| Objective This study is to explore the mechanism of Qingxin Lianzi Drink on SIRT1/MTOR/p70S6K pathway in perimenopausal syndrome model rats through animal experimental technology and network pharmacological method.Methods 1.Sixty female SD rats were randomly divided into normal and model groups,10 in the normal group and 50 in the model group;Rats in the model group were injected intraperitoneally with 80 mg/kg of 4-VCD for 15 days,and the model was tested by taking vaginal smears to see if it was established.After successful modeling,the model group was randomly divided into model group,estradiol valerate group,Qingxinlianzi Drink low,medium and high dose group,a total of 5groups,10 animals in each group,and the normal group for control observation.The normal group and the model group were treated with equal doses of pure water by oral gavage,while the estradiol valerate group and the Qingxinlianzi Drink low,medium and high dose groups were treated with oral gavage for 28 days,and the general condition of rats in each group was observed.Observe the pathological changes of ovaries and uterus of each group of rats by HE staining after the treatment;The levels of serum E2,FSH,LH and AMH in each group of rats were measured by Elisa;The expression levels of SIRT1,MTOR and p70S6K mRNA gene in the ovaries of each group of rats were detected by RT-qPCR;the expression of SIRT1,MTOR and p70S6K protein in the ovaries of each group of rats were detected by WB and immunohistochemistry.2.The chemical components and targets of Qingxinlianzi drink were searched by TCMSP and TCMID;the targets of perimenopausal syndrome were screened by Gene Cards,OMIM and DrugBank databases;the drug targets and disease targets were intersected and the common genes were obtained by Venn diagram;the common genes and SIRT1,MTOR and p70S6K targets were imported into STING database to obtain their correlations and draw protein interactions.SIRT1,MTOR and p70S6K were imported into STING database,and their correlations were obtained and protein interactions were mapped;Cytoscape 3.7.2 software was used to map the "Qingxin Lianzi Drink-Active Ingredients-Target of Action-PMS" network;GO and KEGG enrichment analysis was performed by R software,and key active ingredients were compared with SIRT1,MTOR and p70S6K.Results 1.The results of animal experiments showed that,compared with the normal group,the rats in the perimenopausal syndrome model had dull hair,reduced diet,lower body weight,slower movement and depression after modeling;after 28 days of treatment,the diet and water intake of each drug group gradually increased,body weight was more stable,and hair and mental status gradually improved,among which the effects of estradiol valerate treatment group and Qingxinlianzi drink medium dose group were more obvious;HE results showed that Qingxinlianzi Drink could improve the pathological morphology of ovaries and uterus,thicken the endometrium and promote follicle development in PMS model rats;ELISA results showed that compared with the normal group,the serum E2 and AMH levels of rats in the model group decreased significantly(P<0.05),and FSH and LH levels increased significantly(P<0.05);compared with the model group,the E2 level in the middle dose group of Qingxinlianzi Drink increased significantly(P<0.05),and AMH in each treatment group of Qingxinlianzi Drink increased significantly(P<0.05),and LH and FSH levels significantly decreased(P<0.05);WB results showed that Qingxinlianzi Drink Qingxinlianzi Drink was able to up-regulate the ovarian SIRT1 protein expression level in PMS model rats(P<0.05)and down-regulate the MTOR and p70S6K protein expression levels(P<0.05);immunohistochemical results showed that Qingxinlianzi Drink Qingxinlianzi Drink was able to up-regulate the ovarian SIRT1 protein expression level in PMS model rats(P<0.05);immunohistochemical results showed that Qingxinlianzi Drink Qingxinlianzi Drink was able to up-regulate the ovarian SIRT1 protein expression level(P<0.05)and down-regulated MTOR,p70S6K protein expression level(P<0.05);RT-qPCR results showed that Qingxinlianzi drink could up-regulate the ovarian SIRT1 mRNA gene expression level(P<0.05)and down-regulate the MTOR and p70S6K mRNA gene expression levels(P<0.05)in PMS model rats.2.The network pharmacology results showed that there were 222 active ingredients of Qingxinlianzi Drink,corresponding to a total of 278 drug targets;the four databases of Gene Cards,DrugBank,OMIM and Disgenet obtained a total of 315 perimenopausal syndrome disease targets;a total of 62 genes were intersected between Qingxinlianzi Drink and perimenopausal syndrome;PPI results showed that MTOR,SIRT1 and RPS6KB1 were closely related to drug disease intersected genes.The PPI results showed that MTOR,SIRT1 and RPS6KB1 were closely related to the drug-disease intersection genes,and the Cytoscape3.7.2 software screened the core targets VEGFA,MTOR,TP53,SIRT1,RPS6KB1,IL1 B,ESR1,etc.The key active ingredients of Qingxin Lianzi Drink are quercetin,luteolin,7-O-methylisomucronulatol,kaempferol,etc;the main pathways involved are chemical carcinogenesis-receptor activation,neuroactive The main pathways involved are chemical carcinogenesis-receptor activation,neuroactive ligand-receptor interaction,PI3K-Akt signaling pathway and 103 other signaling pathways.The molecular docking results showed that the active ingredients quercetin,luteolin and 7-O-methylisomucronulatol could bind well to the target MTOR,SIRT1 and p70S6K.Conclusion Qingxinlianzi drink can improve the general condition,ovarian and uterine functions of rats in the perimenopausal syndrome model;it can increase the serum levels of E2 and AMH and decrease the levels of FSH and LH in the perimenopausal model rats;Qingxinlianzi drink can regulate the expression levels of SIRT1,MTOR and p70S6K protein in ovarian tissues of rats with perimenopausal syndrome model,and regulate the expression levels of SIRT1 mRNA,MTORmRNA and p70S6KmRNA gene,and its mechanism of action may be related to the regulation of SIRT1/MTOR/p70S6K pathway. |
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