姜黄素通过EZH2/PPARγ途径抑制肝星状细胞活化

Curcumin inhibits activation of hepatic stellate cells through EZH2/PPARγ pathway

目的 研究姜黄素抑制肝星状细胞(hepatic stellate cells,HSCs)活化的分子机制。方法 大鼠肝星状细胞HSC-T6分为对照组、转化生长因子-β1(transforming growth factor-β1,TGF-β1)组、姜黄素组、EZH2过表达组、EZH2敲降组和EZH2抑制剂组。对照组正常培养,TGF-β1组用TGF-β1处理,姜黄素组用TGF-β1与姜黄素共处理,EZH2过表达组用TGF-β1处理同时转染EZH2真核表达载体,EZH2敲降组用TGF-β1处理同时转染EZH2干扰RNA,EZH2抑制剂组用TGF-β1与EZH2抑制剂DZNep共处理。提取RNA和蛋白质,采用real-time PCR和Western blot技术检测肝纤维化相关基因α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、Ⅰ型胶原蛋白a1(typeⅠcollagen a1,Col1a1)、组织金属蛋白酶抑制剂1(tissue inhibitor of metalloproteinase 1,Timp1)、组蛋白甲基转移酶EZH2和纤维化抑制因子PPARγ的表达,免疫荧光技术检测细胞内H3K27me3水平。结果 与对照组相比,TGF-β1组HSC-T6细胞中α-SMA、Col1a1、Timp1和EZH2的表达升高(P<0.01),H3K27me3水平升高,PPARγ的表达下降(P<0.01);而姜黄素组的结果与TGF-β1组相反(P<0.01或P<0.05)。与TGF-β1组相比,EZH2过表达组PPARγ的表达降低(P<0.05);而EZH2敲降组和EZH2抑制剂组PPARγ表达上调(P<0.05)。结论 姜黄素可通过抑制EZH2表达进而上调PPARγ表达水平,从而起到抑制HSCs活化的作用。

Objective To investigate the molecular mechanism of curcumin inhibiting the activation of hepatic stellate cells(HSCs).Methods Rat hepatic stellate cells HSC-T6 were divided into control group,transforming growth factor-β1(TGF-β1) group,curcumin group,EZH2 overexpression group,EZH2 knockdown group and EZH2 inhibitor group.HSC-T6 cells in control group were cultured normally,HSC-T6 cells in TGF-β1 group was treated with TGF-β1,HSC-T6 cells in curcumin group were treated with TGF-β1 and curcumin,HSC-T6 cells in EZH2 overexpression group were treated with TGF-β1 and EZH2 eukaryotic expression vector,HSC-T6 cells in EZH2 knockdown group were treated with TGF-β1 and EZH2 interfering RNA,and HSC-T6 cells in EZH2 inhibitor group were treated with TGF-β1 and EZH2 inhibitor DZNep.RNA and proteins were extracted,the expressions of liver fibrosis related genes α-smooth muscle actin(α-SMA),type Ⅰ collagen a1(Col1a1),tissue inhibitor of metalloproteinase 1(Timp1),histone transmethylase EZH2 and liver fibrosis inhibitory factor PPARγ were detected by real-time PCR and Western blot,respectively.H3K27me3 level was detected by immunofluorescence.Results Compared with control group,the expressions of α-SMA,Col1a1,Timp1,EZH2 were increased in TGF-β1 group(P<0.01),H3K27me3 level was increased,and the expression of PPARγ was decreased(P<0.01),while the results in curcumin group were opposite to those in TGF-β1 group(P<0.01 or P<0.05).Compared with TGF-β1 group,the expression of PPARγ was decreased in EZH2 overexpression group(P<0.05),while the PPARγ expression was increased in EZH2 knockdown group and EZH2 inhibitor group(P<0.05).Conclusion Curcumin can inhibit the expression of EHZ2,then increase PPARγ expression,thus suppressing HSCs activation.