中药新药与临床药理2025，Vol.36Issue(4)：489-498,10.DOI:10.19378/j.issn.1003-9783.2025.04.001
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
摘要
Abstract
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(10 µmol·L-1);the other groups were treated with 50 ng of corresponding exosomes(final concentration:100 µg·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 of α-SMA,SM22 α,OPN,Wnt1,β-catenin,LRP6,and GSK-3β were measured by Western Blot.Results Exosomes were successfully extracted from HUVECs,with an average particle size of 140.6 nm and a concentration of 1.6×108 particles/mL.Compared with the control group,the model exosome group showed significantly increased HAVSMCs proliferation(P<0.05),enhanced cell migration(P<0.01),elevated apoptosis rate(P<0.01),and increased S-phase cell proportion(P<0.05).HAVSMCs miR-145 expression was significantly downregulated(P<0.01),while WNT2B expression was significantly upregulated(P<0.01).Contractile markers of HAVSMCs α-SMA and SM22α were significantly downregulated(P<0.01),while secretory marker OPN and Wnt/β-catenin signaling pathway proteins Wnt1,β-catenin,LRP6,and GSK-3β were significantly upregulated(P<0.01).Compared with the model exosome group,the Wenxin Formula exosome group showed significantly reduced HAVSMCs proliferation(P<0.05),while the miR-145 inhibitor exosome group exhibited increased proliferation(P<0.05).The Wenxin Formula exosome and miR-145 mimic exosome groups showed significantly reduced HAVSMCs cell migration(P<0.05),while the miR-145 inhibitor exosome group exhibited increased migration(P<0.05).The Wenxin Formula exosome and Wnt inhibitor groups showed reduced S-phase cell proportion(P<0.05).The Wenxin Formula exosome and miR-145 mimic exosome groups showed upregulated HAVSMCs miR-145 expression(P<0.05)and downregulated WNT2B expression(P<0.05).The Wenxin Formula exosome,miR-145 mimic exosome,and Wnt inhibitor groups showed upregulated α-SMA and SM22α protein expression(P<0.05)and downregulated OPN,Wnt1,β-catenin,LRP6,and GSK-3β protein expression(P<0.05).The miR-145 inhibitor exosome group showed downregulated α-SMA and SM22α protein expression(P<0.05)and upregulated OPN expression(P<0.05).Compared with the miR-145 inhibitor exosome group,the miR-145 inhibitor+Wenxin Formula exosome and Wnt inhibitor groups showed significantly reduced HAVSMCs proliferation(P<0.05)and cell migration(P<0.05).The miR-145 inhibitor+Wenxin Formula exosome group showed upregulated α-SMA and SM22α protein expression(P<0.05)and downregulated OPN,Wnt1,β-catenin,LRP6,and GSK-3β protein expression(P<0.05).Conclusion Exosomes derived from HUVECs treated with Wenxin Formula upregulate miR-145 expression in HAVSMCs,targeting WNT2B and inhibiting the Wnt/β-catenin signaling pathway to promote HAVSMCs contractile phenotype transformation,inhibit proliferation and migration,and alleviate vascular intimal hyperplasia,thereby exerting preventive and therapeutic effects on coronary atherosclerosis.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Key words
Wenxin Formula/coronary atherosclerosis/vascular endothelial cells/exosome/vascular smooth muscle cells/miR-145/Wnt/β-catenin signaling pathway/rat serum分类
医药卫生引用本文复制引用
李金霞,张鑫芸,杨力鉴,王怡萱,宗文静,刘旺华,郑彩杏,曹洪欣..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[J].中药新药与临床药理,2025,36(4):489-498,10.基金项目
国家自然科学基金项目(82104775) （82104775）
湖南省科技创新计划项目(2024RC3198) （2024RC3198）
湖南省自然科学基金项目(2025JJ50567) （2025JJ50567）
湖南省教育厅科研项目(23B0357) （23B0357）
湖南中医药大学科研项目(Z2023XJYQ04) （Z2023XJYQ04）
中国科学技术学会青年人才托举工程项目(2022QNRC001). （2022QNRC001）
