目的 基于网络药理学和睾丸间质细胞模型探究恩施巴戟对睾丸间质细胞睾酮分泌的影响和潜在作用机制。方法利用网络药理学和分子对接预测恩施巴戟治疗睾酮缺乏症的主要作用靶点及通路；通过LC-MS/MS技术分析恩施巴戟主要化学成分；构建睾丸间质细胞模型，采用MTT法检测睾丸间质细胞的存活率，采用ELISA法检测睾酮分泌量，采用qRTPCR检测睾酮合成和凋亡相关基因表达，采用Western blotting检测睾酮合成蛋白表达。结果 网络药理学和分子对接结果显示，恩施巴戟主要通过丝裂原活化蛋白激酶1（mitogen-activated protein kinase 1，MAPK1）、磷脂酰肌醇3-激酶调节亚基1（phosphatidylinositol 3-kinaseregulatory subunit 1，PIK3R1）等靶点和环磷酸腺苷（cyclic adenosine monophosphate，cAMP）、MAPK、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）/蛋白激酶B（protein kinase B，Akt）等信号通路发挥作用，水晶兰苷为其主要有效成分。LC-MS/MS分析和睾丸间质细胞实验结果验证了网络药理学预测分析，恩施巴戟提取物和水晶兰苷均具有促进睾丸间质细胞生长和睾酮分泌的作用，其作用机制与促进类固醇生成急性调控蛋白（steroidogenic acute regulatory protein，StAR）、胆固醇侧链裂解酶（cholesterol side-chain cleavage enzyme，Cyp11a1）、B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）/Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）mRNA表达（P＜0.05、0.01）并促进StAR蛋白表达有关（P＜0.01）。结论 恩施巴戟具有促进睾酮分泌的作用，水晶兰苷为其主要活性成分，其作用机制与作用于MAPK、PI3K/Akt通路调控睾酮合成有关。

Objective To investigate the effect and potential mechanism of Enshi Baji (Damnacanthi Officinari Radix) on testosterone secretion in Leydig cells based on network pharmacology and Leydig cells model.Methods Network pharmacology and molecular docking were used to predict the potential targets and pathways of Damnacanthi Officinari Radix in treating testosterone deficiency. LCMS/MS was used to analyze the main chemical components of Damnacanthi Officinari Radix. In Leydig cells model, MTT assay was used to detected cells survival rate, ELISA kit was used to detect testosterone secretio, qRT-PCR was used to detect the related mRNA expressions of testosterone synthesis and apoptosis, and Western blotting was used to detect the expression of testosterone synthetic protein.Results Network pharmacology and molecular docking results showed that Damnacanthi Officinari Radix mainly played a role through targets such as mitogen-activated protein kinase 1 (MAPK1), phosphatidylinositol 3-kinase regulatory subunit 1 (PIK3R1), and pathways such as cyclic adenosine monophosphate (cAMP), MAPK, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt). Monotropein was the main active ingredient. LC-MS/MS analysis results and Leydig cells experiment validated the predictive analysis of network pharmacology. Extract of Damnacanthi Officinari Radix and monotropein could promote Leydig cells growth and testosterone secretion. Its mechanism was related to promoting steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (Cyp11a1), B-cell lymphoma-2 (Bcl-2)/Bcl-2 associated X protein (Bax) mRNA expressions (P<0.05, 0.01), and StAR protein expression (P<0.01).Conclusion Damnacanthi Officinari Radix can promote testosterone secretion, and monotropein is the main active ingredient. Its mechanism is related to the regulation of testosterone synthesis by MAPK and PI3K/Akt pathways.

R285.5

湖北省重点研发计划项目(2020ACA007)