Objective: 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 μmol/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.
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+10 μmol/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。.
Keywords: Cardiac fibrosis; Endothelial cells; Fibroblasts; Piperine.