滋肾清热利湿化瘀方治疗多囊卵巢综合征的网络药理学作用机制及实验研究

Network pharmacological mechanism of action and experimental study of Zishen Qingre Lishi Huayu Prescription in treatment of polycystic ovary syndrome

目的采用网络药理学探讨滋肾清热利湿化瘀方(Zishen Qingre Lishi Huayu Prescription,ZQLHP)治疗多囊卵巢综合征(polycystic ovary syndrome,PCOS)的作用机制,并进行实验验证。方法基于网络药理学预测滋肾方的核心成分、靶基因和主要通路,对其中PCOS相关成分、靶基因、通路进行筛选;采用来曲唑(letrozole,LET)诱导PCOS小鼠模型,观察滋肾方对PCOS小鼠的作用,并对网络药理学结果进行机制验证。结果网络药理学结果表明,滋肾方456个成分中含有361个相关靶点,PCOS相关靶点1397个,交集靶点125个;获得槲皮素、豆甾醇、β-谷甾醇等主要活性成分与丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAPK3)、蛋白激酶1(AKT serine/threonine kinase 1,AKT1)和信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)等核心靶点;GO分析得到BP 663条目,CC 64条目,MF 119条目,其中富集基因数量较多的条目为negative regulation of apoptotic process,plasma membrane,protein binding;KEGG分析显示潜在作用靶点主要富集在癌症信号通路、PI3K-AKT和TNF等信号通路;PPI网络分析结果显示,MAPK3、AKT1和STAT3可能是滋肾方治疗PCOS的关键靶点。动物实验结果表明,与模型组比较,滋肾方组的小鼠的体重降低(P<0.05),且能有效改善PCOS模型小鼠的组织病理学表现,囊性卵泡数量降低(P<0.05),黄体数量增加(P<0.01)。降低卵巢组织中IL-10水平(P<0.01),升高IL-6、TNF-α水平(P<0.05,P<0.01)。Western blot和免疫组织化学(immunohistochemistry,IHC)结果显示,PCOS小鼠卵巢组织p-AKT蛋白表达量下降(P<0.05,P<0.01),p-p38MAPK和p-STAT3蛋白表达较正常组升高(P<0.05,P<0.01),而经滋肾方治疗后p-AKT蛋白表达上升(P<0.05),p-p38MAPK和p-STAT3蛋白表达明显下降(P<0.05,P<0.01)。结论滋肾方可能通过调控AKT/p38MAPK/STAT3信号改善来曲唑诱导的PCOS小鼠的炎性损伤。

Aim To investigate the mechanism of action of Zishen Qingre Lishi Huayu Prescription(ZQLHP)in the treatment of polycystic ovary syndrome(PCOS)using network pharmacology and to experimentally validate it.Methods The core components,target genes and major pathways of ZQLHP were predicted based on network pharmacology,and the PCOS-related components,target genes and pathways were screened;Letrozole(LET)was used to induce PCOS in a mouse model to observe the effects of ZQLHP on PCOS mouse,and the network pharmacology results were validated mechanistically.Results The network pharmacology results showed that the 456 components of ZQLHP contained 361 relevant targets,1397 PCOS-related targets and 125 intersecting targets;the main active ingredients such as quercetin,dousterol andβ-sitosterol were obtained with MAPK3(mitogen-activated protein kinase 3,mitogen-activated protein kinase 3)and AKT1(protein kinase 1,AKT serine/threonine kinase 1)and STAT3(signal transducer and activator of transcription 3)and other core targets;GO analysis obtained BP 663 entries,CC 64 entries and MF 119 entries,where the entries with a large number of enriched genes were negative regulation of apoptotic process,plasma membrane,and protein binding;KEGG analysis showed that the potential targets were mainly enriched in signaling pathways such as cancer,PI3K-AKT and TNF;the results of PPI network analysis showed that MAPK3,AKT1 and STAT3 might be the key targets for the treatment of PCOS with ZQLHP.The results of animal experiments showed that,compared with the model group,the mice in the ZQLHP group had lower body weight(P<0.05)and could effectively improve the histopathological manifestations of PCOS model mice,with lower number of cystic follicles(P<0.05)and increased number of corpus luteum(P<0.01).The levels of IL-10 in ovarian tissues decreased(P<0.01)and the levels of IL-6 and TNF-αincreased(P<0.05,P<0.01).Western blot and IHC observed that the expression of p-AKT protein in ovarian tissue of PCOS mice decreased(P<0.05,P<0.01)and the expression of p-p38MAPK and p-STAT3 protein increased compared with normal group(P<0.05,P<0.01),while p-AKT protein expression increased(P<0.05)and p-p38MAPK and p-STAT3 protein expression decreased significantly(P<0.05,P<0.01)after treatment with ZQLHP.Conclusions ZQLHP may improve letrozole-induced inflammatory injury in PCOS mice by regulating AKT/p38MAPK/STAT3 pathway.