脂多糖促进大鼠肝星状细胞系HSC-T6细胞自噬

LPS promotes autophagy in rat hepatic stellate cell line HSC-T6

目的研究脂多糖(LPS)对大鼠肝星状细胞系HSC-T6自噬的影响及NF-κB通路在其中的作用。方法 1)常规培养的HSC-T6细胞以不同质量浓度(0、0.01、0.1、1和10 mg/L)的LPS分别处理不同时间(0、3、6、12、18和24 h)后,Western blot检测细胞内微管相关蛋白轻链Ⅱ(LC3Ⅱ)及Beclin1含量;2)常规培养的HSC-T6细胞随机分为对照组、LPS组、PDTC组、LPS+PDTC组、PMA组和LPS+PMA组,各组经相应处理后,Western blot检测各组LC3Ⅱ及Beclin1含量;免疫荧光法观察NF-κB P65细胞内分布情况;比色法检测各组细胞培养上清羟脯氨酸(Hyp)含量;ELISA检测细胞培养上清层黏蛋白(LN)及透明质酸(HA)含量。结果经LPS处理后HSC-T6细胞中LC3Ⅱ及Beclin1含量增加且在LPS质量浓度为0.1 mg/L作用6 h达到峰值,同时细胞培养上清中肝纤维化指标Hyp、LN和HA含量明显增加(P<0.05);经PDTC预处理后,LPS刺激的HSC-T6中LC3Ⅱ及Beclin1含量增加,上清中Hyp、LN和HA含量明显降低(P<0.05);而经PMA预处理则LC3Ⅱ、Beclin1含量降低而Hyp、LN和HA含量增加(P<0.05)。结论 LPS可促进HSC-T6细胞自噬及NF-κB通路的活化,NF-κB的活化可能抑制HSC-T6细胞自噬。

Objective To investigate the effects of lipopolysaccharide（LPS） on autophagy in rat hepatic stellate cells（HSC-T6） and involvement of NF-κB pathway in it.Methods 1） HSC-T6 cells were treated with LPS at the concentraction of 0,0.01,0.1,1,and 10 mg/L for 0,3,6,12,18,and 24 h respectively,Microtubule-associated protein light chain Ⅱ（LC3Ⅱ） and Beclin1 levels were detected by Western blot; 2） HSC-T6 cells were randomized into groups of control group,LPS group,PDTC group,LPS ＋ PDTC group,PMA group and LPS ＋ PMA group,Western blot assay was used to measure the levels of LC3 Ⅱand Beclin1,immunofluoresence was used to measure NF-κB P65 intracellular distribution.The levels of hydroxyproline（Hyp） were determined by the method of colorimetry and the levels of laminin（LN） and hyaluronic acid（HA） were determined by ELISA in the culturesupernatants after corresponding processing.Results The levels of LC3Ⅱ and Beclin1 were significantly increased after HSC-T6 cells were treated with LPS at the concentraction of 0.1 mg/L for 6 h and the levels of Hyp,LN,and HA in the culture supernatants increased remarkably as well（P〈0.05）.PDTC pretreatment increased the levels of LC3Ⅱand Beclin1 in the LPS-treated HSC-T6 cells while decreased the levels of Hyp,LN,and HA significantly（P〈0.05）.PMA pretreatmen decreased the levels of LC3Ⅱand Beclin1 in the LPS-treated HSC-T6 cells while increased the levels of Hyp,LN,and HA（P〈0.05）.Conclusions LPS can promote autophagy and activation of NF-κB pathway in HSC-T6 cells.Activation of NF-κB pathway may inhibit the LPS-induced autophagy of HSC-T6 cells.