| Objective:1.Through digging,selecting and integrating the main chemical components and related targets of Guishen Pills and related disease targets of premature ovarian insufficiency,to predict the common targets of Guishen Pills in treating premature ovarian insufficiency.Core targets through the STRING website were selected and Cytoscape software was used to construct a network diagram of the interaction relationship between the core targets.DAVID database was used to perform GO function enrichment analysis and KEGG signal pathway enrichment analysis on core targets and key biological processes and signal pathways were obtained,laying the bioinformatics foundation of Guishen Pills in treating POI in subsequent in vivo and in vitro research.2.The changes of premature ovarian insufficiency patients in TCM syndrome scores,serum sex hormone levels and serum oxidative stress related indicators before and after three-month treatment with Guishen Pills were observed.The clinical effectiveness of the method of invigorating the kidney and strengthening the spleen,soothing the liver and promoting blood circulation on POI were verified.At the same time,the effects whether Guishen Pills can improve the serum oxidative stress levels in POI patients and lay a clinical foundation for its anti-ovarian hypofunction mechanism were explored.3.The improvement on the oxidative stress state of human ovarian granulosa cell line KGN and the effect on PI3K/AKT/Nrf2 signaling pathway of epicatechin,an important ingredient in Guishen pills,were explored.The antioxidant effect of epicatechin on ovarian granulosa cells and the up-regulation and down-regulation of core proteins in PI3K/AKT/Nrf2 signaling pathway were preliminary explored,constructing the foundation of Guishen Pills in treating POI in vitro research.Method:1.Network pharmacology research:The effective ingredients and the ingredient-related targets of eight herbs in Guishen Pills were digged,selected and integrated through TCMSP,TCM-ID and BATMAN databases.The disease targets related to premature ovarian insufficiency were searched in OMIM and Gene Cards databases.The common targets after intersection were imported into the STRING website to obtain the core targets of Guishen Pills in treating POI.The core targets in DAVID database were analyzed for GO biological process and KEGG signal pathway enrichment analysis,and the core biological process and signal pathway were screened out.Meanwhile,Cytoscape software was used to construct various visual network diagrams.2.Clinical research:Using the before-and-after comparison method,20 POI patients who were treated at the gynecological clinic of Dongzhimen Hospital of Beijing University of Chinese Medicine were given Guishen Pills orally for 3 menstrual cycles(amenorrhea were treated for 3 months).The scores of TCM syndromes,modified Kupperman scale scores,sex hormone related indexes(FSH,LH,E2)and serum oxidative stress related indexes(SOD,GSH)were evaluated before and after the treatment to verify the clinical efficacy of Guishen Pills in treating POI.3.In vitro experiment:The human ovarian granulosa cell line KGN was adaptively cultured,and the optimal concentration of H2O2 and epicatechin was found using the CCK-8 assay.The experiment is divided into 5 groups,which namely the blank control group,the model group and the epicatechin low,medium and high dose group.First day,the cell line was seeded at an appropriate concentration.Next day was used for modeling with H2O2 except the blank control group.The third day,the cells were used for intervention with low,medium and high epicatechin concentrations and the last day is used for taking material.Result:1.Network pharmacology research:162 active ingredients in Guishen Pills were retrieved,including 2 of shudi,18 of shanyao,23 of shanzhuyu,16 of fuling,6 of danggui,42 of duzhong,43 of gouqizi,12 of tuszi,and a total of 829 potential targets for the ingredients are predicted.A total of 4854 POI disease-related targets were retrieved,and the two target data sets were intersected to obtain a total of 152 core targets,including AKT1,CASP3,CASP8,CASP9,FAS and EGF.The results suggest that quercetin,stigmasterol,kaempferol,β-carotene and epicatechin are among the top five through constructing a "component-target" network.The PPI protein interaction network was constructed,and the results suggested that TP53,AKT1,MMP9,ASP3 and IL6 are at the core situations.GO enrichment analysis results suggest that biological processes are significantly enriched in oxygen-containing compound reactions,cell responses to chemical stimuli;molecular functions are mainly enriched in enzyme binding,signal receptor binding;cell components are mainly enriched in extracellular space,extracellular area,etc.KEGG enrichment analysis results suggested that the core targets are mainly enriched in TNF,PI3K/AKT,apoptosis-related,FoxO,Toll-like receptor,VEGF and MAPK signaling pathway.2.Clinical research:The various situations of patients before and after treatment with Guishen Pills for 3 menstrual cycles were compared.Scores of TCM syndrome:most patients have improved menstrual conditions to varying degrees,including menstrual cycle recovery and menstrual blood volume increase.Scores of symptoms:Guishen Pills also has significant improvement effect on the symptoms such as waist and knee weakness,dizziness,tinnitus,loss of libido,hot flashes and night sweats,irritability,chest tightness,hypochondriac pain,insomnia and dreams,fatigue and dark menstruation with blood clots.Scores of modified Kupperman scale:there are significant differences in the scores of hot flashes,sweating,insomnia,anxiety,depression,fatigue,and dizziness before and after treatment,while paresthesias,muscle and joint pain,headache,heart palpitations,skin ant behavior sensation,sexual pain,urinary system irritation symptoms are not significant different before and after treatment,and the total effective rate of treatment is 70%.Sex hormone-related indicators:there are significant differences in serum FSH and LH values of patients before and after treatment,and E2 values are higher than before treatment,but there is no significant difference.Serum OS related indicators:the SOD and GSH were all higher than before treatment but there was a significant difference in GSH,and no significant difference in SOD.3.In vitro experiment:The results of CCK-8 method indicated that the optimal concentration of H2O2 for modeling is 50μM/L,and the safe concentration range of epicatechin is 100μM/L to 300μM/L.Related indicators of oxidative stress:Compared with the blank control group,the SOD activity of the model group was significantly lower(p<0.05).After the intervention of three doses of epicatechin,only the low dose(100μM/L)did not show significant difference(p≥0.05).There are significant differences in SOD activity at medium and high doses(200μM/L,300μM/L)(p<0.05,p<0.01),and the oxidative stress damage to cells is reduced with the increase of EC concentration.Western Blot related indicators:Compared with the blank control group,the protein concentrations of PI3K,AKT,and Nrf2 in the model group were significantly decreased(p<0.05,p<0.01),and the three protein concentrations all increased after the intervention of three doses of epicatechin,except for the PI3K protein of low dose group(100μM/L)was outside(p≥0.05),and the rest were all significantly different(p<0.05,p<0.01).The protein concentration of HO-1 in the model group was significantly lower than that in the blank control group(p<0.01),and HO-1 expression level was significantly reversed after the intervention of three doses of epicatechin(p<0.001).The eNOS protein concentration in the model group was significantly higher than that in the blank control group(p<0.01).The protein concentration gradually decreased after the intervention of three doses epicatechin,but it showed a significant difference with the model group(p<0.01)only in the high-dose group(300μM/L).PCR related indicators:Compared with the control group,the mRNA expression levels of PI3K,AKT and Nrf2 proteins in the model group decreased significantly(p<0.001).The mRNA expression levels of these three proteins gradually increased after intervention with three doses epicatechin and all showed significant differences(p<0.001).The mRNA expression levels of HO-1 and NQO1 in the model group were significantly lower than those in the control group(p<0.001).The mRNA expression levels of these two types proteins gradually increased after intervention with three doses epicatechin,and both showed significant differences.(p<0.01,p<0.001).Compared with the control group,the mRNA expression level of NADPH in the model group increased significantly(p<0.001),and the mRNA expression level gradually decreased after the intervention of three doses epicatechin,all showing significant differences(p<0.05,p<0.001).Conclusion:1.It is predicted that Guishen Pills can treat premature ovarian insufficiency through multiple signaling pathways and multiple targets.The inner mechanism is related to the regulation of inflammation,improvement of oxidative stress,inhibition of apoptosis and estrogen receptor regulation2.Guishen Pills can reduce serum FSH and LH levels in POI patients,adjust menstrual cycle,increase menstrual blood volume,and have certain adjustments to TCM syndrome scores,perimenopausal symptoms and oxidative stress level.The effect indicates that Guishen Pills can certain improve the ovarian function of POI patients.3.Epicatechin can improve the H2O2-mediated oxidative stress state of human ovarian granulosa cell line KGN,and the inner mechanism may be related to the PI3K/AKT/Nrf2 signaling pathway. |
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