温心方调控人脐静脉内皮细胞外泌体miR-145表达抑制人主动脉血管平滑肌细胞表型转化及增殖、迁移的作用机制  

Mechanism of Wenxin Formula Regulating HUVECs Exosome miR-145 Expression to Inhibit HAVSMCs Phenotypic Transformation,Proliferation and Migration

作　　者：李金霞[1,2] 张鑫芸 杨力鉴 王怡萱 宗文静 刘旺华 郑彩杏[1] 曹洪欣 LI Jinxia;ZHANG Xinyun;YANG Lijian;WANG Yixuan;ZONG Wenjing;LIU Wanghua;ZHENGCaixing;CAO Hongxin(Hunan University of Chinese Medicine,Changsha 410208 Hunan,China;Key Laboratory of TCM Diagnostics in Hunan Province,Changsha 410208 Hunan,China;China Academy of Chinese Medical Sciences,Beijing 100700,China;China Association of Chinese Medicine,Beijing 100029,China)

机构地区：[1]湖南中医药大学,湖南长沙410208 [2]中医诊断学湖南省重点实验室,湖南长沙410208 [3]中国中医科学院中药研究所,北京100700 [4]中华中医药学会,北京100029

出　　处：《中药新药与临床药理》2025年第4期489-498,共10页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：国家自然科学基金项目(82104775);湖南省科技创新计划项目(2024RC3198);湖南省自然科学基金项目(2025JJ50567);湖南省教育厅科研项目(23B0357);湖南中医药大学科研项目(Z2023XJYQ04);中国科学技术学会青年人才托举工程项目(2022QNRC001)。

摘　　要：目的基于人脐静脉内皮细胞(HUVECs)外泌体miR-145调控人主动脉血管平滑肌细胞(HAVSMCs)表型转化角度探讨温心方治疗冠心病的作用机制。方法给予大鼠温心方水煎剂33.75 g·kg^(-1)灌胃,每天1次,连续7 d,制备含药血清。(1)取对数生长期的HUVECs,分别设立模型组、温心方组、miR-145 mimic组、miR-145 inhibitor组、miR-145 inhibitor+温心方组。模型组加入50 mg·L^(-1)ox-LDL培养24 h;温心方组加入25%温心方含药血清培养24 h后,再加入ox-LDL培养24 h;miR-145 mimic组、miR-145 inhibitor组分别用miR-145过表达质粒及小干扰RNA转染后,再加入ox-LDL培养24 h;miR-145 inhibitor+温心方组在转染后加入25%温心方含药血清培养24 h,再加入50 mg·L^(-1)ox-LDL培养24 h。采用高速离心分离获取HUVECs外泌体,电镜观察外泌体形态,BCA法及NTA法对外泌体蛋白浓度及粒径进行测定。(2)取对数生长期HAVSMCs,分别设立对照组、模型外泌体组、温心方外泌体组、miR-145 mimic外泌体组、miR-145 inhibitor外泌体组、miR-145 inhibitor+温心方外泌体组、Wnt抑制剂组。对照组继续使用培养基培养;Wnt抑制剂组加入50 ng XAV-939(10μmol·L^(-1));其余各组加入对应外泌体50 ng(外泌体终浓度为100μg·mL^(-1)),培养24 h。采用MTT法检测HAVSMCs细胞增殖率;Transwell法检测细胞迁移能力;流式细胞术检测细胞凋亡及细胞周期;qRT-PCR法检测细胞miR-145、WNT2B基因表达水平;Western Blot法检测细胞α平滑肌肌动蛋白(α-SMA)、平滑肌22α蛋白(SM22α)、骨桥蛋白(OPN)、Wnt1、β-catenin、LRP6、GSK-3β蛋白表达水平。结果成功提取HUVECs外泌体,平均粒径为140.6 nm,浓度为1.6×10^(8)个/mL。与对照组比较,模型外泌体组的HAVSMCs细胞增殖率明显升高(P<0.05),细胞迁移数显著增加(P<0.01);细胞凋亡率显著升高(P<0.01),S期细胞占比明显增加(P<0.05);HAVSMCs miR-145基因表达显著下调(P<0.01),WNT2B基因表达显著上调(P<0.01);HAVSMCs�Objective To explore the mechanism of Wenxin Formula in the treatment of coronary heart disease from the perspective of regulating human umbilical vein endothelial cell(HUVECs)exosome miR-145 expression to inhibit phenotype transformation of human aortic vascular smooth muscle cells(HAVSMCs).Methods Rats were administered Wenxin Formula decoction(33.75 g·kg^(-1))by gavage once daily for 7 days to prepare drug-containing serum.(1)HUVECs in the logarithmic growth phase were divided into model,Wenxin Formula,miR-145 mimic,miR-145 inhibitor,and miR-145 inhibitor+Wenxin Formula groups.The model group was treated with 50 mg·L^(-1)ox-LDL for 24 hours;the Wenxin Formula group was treated with 25%Wenxin Formula drug-containing serum for 24 hours,followed by ox-LDL for 24 hours;the miR-145 mimic and miR-145 inhibitor groups were transfected with miR-145 overexpression plasmid or siRNA,respectively,followed by ox-LDL treatment for 24 hours;the miR-145 inhibitor+Wenxin Formula group was transfected and then treated with 25%Wenxin Formula drug-containing serum for 24 hours,followed by 50 mg·L^(-1)ox-LDL for 24 hours.Exosomes were isolated from HUVECs by ultracentrifugation,and their morphology was observed by electron microscopy.Exosome protein concentration and particle size were measured using BCA and NTA assays.(2)HA-VSMCs in the logarithmic growth phase were divided into control,model exosome,Wenxin Formula exosome,miR-145 mimic exosome,miR-145 inhibitor exosome,miR-145 inhibitor+Wenxin Formula exosome,and Wnt inhibitor groups.The control group was cultured in medium;the Wnt inhibitor group was treated with 50 ng XAV-939(10μmol·L^(-1));the other groups were treated with 50 ng of corresponding exosomes(final concentration:100μg·mL^(-1))for 24 hours.HA-VSMC proliferation was assessed by MTT assay;cell migration was evaluated using Transwell assay;apoptosis and cell cycle were analyzed by flow cytometry;miR-145 and WNT2B gene expression levels were detected by qRT-PCR;and protein expression levels ofα-SMA,SM22α,OPN,

关 键 词：温心方 冠状动脉粥样硬化 血管内皮细胞 外泌体 血管平滑肌细胞 MIR-145 WNT/Β-CATENIN信号通路 大鼠血清 

分 类 号：R285.5[医药卫生—中药学]