each mouse was recorded.Immunofluorescence assay was used to detect the levels of ZP antibodies in the mouse ovaries.The expression of genes and proteins of Nrf2?Keap1?HO-1?NQO1 involved in the Nrf2/Keap1 signaling pathway were measured by Q-PCR and IHC respectively.Results:1.Clinical research:The average age of all enrolled patients was 34.65±3.78 years old.Patients aged 36-40 accounted for 50%.There were 4 cases of POI patients with immune diseases,9 cases with psychological stress,4 cases with environmental stress,6 cases with surgery,2 cases with infection,and 1 case with family history.After 3 cycles of treatment,in terms of comprehensive efficacy,TCM symptom efficacy,and Kupperman score,the BSHXT group(88%,82%,30.07±3.74)was as effective as same as the fenmatol group(78%,72%,31.61±3.18)and the combined treatment group of BSHXT and fenmatol(94%,88%,28.94±3.83).Symptoms of hot flashes,sweating,insomnia,and fatigue were significantly improved after treatment with BSHXT(P<0.05).The symptom of muscle pain improved after fenmetone treatment(P<0.05),while the symptoms of hot flashes,sweating,insomnia,dizziness,fatigue,and joint pain were significantly improved after the combination of Chinese and western treatment(P<0.05).The three groups had a significant effect in down-regulating FSH levels(P<0.05).There were no changes in E2,OV and AFC agmong the three groups.Compared with before treatment,RI was decreased significantly after treatment in BSHXT group(P<0.05)and not changed in the fenmatol group and the combined treatment group.After treatment,SOD increased in the BSHXTgroup(P<0.05)and the combined treatment group(P<0.05).The MDA level also decreased significantly in the BSHXT group after treatment(P<0.05).The MDA was not changed in the fenmatol group and the combined treatment group.During the clinical study,patients of three groups had no adverse reaction.2.Experimental research2.1 Twenty-one compounds were identified and quantitatively analyzed in the BSHXT water extract.2.2 Compared with the Control group,the weight of the mice in the Model group decreased significantly(P<0.01).Compared with the Model group,the weight of the mice in the BSHXT group increased significantly(P<0.05).The estrous cycle disorder rate of the Control group mice was 0%,the estrus cycle disorder rate of the Model group mice was 100%,the positive group mice estrous cycle disorder rate was 60%,and the estrus cycle disorder rate of the BSHXT mice was 80%.After 28 days administration,specimens were collectted and relevant indicators were measured.Compared with the Control group,the ovarian index,uterine index and thymus index of the Model group decreased(P<0.01),and the spleen index increased(P<0.01);Compared with the Model group,the ovarian index increased(P<0.01)in the Positive group and the BSHXT group(P<0.01,P<0.05),and the spleen index in the BSHXT group decreased(P<0.05).Ovarian tissue H&E staining and ZP immunofluorescence staining showed that BSHXT not only significantly reduced the infiltration of inflammatory cells and the formation of zona pellucida in the ovaries of mice but also increased the number of primordial follicles and secondary follicles,and reduced the number of atresia follicles.While Progynat has no such effect.ELISA method was used to detect the serum levels of sex hormones(E2,FSH,LH,AMH),AzpAb,SOD and MDA.It was found that sex hormones of the model group mice were disturbed,Estradiol valerate and BSHXT could improved abnormal sex hormone status.BSHXT group down-regulated the AzpAb level(P<0.01)was better than the Positive group(P<0.05).The SOD and MDA levels in the Model group were significantly decreased,the Positive and BSHXT groups were improved after drug treatment.The results of immunohistochemistry and Q-PCR indicated that,compared with the Control group,the expression of Nrf2,Keapl,NQO1 and their mRNA level in the Model group decreased significantly.Although the expression of HO-1 did not change significantly,its mRNA level decreased significantly(P<0.05).Compared with the Model group,the expression of Nrf2 and its genes in the BSHXT group were upregulated,and the Positive group could increased the levels of Keap1 mRNA and NQO1 mRNA.Conclusions:1.BSHXT can significantly improve TCM syndrome scores,clinical symptom scores,and sex hormone levels.In improving ovarian RI and oxidative stress BSHXT was better than fenmatol 1/10 and the combination therapy.2.Twenty-one kinds of Chinese medicine monomer compounds were identified and quantitatively analyzed in the water extract of BSHXT.3.The POI immunity model successfully induced by ZP3 showed that the mouse ovary had inflammation and its function declined.BSHXT not only reduced the infiltration of inflammatory cells and the formation of zona pellucida in the ovaries,reversed the abnormal sex hormone state,but also down-regulated serum AzpAb and activated the Keap1/Nrf2/ARE signaling pathway and the downstream antioxidant enzyme SOD,HO-1 and NQO1 activity.Therefore,BSHXT improved the ovarian function of model mice,reduced oxidative stress and regulated the immunity through Keap1/Nrf2/ARE signaling pathway.