白花丹醌对大鼠肝星状细胞活性氧及TGF-β1/Smad信号通路的影响

Effects of plumbagin on ROS and TGF-β1/Smad signaling pathway in hepatic stellate cells

目的 研究白花丹醌对大鼠原代分离肝星状细胞(HSC)活性氧(ROS)水平、α-平滑肌肌动蛋白(α-SMA)、Smad2/3、p-Smad2/3、Smad7蛋白表达的影响。方法 分离原代大鼠HSC,接种活化期HSC,进行α-SMA免疫荧光鉴定,分为空白组、TGF-β1组、NADPH氧化酶抑制剂组(DPI)、白花丹醌低、中、高剂量(2、4、8 μmol·L -1 )组。MTT检测白花丹醌对HSC增殖的影响;显微镜下观察白花丹醌对HSC形态学的影响;荧光探针法测定HSC内ROS表达水平;Western blot检测HSC中Smad2/3、p-Smad2/3和Smad7蛋白表达。结果 经α-SMA免疫组织荧光鉴定,阳性率达91%,表明获得高纯度的大鼠原代HSC。MTT测定白花丹醌最佳给药浓度为2、4、8 μmol·L -1;DPI组和白花丹醌各浓度组可不同程度下调HSC内ROS的表达;Western blot结果显示,与模型组比较,DPI和白花丹醌给药组Smad2/3、p-Smad2/3的蛋白表达降低,Smad7蛋白表达增加。结论 白花丹醌可能是通过下调ROS水平,进而降低Smad2/3和p-Smad2/3的表达,从而发挥抗HSC活化的作用。

Aim To investigate the effects of plumbagin on reactive oxygen species (ROS) level and expressions of α-smooth muscle actin (α-SMA),Smad2/3,p-Smad2/3,and Smad7 proteins. Methods We isolated primary hepatic stellate cell(HSC) of rat and inoculated the activated HSC. Then these cells would be divided into TGF-β1 treatment group,NADPH oxidase inhibitor (DPI) group,plumbagin treatment group, and control group. MTT was used to test cell proliferation and fluorescent probe was used to detect ROS level. Moreover,the protein expressions of Smad2/3,p-Smad2/3,and Smad7 were determined using Western blot. Results Results of fluorescent probe showed that the positive rate of α-SMA was up to 91%. MTT test indicated the optimal concentrations of plumbagin were 2,4,and 8 μmol·L -1 . A decreased level of ROS could be found in DPI and plumbagin treatment groups. In addition,results of Western blot showed that a decreased expression of Smad2/3,p-Smad2/3 and an increased expression of Smad7 could be found in DPI and plumbagin treatment groups compared to control group. Conclusions Plumbagin may decrease the expression of smad2/3 and p-smad2/3 by down-regulating the level of ROS,and thus play an anti-HSC activation role.