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+1 μmol/L胡椒碱组、TGF-β+5 μmol/L胡椒碱组、TGF-β+ 10 μmol/L胡椒碱组和TGF-β+20 μmol/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.Western blot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+1 μmol/L胡椒碱组和TGF-β+5 μmol/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+10 μmol/L胡椒碱组和TGF-β+20 μmol/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+20 μmol/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+ 10 μmol/L胡椒碱组和TGF-β+20 μmol/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+ 10 μmol/L胡椒碱组和TGF-β+20 μmol/L胡椒碱组则均明显低于TGF-β组(P均＜0.05).(5)胡椒碱对内皮向间质转化标志物转录和表达的影响:TGF-β组细胞内皮向间质转化标志物snail1、2和twist1、2的mRNA表达水平均明显高于对照组(P均＜0.05),而TGF-β+ 10 μmol/L胡椒碱组和TGF-β+20 μmol/L胡椒碱组则均明显低于TGF-β组(P均＜0.05).TGF-β组细胞内皮向间质转化标志物snail和twist蛋白的表达水平均明显高于对照组(P均＜0.05),而TGF-β+20 μmol/L胡椒碱组则均明显低于TGF-β组(P均＜0.05).结论 胡椒碱可抑制内皮细胞向成纤维细胞转化,这可能是胡椒碱抗心肌纤维化的机制之一.

abstractsObjective 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 factor β (TGF-β) stimulation.HUVECs were divided into 6 groups:control group,TGF-β group and 4 groups treated with various concentrations of piperine (1,5,10,20 μmol/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-β increased HUVECs proliferation (P＜0.05),which could be significantly inhibited by 10 and 20 μmol/L of piperine,but not by 1 and 5 μmol/L of piperine.Immunofluorescence results demonstrated that TGF-β increased HUVECs transformation to fibroblasts as shown by downregulated expression of endothelial markers CD31,VE-cadherin,and upregulated expression of α-SMA and vimentin,again,these effects could be attenuated by 10 and 20 μmol/L piperine.The expression levels of collagen type Ⅰ and type Ⅲ were significantly higher in TGF-β group than in control group (P＜0.05),significantly lower in TGF-β+ 10 μ mol/L piperine group and TGF-β+20 μmol/L piperine group than in TGF-β group (P＜0.05).In addition,RT-PCR results showed that TGF-β increased mRNA expression of transformation markers (snail1,snail2,twist1,twist2),while 10 and 20 μmol/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-β group than in control group (both P＜0.05),which was significantly lower in TGF-β+20 μmol/L piperine group than in TGF-β group (both P＜0.05).Conclusions Piperine can inhibit the transformation of endothelial cells into fibroblasts.This effect might be viewed as one of the potential mechanisms of reduced myocardial fibrosis post piperine treatment.