护肝布祖热对CCl4诱导人肝星状细胞LX-2活化的作用及机制研究

Study on the Effect and Mechanism of Hugan Buzure on CCl4-induced Activation in Human Hepatic Stellate Cells LX-2

目的探讨护肝布祖热(HGBZR)抗肝损伤的作用及机制。方法体外培养人肝星状细胞LX-2,采用MTT法检测LX-2细胞存活率以确定四氯化碳(CCl)造模浓度和药物干预浓度。细胞实验分为6组:空白组、模型组、阳性组(水飞蓟素,10μg·mL)及HGBZR低、中、高剂量组(5、10、20μg·mL),除空白组以外,其余各组分别加入终浓度5 mmol·L^(-1)CCl损伤干预24 h,然后再进行药物干预24 h。采用MTT法检测各组细胞存活率;Annexin V/PE双染法检测细胞凋亡率;qRT-PCR法检测各组细胞α-平滑肌动蛋白(SMA)、TIMP-1、PTP1B、转化生长因子(TGF)-β、Smad3、Smad4、Smad7、CHOP、PERK、IRE1、ATF6 mRNA表达情况;Western Blot法检测各组细胞Jak1、Stat3、TGF-β、Smad3、Smad4、Smad7、CHOP、Caspase12、核因子(NF)-κB p65蛋白表达情况。结果随着CCl浓度(5~30 mmol·L^(-1))升高,LX-2细胞存活率逐渐下降,当CCl浓度为5 mmol·L^(-1)时细胞呈现增殖状态,故选择5 mmol·L^(-1)CCl处理24 h作为肝损伤模型复制条件。与模型组比较,HGBZR中、高剂量组的细胞存活率明显降低(P<0.05),细胞早期凋亡率和晚期凋亡率均明显升高(P<0.05);LX-2细胞的α-SMA、TIMP-1、TGF-β、Smad3、Smad4、CHOP、PERK、IRE1、ATF6 mRNA表达水平明显降低(P<0.05),PTP1B、Smad7 mRNA表达水平明显升高(P<0.05);LX-2细胞的Jak1、Stat3、TGF-β、Smad4、CHOP、Caspase12及NF-κB p65(核蛋白)蛋白表达水平明显降低(P<0.05),Smad7、NF-κB p65(质蛋白)蛋白表达水平明显升高(P<0.05),Smad3蛋白表达水平无明显变化(P>0.05)。结论HGBZR可能通过调控TGF-β/Smad、Jak/Stat和内质网应激信号通路相关目标基因转录水平及蛋白表达,促进活化LX-2细胞的凋亡,对CCl诱导的化学性肝损伤具有一定保护作用。

Objective To investigate the anti-liver injury effect of Hugan Buzure(HGBZR) and its mechanism through in vitro experiments. Methods Human hepatic stellate cells LX-2 were cultured in vitro and the survival rate of LX-2 cells was detected by MTT assay to determine the CClmodeling concentration and drug intervention concentration. The cell experiments were divided into 6 groups: blank group, model group, positive group(silymarin,10 μg·mL-1)and HGBZR low-,medium-and high-dose groups(5,10,20 μg·mL-1);except for the blank group, each group was added 5 mmol·L^(-1)CClinjury intervention for 24 hours, followed by drug intervention for 24 hours. The cell survival rate of each group was detected by MTT method;the apoptosis rate was detected by Annexin V/PE double-staining method;the mRNA expressions of α-SMA,TIMP-1,PTP1B,TGF-β,Smad3,Smad4,Smad7,CHOP,PERK,IRE1,ATF6 were detected by qRT-PCR;the protein expressions of Jak1,Stat3,TGF-β,Smad3,Smad4,Smad7,CHOP,Caspase12,NF-κB p65 were detected by Western Blot.Results The survival rate of LX-2 cells was gradually decreased with the increase of CClconcentration(5-30 mmol·L^(-1)),and the cells showed proliferation when the CClconcentration was 5 mmol·L^(-1). The 5 mmol·L^(-1)CCltreatment for24 hours was therefore selected as the replication condition of the liver injury model. Compared with the model group,the cell survival rate was significantly decreased(P<0.05),and the early apoptosis rate and late apoptosis rate were both significantly increased(P<0.05) in the HGBZR medium-and high-dose groups;the mRNA expression levels of α-SMA,TIMP-1,TGF-β,Smad3,Smad4,CHOP,PERK,IRE1,ATF6 in LX-2 cells were significantly decreased(P<0.05),and the mRNA expression levels of PTP1B and Smad7 were significantly increased in LX-2 cells(P<0.05);the protein expression levels of Jak1, Stat3, TGF-β, Smad4, CHOP,Caspase12 and NF-κB p65(nuclear protein)were significantly decreased(P<0.05),the protein expression levels of Smad7 and NF-κB p65(plasmin)were significantly increased in LX-2 cells(P<0.05),and the protein expression level of Smad3 was not significantly changed(P>0.05). Conclusion HGBZR may have a protective effect against CCl-induced chemical liver injury by promoting apoptosis in activated LX-2 cells through regulating the transcript levels and protein expressions of target genes related to TGF-β/Smad,Jak/Stat and endoplasmic reticulum stress(ERS)signaling pathways.