丙型肝炎病毒核心蛋白下调沉默信息调节因子1导致肝星状细胞活化

目的研究丙型肝炎病毒(HCV)核心蛋白对肝星状细胞沉默信息调节因子1(SIRT1)表达及肝星状细胞活化的影响。方法 HepG2细胞或表达HCV核心蛋白的HepG2细胞与肝星状细胞(LX-2细胞)共培养。应用液体闪烁计数仪、实时荧光定量-PCR、Western印迹检测LX-2细胞SIRT1活性、mRNA及蛋白的表达。Western印迹检测LX-2细胞磷酸化AMP激活的蛋白激酶(p-AMPK)、脂联素受体2(AdipoR2)、转化生长因子-β1(TGF-β1)蛋白的表达。ELISA法检测共培养上清液中人Ⅳ胶原(ColⅣ)、Ⅲ型前胶原肽(PⅢNP)、透明质酸(HA)和人层黏连蛋白(LN)的水平。计量资料采用t检验。结果与LX-2细胞和HepG2细胞共培养组相比,LX-2细胞和表达HCV核心蛋白HepG2细胞共培养组SIRT1活性(0.4±0.1比1.0±0.2,t=6.573,P0.01)、mRNA(0.3±0.1比1.0±0.3,t=5.422,P0.01)和蛋白(0.4±0.1比0.8±0.2,t=4.382,P0.01)水平下降;p-AMPK(0.3±0.1比0.8±0.2,t=5.477,P0.01)和AdipoR2(0.4±0.1比0.8±0.2,t=4.382,P0.01)表达下降;TGF-β1(2.3±0.5比0.8±0.2,t=6.823,P0.01)表达增加;共培养上清液中ColⅣ、PⅢNP、HA和LN的水平增加。SIRT1激动剂白藜芦醇降低TGF-β1的表达。结论 HCV核心蛋白下调肝星状细胞SIRT1活性及表达,下调AdipoR2表达,上调TGF-βl表达,活化肝星状细胞。

Hepatitis C virus core protein induces activation of hepatic stellate cell by down-regulation of silent information regulator 1

Objective To investigate the effects of hepatitis C virus (HCV) core protein on expression of silent information regulator 1 (SIRT1) and activation of hepatic stellate cells (HSC).Methods HSC (LX-2 cells) were co-cultured with HepG2 cells or HCV core protein-positive HepG2 cells.Activity, mRNA and protein expressions of SIRT1 in LX-2 cells were detected using scintillation counter, real time-PCR (RT-PCR) and western blot, respectively.Expressions of phosphorylated adenosine monophosphate activated protein kinase (p-AMPK), adiponectin receptor 2 (AdipoR2) and transforming growth factor β1 (TGF-β1) were measured using western blot.Levels of collagen Ⅳ (ColⅣ), procollagen Ⅲ peptide (PⅢNP), hyaluronan (HA) and laminin (LN) in the supernatant were measured using enzyme-linked immunosorbent assay (ELISA).The quantitative data was analyzed using t-test.Results Compared with LX-2 cells co-cultured with HepG2 cells, the activity (0.4±0.1 vs.1.0±0.2, t=6.573, P<0.01), mRNA (0.3±0.1 vs.1.0±0.3, t=5.422, P<0.01) and protein expressions (0.4±0.1 vs.0.8±0.2, t=4.382, P<0.01) of SIRT1 were both reduced in LX-2 cells co-cultured with HCV core-positive HepG2 cells.In LX-2 cells co-cultured with HCV core-positive HepG2 cells, the expression levels of p-AMPK protein (0.3±0.1 vs.0.8±0.2, t=5.477, P<0.01) and AdipoR2 protein (0.4±0.1 vs.0.8±0.2, t=4.382, P<0.01) were decreased comparing with those in LX-2 cells co-cultured with HepG2 cells, while TGF-β1 protein expression (2.3±0.5 vs 0.8±0.2, t=6.823, P<0.01) was increased.Moreover, the levels of ColⅣ, PⅢNP, HA and LN in the supernatant were increased in LX-2 cells co-cultured with HCV core-positive HepG2 cells.SIRT1 activator resveratrol decreased the expression of TGF-β1 protein.Conclusion HCV core protein might decrease the expression of AdipoR2 and increase the expression of TGF-β1 through down-regulating the activity and expression of SIRT1, and ultimately cause the activation of HSC.