褪黑素调控自噬抑制肝星状细胞增殖的作用及机制研究

Effects and mechanisms of melatonin on autophagy and inhibition of hepatic stellate cell proliferation

目的研究褪黑素(MEL)对血小板源性生长因子(PDGF-BB)诱导的肝星状细胞(HSCs)增殖的影响,并探索其与自噬水平调控的相关性。方法HSC-T6共设置5组:对照组、模型组、MEL(低、中、高)处理组。培养24 h后细胞贴壁,换用无血清的DMEM培养基,使细胞同步化于G_(0)期,再培养24 h后除对照组外各组加入PDGF-BB(终浓度为10 ng/ml),MEL处理组加入MEL(低剂量组为1 nmol/L,中剂量组1μmol/L,高剂量组为0.1 mmol/L),孵育48 h。细胞计数试剂盒-8(CCK-8)检测MEL对PDGF-BB激活的HSCs增殖的影响。Western blot测定HSCs的微管相关蛋白1轻链3b(LC3b)、α-平滑肌肌动蛋白(α-SMA)蛋白表达水平。实时荧光定量逆转录聚合酶链式反应(qRT-PCR)测定HSCs LC3b mRNA、α-SMA mRNA表达水平。透射电镜观察HSCs的超微结构以了解自噬水平。结果与对照组比较,PDGF-BB能诱导HSCs的增值(P<0.01)。与模型组比较,MEL能抑制PDGF-BB激活的HSCs增殖(P<0.01);与对照组比较,模型组LC3b、α-SMA蛋白表达明显增强(均P<0.05),LC3b mRNA、α-SMA mRNA表达明显增强(P<0.05,P<0.01),与模型组比较,MEL能抑制这种作用(LC3b:P<0.05,P<0.01;α-SMA:P<0.01)。透射电镜显示,与对照组比较,模型组自噬溶酶体显著增加(P<0.05);与模型组比较,MEL处理组自噬溶酶体显著减少(P<0.01)。结论HSCs的增殖可能与自噬激活有关,MEL抑制HSCs增殖可能与其下调自噬水平相关。

Objective To investigate the effects of melatonin(MEL)on the proliferation of hepatic stellate cells(HSCs)induced by platelet-derived growth factor(PDGF-BB)and explore its correlation with the regulation of autophagy levels.Methods The HSC-T6 cells were divided into the following groups:control group,model group and MEL(low,medium and high)treatment groups.After 24 hours culture,the cells adhered to the wall and were changed into serum-free DMEM medium to synchronize the cells to the G_(0)stage.After 24 hours culture,all groups were given with PDGF-BB(10 ng/ml)excepted the control group.Besides,melatonin of different concentrations(1 nmol/L,1μmol/L and 0.1 mmol/L)were added immediately in three treated groups.After incubated for 48 hours,the effect of MEL on the proliferation of hepatic stellate cells activated by PDGF-BB was detected by CCK-8 method.The protein expression levels of LC3b andα-SMA in hepatic stellate cells were determined by Western blot.The expression levels of LC3b mRNA andα-SMA mRNA in hepatic stellate cells were determined by qRT-PCR.The ultrastructure of HSCs was observed by transmission electron microscopy to understand the autophagy level.Results Compared with control group,PDGF-BB could induce the proliferation of HSCs(P<0.01).Compared with model group,MEL inhibited the proliferation of HSCs activated by PDGF-BB(P<0.01).Compared with the control group,LC3b andα-SMA protein expressions significantly increased in the model group(all P<0.05),and LC3b mRNA andα-SMA mRNA expressions significantly increased in the model group(P<0.05,P<0.01).Compared with the model group,MEL could inhibit such effects(LC3b:P<0.05,P<0.01;α-SMA:P<0.01).Transmission electron microscopy(TEM)showed that compared with the control group,autopolysosome significantly increased in the model group(P<0.05).Compared with model group,autopolysosome significantly decreased in MEL treatment group(P<0.01).Conclusion The up-regulation of autophagy level can promote the proliferation of hepatic stellate cells and the inhibition of hepatic stellate cell proliferation by MEL may be related to the down-regulation of autophagy level.