胡椒碱抑制内皮细胞向成纤维细胞转化  

Piperine inhibits the transformation of endothelial cells into fibroblasts

作　　者：李亚彭[1] 高路[1] 史慧婷 冯胜东 田欣雨 孔令尧[1] 张彦周[1] Li Yapeng;Gao Lu;Shi Huiting;Feng Shengdong;Tian Xinyu;Kong Lingyao;Zhang Yanzhou(Department of Cardiology,First Affiliated Hospital of Zhengzhou University,Zhengzhou 450000,China)

机构地区：[1]郑州大学第一附属医院心内科,450000

出　　处：《中华心血管病杂志》2019年第7期554-560,共7页Chinese Journal of Cardiology

基　　金：河南省医学科技攻关计划(201702063).

摘　　要：目的探讨胡椒碱在内皮细胞向成纤维细胞转化中的作用.方法培养人脐静脉内皮细胞(HUVEC),取第4~6代细胞用于实验.采用转化生长因子β(TGF-β)诱导内皮细胞向成纤维细胞转化模型.将HUVEC分为6组,即对照组、TGF-β组、TGF-β+1μmol/L胡椒碱组、TGF-β+5μmol/L胡椒碱组、TGF-β+10μmol/L胡椒碱组和TGF-β+20μmol/L胡椒碱组.采用CKK-8试剂盒检测细胞增殖率.采用CD31/波形蛋白(vimentin)荧光染色检测2种蛋白的表达水平.采用血管内皮钙黏蛋白(VE-cadherin)/α-平滑肌肌动蛋白(α-SMA)免疫荧光染色检测2种蛋白的表达水平.采用实时定量PCR法检测内皮向间质细胞转化标志物的转录水平.Westernblot法检测snail和twist的蛋白表达水平.结果(1)胡椒碱对TGF-β诱导的细胞增殖率的影响:TGF-β组细胞增殖率明显高于对照组(P<0.05),TGF-β+1μmol/L胡椒碱组和TGF-β+5μmol/L胡椒碱组细胞增殖率与TGF-β组比较差异均无统计意义(P均>0.05),TGF-β+10μmol/L胡椒碱组和TGF-β+20μmol/L胡椒碱组细胞增殖率则明显低于TGF-β组(P均<0.05).(2)胡椒碱对TGF-β诱导的CD31/vimentin表达的影响:TGF-β组细胞CD31的表达水平明显低于对照组(P<0.05),而vimentin的表达水平则明显高于对照组(P<0.05).TGF-β+10μmol/L胡椒碱组和TGF-β+20μmol/L胡椒碱组细胞CD31的表达水平则均明显高于TGF-β组(P均<0.05),而vimentin的表达水平则均明显低于TGF-β组(P均<0.05).(3)胡椒碱对TGF-β诱导的VE-cadherin/α-SMA表达的影响:TGF-β组细胞VE-cadherin的表达水平明显低于对照组(P<0.05),而α-SMA的表达水平则明显高于对照组(P<0.05).TGF-β+10μmol/L胡椒碱组和TGF-β+20μmol/L胡椒碱组细胞的VE-cadherin表达水平则明显高于TGF-β组(P<0.05),而α-SMA的表达水平则明显低于TGF-β组(P<0.05).(4)胡椒碱对胶原蛋白转录的影响:TGF-β组细胞Ⅰ型胶原和Ⅲ型胶原的mRNA表达水平均明显高于对照组(P均<0.05),而TGF-β+10μmol/L胡椒碱组和TObjective To investigate the role of piperine on the transformation of endothelial cells into fibroblasts.Methods Cultured human umbilical vein endothelial cells(HUVECs,4-6 passage)were used for the main experiments.The transformation models of endothelial cells into fibroblasts were induced by transforming growth factorβ(TGF-β)stimulation.HUVECs were divided into 6 groups:control group,TGF-βgroup and 4 groups treated with various concentrations of piperine(1,5,10,20μmol/L).CKK-8 was used to detect cell proliferation.The CD31/α-smooth muscle actin(α-SMA)expression level was detected by fluorescent staining.The vascular endothelial cadherin(VE-cadherin)/vimentin expression was detected by immunofluorescence staining.RT-PCR was used detect the mRNA expressions of transformation markers.Western blot was used to detect the protein expression of snail and twist.Results TGF-βincreased HUVECs proliferation(P<0.05),which could be significantly inhibited by 10 and 20μmol/L of piperine,but not by 1 and 5μmol/L of piperine.Immunofluorescence results demonstrated that TGF-βincreased HUVECs transformation to fibroblasts as shown by downregulated expression of endothelial markers CD31,VE-cadherin,and upregulated expression ofα-SMA and vimentin,again,these effects could be attenuated by 10 and 20μmol/L piperine.The expression levels of collagen typeⅠand typeⅢwere significantly higher in TGF-βgroup than in control group(P<0.05),significantly lower in TGF-β+10μmol/L piperine group and TGF-β+20μmol/L piperine group than in TGF-βgroup(P<0.05).In addition,RT-PCR results showed that TGF-βincreased mRNA expression of transformation markers(snail1,snail2,twist1,twist2),while 10 and 20μmol/L of piperine could significantly downregulated the mRNA expressions of these markers.The protein expression levels of snail and twist were significantly higher in TGF-βgroup than in control group(both P<0.05),which was significantly lower in TGF-β+20μmol/L piperine group than in TGF-βgroup(both P<0.05).Conclusions Piperine

关 键 词：内皮细胞 成纤维细胞 胡椒碱 心肌纤维化 

分 类 号：R54[医药卫生—心血管疾病]