白首乌C-21甾体总苷通过TGF-β_(1)/Smad信号通路干预人肝星状细胞活化的机制  

Mechanism of total C-21 steroidal glucosides from root of Cynanchum auriculatum in activation of human hepatic stellate cells via TGF-β_(1)/Smad signaling pathway

作　　者：张婷 丁永芳 彭蕴茹[1,2] ZHANG Ting;DING Yong-fang;PENG Yun-ru(Affiliated Hospital of Integrated Traditional Chinese and Western Medicine,Nanjing University of Chinese Medicine,Nanjing 210028,China;Jiangsu Province Academy of Traditional Chinese Medicine,Nanjing 210028,China)

机构地区：[1]南京中医药大学附属中西医结合医院 [2]江苏省中医药研究院,江苏南京210028

出　　处：《中国药理学通报》2021年第9期1231-1236,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81774178,81373888)。

摘　　要：目的研究白首乌C-21甾体总苷(TCSG)体外对LX-2活化的影响及其作用机制。方法实验分为:对照组、TGF-β_(1)+LPS组、TGF-β_(1)+LPS联合TCSG药物干预低、中、高浓度(10,20,40 mg·L^(-1))组。MTT法检测细胞活力,试剂盒检测细胞上清液中羟脯氨酸(hydroxyproline,Hyp)含量,免疫荧光染色观察LX-2细胞活化标志物α-SMA表达情况,Western blot法检测TCSG干预前后LX-2细胞中TGF-β_(1),Smads蛋白相对表达量变化。结果MTT实验结果显示40-100 mg·L^(-1) TCSG处理细胞48 h后,均对细胞增殖有明显的抑制作用(P<0.05,P<0.01)。与对照组相比,TGF-β_(1)+LPS组HYP含量显著升高,而经TCSG处理后HYP含量有明显降低(P<0.05,P<0.01)。免疫荧光染色结果显示,TGF-β_(1)+LPS诱导组α-SMA表达较对照组有明显增强,而经TCSG处理后降低。与TGF-β_(1)+LPS组相比,TCSG处理组下调LX-2细胞内TGF-β_(1),α-SMA,p-Smad2/Smad2,p-Smad3/Smad3及NF-κB蛋白表达,显著上调IκB-α表达(P<0.05,P<0.01)。结论TCSG体外能明显抑制TGF-β_(1)联合LPS诱导的人肝星状细胞的活化,且与调控TGF-β_(1)/Smad信号通路有关。Aim To investigate the inhibitory effect of total C-21 steroidal glucosides(TCSG)from the root of Cynanchum auriculatum on activation of human hepatic stellate cells and the underlying mechanism.Methods The fibrosis model in vitro was established by treating LX-2 cells with TGF-β_(1)+LPS.The experiment was divided into control group,model group(TGF-β_(1)+LPS)and three TCSG treated groups(10,20,40 mg·L^(-1)).Cell proliferation was measured by MTT assay.The content of hydroxyproline in cell supernatant was measured by alkaline hydrolysis method.α-SMA expression was determined using immunofluorescence staining.The protein expression levels of TGF-β_(1) and Smads in cells were detected by Western blot.Results MTT results showed that TCSG at the concentrations exceeding 40 mg·L^(-1) for 48 h displayed obvious inhibitory effects on LX-2 cell proliferation(P<0.05,P<0.01).Compared with control group,HYP content in cell supernatant in the TGF-β_(1) and LPS treated group markedly increased(P<0.01),while TCSG treatment led to a significant reduction(P<0.05).Immunofluorescence result showed that the TGF-β_(1) and LPS treatment significantly increased theα-SMA expression in LX-2 cells(P<0.01),which markedly decreased by TCSG.Western blot results revealed that the protein expression levels of TGF-β_(1),α-SMA,NF-κB,p-Smad2/Smad2 and p-Smad3/Smad3 in TGF-β_(1) and LPS treated cells significantly increased while IκB-αexpression decreased as compared with control group(P<0.01).Treatment with TCSG clearly suppressed the protein expression of TGF-β_(1),α-SMA,NF-κB,p-Smad2/Smad2 and p-Smad3/Smad3 and increased IκB-αexpression(P<0.05,P<0.01).Conclusions TCSG could suppress TGF-β_(1) and LPS induced activation of LX-2 cells,which may be related to the regulation of TGF-β_(1)/Smad signaling pathway.

关 键 词：人肝星状细胞 纤维化 TGF-β_(1) SMAD NF-κB α-SMA 白首乌 

分 类 号：R282.71[医药卫生—中药学] R284.1[医药卫生—中医学]