摘要
目的探讨还原型谷胱甘肽(GSH)对大鼠肝星状细胞HSC—T6增殖及核转录因子NF—E2相关因子2(Nrf2)/血红素加氧酶-1(HO-1)信号通路的影响。方法分别用浓度为0、1、2、5、10mmol/L的GSH作用于经0.1μg/ml脂多糖活化的大鼠HSC-T6细胞24h后,四甲基偶氮唑盐比色法检测HSC-T6增殖情况,放射免疫法检测细胞上清液中透明质酸(HA)及Ⅳ型胶原的含量,实时荧光定量PCR检测Nrf2和HO-1的mRNA表达水平,免疫细胞化学染色法检测HSC-T6中Nrf2和HO-1的蛋白质表达情况,分光光度计检测HO-1的活性变化。两两比较采用t检验,两变量间的关系采用曲线拟合方法。结果GSH能抑制HSC—T6增殖,1、2、5、10mmol/L组的吸光度值分别为0.79±0.02、0.74±0.03、0.70±0.02、0.62±0.01,均低于0mmol/L组的0.88±0.03(t值分别为3.16、6.09、7.17、11.94,P值均〈0.05)。GSH1、2、5、10mmol/L组的HA表达量分别为(372.98±11.01)μg/L、(320.76±16.37)μg/L、(284.46±13.17)μg/L、(239.08±16.95)μg/L,明显低于0mmol/L组的(415.74±14.52)μg/L(t值分别为4.07、7.52、11.59、13.71,P值均〈0.05);Ⅳ型胶原表达量分别为(191.27±17.49)μg/L、(163.85±16.26)μg/L、(133.03±13.14)μg/L、(103.31±12.52)μg/L,也低于0mmol/L组的(251.47±14.06)μg/L(t值分别为4.65、7.58、10.66、13.63,P值均〈0.05)。0mmol/L组HSC-T6中Nrf2 mRNA相对表达量(1.21±0.11)低于1、2、5、10mmol/L组(分别为1.51±0.06、1.92±0.08、2.69±0.07、3.43±0.07),Nrf2蛋白表达的累积吸光度值(17.84±0.61)也低于1、2、5、10mmol/L组(分别为23.85±0.20、27.90±0.32、33.69±0.75、38.64±0.38);HO-1 mRNA相对表达量(1.25±0.09)低于1、2、5、10mmol/L组(分别为1.43±0.08、1.73±0.07、2.10±0.08、2.64±0.07),HO-1蛋白表达的累积吸光度值(16.77±0.31)也低于1、2、5、10mmol/L组(20.75±0.30、24.84±0.24、28.89±0.19、33.88±0.19),差异均有统计学意义(P值均〈0.05)。HO-1的活陛也随GSH浓度增加而上升。结论GSH可抑制HSC—T6增殖,减少其细胞外基质HA及Ⅳ型胶原分泌,其机制可能与GSH对HSC-T6的Nrf2/HO-1信号通路调控有关。
Objective To explore the effects of reduced glutathione (GSH) on the proliferation of hepatic stellate cells and the nuclear factor erythroid 2-related factor 2 (Nrf2)/hemeoxygenase-1 (HO-1) signaling pathway. Methods The rat HSC-T6 cell line was activated by lipopolysaccharide (LPS, 0.1 μg/ml) and incubated with various concentrations of GSH (0, 1, 2, 5 and 10 mmol/L) for 24 h. Cell proliferation was evaluated by the MTT colorimetric assay. Collagen Ⅳ and hyaluronic acid (HA) contents were measured by chemiluminescence immunoassay of cell supernatants. Nrf2 and HO-1 protein expression was observed by immunohistochemistry. Nrf2 and HO-1 mRNA expression was assessed by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). HO-1 activity was analyzed by spectrophotometer. Results GSH treatment inhibited HSC-T6 proliferation and decreased the secretion of HA and collagen Ⅳ (P〈 0.05); GSH treatment of HSC-T6 cells also led to increased expression of Nrf2 and HO-1, and increased activity of HO-1 (P〈0.05). Conclusion GSH can inhibit the proliferation of hepatic stellate cells in vitro and reduce secretion of hyaluronic acid and collagen Ⅳ. The underlying mechanism in HSC-T6 cells may involve regulation of the Nrf2/HO-1 signaling pathway.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2012年第7期507-511,共5页
Chinese Journal of Hepatology