| 1 BackgroundDiminished ovarian reserve(DOR)refers to the decrease in the number and quality of ovarian follicles in women of reproductive age.The main clinical manifestations are menstrual disorders,amenorrhea and infertility.Its pathogenesis is affected by many factors such as age,genetics,immunity,psychology and iatrogenic factors.In recent years,the onset population is getting younger and younger,which has become a research hotspot for clinicians.Traditional Chinese medicine(TCM)has the characteristics of multisystem,multi-link,multi-target and holistic regulation,which has significant advantages in the treatment of DOR,Bushen Shengjing-Tiaohe Qixue Formula has been used in clinic for a long time,but its specific mechanism is not clear.Based on this,this study firstly integrated the existing clinical data by systematic review to demonstrate the clinical efficacy of TCM compound in the treatment of DOR,proved its safety and effectiveness,and summarized the commonly used TCM in the treatment of DOR by cluster analysis;Secondly,the network pharmacology method was used to explore the main components and the core genes acting on the disease of the Bushen Shengjing-Tiaohe Qixue Formula,and the molecular docking method was used to verify and analyze the possible mechanism of action.Finally,according to the results of network pharmacology,animal experiments were set up to further discuss the effect of this Chinese herbal formula on the apoptosis and oxidative stress of ovary in DOR model rats,so as to enrich the theoretical system of the formula in the treatment of DOR,and to prove the effectiveness of clinical treatment.This project is divided into three parts: systematic review,network pharmacology and experimental study.2 Objective2.1 By systematic review,to evaluate the clinical efficacy and adverse reactions of the TCM formula in the treatment of DOR,and by cluster analysis,to summary the common herb in the clinical treatment of DOR.2.2 By network pharmacology method,to explore the possible pharmacological mechanism of the Bushen Shengjing-Tiaohe Qixue Formula in the treatment of DOR.2.3 By using animal experiments,to evaluate the curative effect of the Bushen Shengjing-Tiaohe Qixue Formula on DOR model rats,and explore its mechanism of action from the aspects of cell apoptosis and oxidative stress.3 Method3.1 Systematic review of the Bushen Shengjing-Tiaohe Qixue Formula in the treatment of DORUsing the Cochrane Collaboration manual to formulate the search strategy and evaluate the quality of the literature.The databases of CNKI,Wanfang,VIP,Pub Med and CBM were searched.Chinese search terms include"卵巢储备功能减退","卵巢储备功能下降","卵巢储备功能降低",etc.English search terms included"Diminished Ovarian Reserve","Ovarian Reserve*","Medicine,Chinese Traditional",etc.To search Randomized controlled trials(RCTs)of the TCM formula in the treatment of DOR from the establishment of the database to February 2023.According to the inclusion and exclusion criteria,to screen the literature,extract the data and assess the risk of bias.By Revman5.3 software,analyzing the relevant outcome indicators.3.2 By network Pharmacology,to research on the mechanism of the Bushen Shengjing-Tiaohe Qixue Formula in the treatment of DORFirstly,searching Traditional Chinese Medicine Systems Pharmacology Databaseand Analysis Platform(TCMSP),to screen the active ingredients and action targets of the Bushen Shengjing-Tiaohe Qixue Formula according to OB≥30% and DL≥0.18,and standard the target information by Uniport database.Secondly,to obtained the related target genes of DOR from Gene Cards and OMIM databases,and to screen the intersection targets of drugs and diseases by R software;To construct the "drug-component-disease-target" network graph by Cytoscape software,to analyse Protein-Protein Interactions(PPI)by the intersection targets which obtained the String platform,and to obtain the final core genes according to the degree value,and then,to creating GO and KEGG enrichment analysis by using the intersection targets.Finally,to docking the core active components of the Bushen Shengjing-Tiaohe Qixue Formula and the core gene of DOR,by molecular docking technology.3.3 To investigate the effect of the Bushen Shengjing-Tiaohe Qixue Formula on ovarian function of DOR model rats,and research on its mechanism to cell apoptosis and oxidative stress of DOR model ratsSixty specific pathogen-free female SD rats were randomly divided into normal group,model control group,western medicine group and low,medium and high dose of the Bushen Shengjing-Tiaohe Qixue Formula,with 10 rats in each group.To induce the DOR model by a single intraperitoneal injection of cyclophosphamide 75mg/kg,After modeling,the rats were treated by intragastric administration for 3 weeks.Experiment 1: During the experiment,the state of the rats was dynamically observed,including general conditions.At the end of the experiment,the serum and ovarian tissue of the rats were collected to detect the contents of FSH,LH,E2 and AMH by enzyme-linked immunosorbent assay(ELISA),and the pathological changes of the ovarian tissue of the rats were observed by light microscopy.Experiment 2: The m RNA expressions of Bcl-2,Bax,Caspase-3,Caspase-8,and Caspase-9 in ovarian tissues were detected by q PCR,and the protein expressions of Bcl-2,Bax,Caspase-3,Caspase-8,and Caspase-9 in ovarian tissues were detected by Western blot.Experiment 3: The contents of MDA,SOD,GSH,and T-AOC in serum were detected by biochemical method,the m RNA expression of HO-1 and NQO-1 in ovarian tissue was detected by q PCR,the protein expression of HO-1 and NQO-1 in ovarian tissue was detected by Western blot,and the expression of HO-1 and NQO-1 in ovarian tissue was detected by immunohistochemistry.4 Result4.1 Systematic review of the TCM formula in the treatment of DORA total of 17 studies were included in the systematic review.Meta-analysis showed that the total effective rate of the TCM formula in the treatment of DOR was better than that of hormone treatment [RR=1.21,95%CI(1.09,1.33),P=0.0002];In terms of sex hormone levels,the TCM formula was better than the hormone treatment group in reducing serum FSH level and FSH/LH,and increasing E2 and AMH levels.The results are [MD=-2.20,95%C(-3.13,-1.27),P<0.00001],[MD=-0.26,95%C(-0.5,-0.02),P=0.04],[SMD=0.74,95%C(0.23,1.24),P=0.004],[MD=0.74,95%CI(0.35,1.13),P=0.0002];In terms of ultrasonic AFC assessment,the TCM formula was superior to the hormone treatment group in increasing the number of ovarian AFC[MD=0.72,95%CI(0.35,1.08),P=0.0001].4.2 Study on the mechanism of the Bushen Shengjing-Tiaohe Qixue Formula in the treatment of DOR based on network pharmacology.From the TCMSP database,116 active ingredients and 453 targets of TCM compounds were obtained.A total of 191 genes related to DOR were obtained from the disease Gene database.A total of 17 intersection targets were obtained by constructing the drug-disease intersection target network.PPI analysis was performed,and 17 core genes such as ESR1 and Bcl-2 were obtained.The 17 intersection targets were subjected to GO and KEGG enrichment analysis.The results of enrichment analysis showed that the Bushen Shengjing-Tiaohe Qixue Formula could treat the DOR through multiple pathways such as apoptosis,anti-oxidative stress,and endocrine resistance,etc.The proteins corresponding to 8 key genes of the disease “ESR1,CASP3,CASP8,BCL-2,BAX,ESR2,CASP9,AKT1” and 6 key active components of the TCM formula“luteolin、quercetin、isorhamnetin、kaempferol、glycitein、beta-sitosterol” were screened,and molecular docking was performed.The docking results showed that there was an efficient affinity.4.3 Study on the effect of the Bushen Shengjing-Tiaohe Qixue Formula on apoptosis and oxidative stress to DOR model ratsExperiment 1:(1)General situation of rats: After modeling,the estrus cycle of the blank group was basically stable,the estrus cycle of the other groups gradually appeared disorder,showing cycle arrest,prolongation or no obvious cyclical changes.After drug intervention,the estrous cycle of each group gradually recovered.After modeling,the increase rate of body weight in CTX group and high dose TCM group was significantly slower than that in the other groups.After 2 weeks of drug intervention,the increase rate of body weight in each group gradually recovered,and after 3 weeks of drug intervention,the increase rate tended to be synchronous.(2)Serum sex hormone levels: compared with the blank group,the serum levels of FSH and LH were increased,and the levels of E2 and AMH were decreased in the CTX group(P<0.05).Compared with the CTX group,the levels of serum FSH and LH were decreased,and the level of E2 was increased in the western medicine group and the medium-and high-dose Chinese medicine group(P<0.05),and the level of serum AMH was increased in the high-dose Chinese medicine group(P<0.05).Compared with the western medicine group,the traditional Chinese medicine group had the same effect on improving serum sex hormones(P>0.05).(3)Ovarian morphology:the blank group showed uniform growth of follicles at all levels(including primary follicles,secondary follicles,and developing follicles);Compared with the control group,the CTX group showed atrophy of the ovarian medulla,fewer growing follicles in the cortex,mainly primary follicles,and some follicles were surrounded by only 1-2 layers of granulosa cells,and the number of atrete follicles increased.The vascular lumen was small.After drug intervention,the ovarian medulla of each group was loose,the number of growing follicles in the cortex was significantly higher than that in the CTX group,the number of granulosa cell layer in the follicular cavity was increased,the atretic follicles were reduced,and the volume of corpus luteum was increased.The lumen of the vessel was slightly enlarged.(4)Ovarian ultrastructure: normal ovarian ultrastructure,normal nucleoli,uniform chromatin distribution,a large number of mitochondria and rich cristae in the cytoplasm of ovarian granulosa cells were observed in the blank group.In the cyclophosphamide group,the ovary ultrastructure was severely damaged,and the GCs nuclear pyknosis,nuclear chromatin aggregation and edge collection were observed.The number of mitochondria in the cytoplasm was significantly reduced,and the cristae was reduced or disappeared,and even vacuolization was observed.The number of mitochondria in the western medicine group was less than that in the TCM compound group.Nuclear fission was observed in the low-dose TCM compound group.The number of mitochondria in the middle dose and high dose TCM compound groups was more,cristas were increased,and autophagolysosomes were relatively reduced.Experiment 2:(1)Westernblot results: compared with the blank group,the expression of Bcl-2 protein was down-regulated and the expression of Bax protein was up-regulated in the CTX group(P<0.05).Compared with CTX,the expression of Bcl-2 protein was up-regulated after drug intervention,and the expression of Bax protein was down-regulated,and the expression of Bax protein in the high dose of Chinese medicine group was significantly downregulated(P<0.05).Compared with the western medicine group,the expression of Bax protein in the high dose of Chinese medicine group was significantly down-regulated(P<0.05).Compared with the blank group,the protein expressions of Cleaved-caspase3,caspase8 and caspase9 were significantly upregulated in the CTX group(P<0.05).Compared with CTX group,the protein expression levels of caspase3,Cleaved-caspase3,caspase8,Cleaved-caspase8,caspase9 and Cleaved-caspase9 showed a downward trend after drug intervention.The expression levels of Cleaved-caspase3,caspase8 and Cleavedcaspase8 proteins were significantly down-regulated in the low dose group(P<0.05),and in the middle dose group(P<0.05),while the content of Caspase protein in the western medicine group was not significantly changed(P>0.05).Compared with the western medicine group,the expression of caspase3,Cleaved-caspase3,caspase8,Cleaved-caspase8,Cleaved-caspase9 protein after the intervention of the TCM formula group showed a downward trend.The levels of caspase3,Cleaved-caspase3,caspase8 and Cleaved-caspase9 in the low dose group were significantly lower than those in the control group(P<0.05).The levels of Cleaved-caspase3,caspase8,and Cleaved-caspase8 were significantly down-regulated in the middle dose group of TCM(P<0.05),and in the high dose group of TCM(P<0.05).(2)q PCR results: compared with the blank group,the expression of Bcl-2 gene in the CTX group was downregulated,and the expression of Bax gene was up-regulated,but the differences were not statistically significant(P>0.05).Compared with CTX,the expression of Bcl-2 gene was up-regulated after drug intervention,and the expression of Bcl-2 gene in the western medicine group and the low and medium dose of Chinese medicine group was significantly up-regulated(P <0.05),and the expression of Bax gene was down-regulated,but the difference was not statistically significant(P>0.05).Compared with the blank group,the gene expression levels of caspase3,caspase8 and caspase9 in the CTX group were significantly up-regulated(P<0.05).Compared with CTX,the gene expressions of caspase3,caspase8 and caspase9 were down-regulated after drug intervention,and the low and middle doses of traditional Chinese medicine were significantly down-regulated(P<0.05).Compared with the western medicine group,the expression of caspase8 gene in the middle dose of Chinese medicine group was significantly down-regulated(P<0.05),and its efficacy was similar to that of western medicine group,and the difference was not statistically significant(P>0.05).Experiment 3:(1)Serum level of oxidative stress factors: compared with the blank group,the serum level of MDA was increased and the serum level of T-AOC was decreased in the CTX group(P<0.05);Compared with the CTX group,the serum MDA level in the TCM medium dose group was significantly decreased,and the serum SOD,GSH and T-AOC levels were significantly increased(P<0.05),and the serum SOD and T-AOC levels in the western medicine group and the TCM low and high dose group were significantly increased(P<0.05).Compared with the western medicine group,the serum GSH level in the middle dose of Chinese medicine group was significantly increased(P<0.05),and there was no significant difference in the serum levels of the other groups(P>0.05).(2)Western blot results: compared with the blank group,the expression of HO-1 and NQO-1 protein in the CTX group was significantly down-regulated(P<0.05).Compared with the CTX group,the expression of HO-1 protein in the high dose of TCM group was significantly up-regulated(P<0.05),and the expression of NQO-1 protein in the low dose of TCM group was significantly up-regulated(P<0.05).Compared with the western medicine group,the expression of HO-1 protein in the low-,middle-and high-dose Chinese medicine groups was significantly up-regulated(P<0.05),and the expression of NQO-1 protein in the low-and high-dose Chinese medicine groups was significantly up-regulated(P<0.05).(3)Immunohistochemical results: HO-1 and NQO-1 were mainly expressed in oocytes,follicular fluid and granulosa cells,which were strongly expressed in oocytes,follicular fluid and granulosa cells,and weakly expressed in ovarian stromal cells.Compared with NQO-1 protein,HO-1 protein was more strongly expressed in ovary.Compared with the control group,the expression of HO-1 and NQO-1 protein in the ovaries of the CTX group was significantly decreased(P<0.05).Compared with the CTX group,the expression of HO-1 protein in the low-,middle-and highdose TCM group was significantly increased(P<0.05),and the expression of NQO-1 protein in the low-dose TCM group was significantly increased(P<0.05).Compared with the western medicine group,there was no significant difference in the protein expression of HO-1 and NQO-1 in the ovaries of the Chinese medicine groups(P>0.05).The results were basically consistent with the general trend of Westernblot.(4)q PCR results: compared with the blank group,the expression of HO-1 and NQO-1 in the CTX was down-regulated(P>0.05);Compared with the CTX group,the expression of HO-1 gene in the middle-dose and high-dose TCM group was significantly up-regulated(P<0.05),and the expression of NQO-1 gene in the western medicine group and the lowmiddle-dose and high-dose TCM group was slightly up-regulated,but the difference was not statistically significant(P>0.05).5 ConclusionThis study used meta-analysis to summarize that TCM formula can regulate the reproductive endocrine of patients with DOR function,reduce the level of serum FSH,increase the level of serum E2,increase the number of ovarian antral follicles,and effectively improve the ovarian function of patients,and the most common TCM formula is tonifying kidney,strengthening spleen,regulating qi and blood.Secondly,through the network pharmacology method,the core targets and signaling pathways of the Bushen Shengjing-Tiaohe Qixue formula for the treatment of DOR were screened out,and to predicted preliminarily the mechanism of action of the TCM formula in the treatment of DOR by regulating cell apoptosis.Finally,the results were verified by animal experiments. |
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