桑色素通过Akt/mTOR/STAT3通路诱导肝癌细胞自噬和凋亡

Morin induces autophagy and apoptosis in hepatocellular carcinoma cells through Akt/mTOR/STAT3 pathway

研究桑色素(morin)通过蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)/信号传导与转录活化因子(STAT3)通路诱导肝癌细胞自噬和凋亡的影响及其机制。分别用0、50、100、125、200、250μmol·L-1桑色素刺激人肝癌SK-HEP-1细胞,通过细胞活性及增殖检测(CCK-8)法,检测桑色素对SK-HEP-1细胞活力的影响;使用浓度0、125、250μmol·L-1桑色素处理SK-HEP-1细胞,通过克隆形成实验、流式细胞术及BeyoClickTMEdU-488检测探究桑色素对SK-HEP-1细胞增殖及凋亡的影响;通过透射电镜、联用自噬抑制剂探究桑色素处理后的细胞自噬水平的变化;运用蛋白免疫印迹法(Western blot),验证桑色素对Akt/mTOR/STAT3通路蛋白表达水平的影响情况。与对照组比较,桑色素组SK-HEP-1细胞存活率明显降低,呈时间-浓度依赖性;凋亡细胞数目增多且凋亡标志物PARP表达水平上升,凋亡调控组蛋白H2AX的磷酸化表达水平上调;桑色素组阳性细胞数及克隆形成集落数明显减少;自噬相关蛋白LC3-Ⅱ、Atg5、Atg7表达呈上升趋势;Akt、mTOR、STAT3的磷酸化水平下降。该研究揭示桑色素可促进肝癌细胞凋亡,抑制肝癌细胞增殖,并诱导自噬,其作用机制可能与Akt/mTOR/STAT3通路有关。

This study investigated the effect and mechanism of morin in inducing autophagy and apoptosis in hepatocellular carcinoma cells through the protein kinase B(Akt)/mammalian target of rapamycin(mTOR)/signal transducer and activator of transcription protein 3(STAT3)pathway.Human hepatocellular carcinoma SK-HEP-1 cells were stimulated with different concentrations of morin(0,50,100,125,200,and 250μmol·L-1).The effect of morin on the viability of SK-HEP-1 cells was detected by Cell Counting Kit-8(CCK-8).The effect of morin on the proliferation and apoptosis of SK-HEP-1 cells was investigated using colony formation assay,flow cytometry,and BeyoClick TM EdU-488 with different concentrations of morin(0,125,and 250μmol·L-1).The changes in the autophagy level of cells treated with morin were examined by transmission electron microscopy and autophagy inhibitors.The impact of morin on the expression levels of proteins related to the Akt/mTOR/STAT3 pathway was verified by Western blot.Compared with the control group,the morin groups showed decreased viability of SK-HEP-1 cells in a time-and concentration-dependent manner,increased number of apoptotic cells,up-regulated expression level of apoptosis marker PARP,up-regulated phosphorylation level of apop tosis-regulating protein H2AX,decreased number of positive cells and the colony formation rate,an upward trend of expression levels of autophagy-related proteins LC3-Ⅱ,Atg5,and Atg7,and decreased phosphorylation levels of Akt,mTOR,and STAT3.These results suggest that morin can promote apoptosis,inhibit proliferation,and induce autophagy in hepatocellular carcinoma cells,and its mechanism of action may be related to the Akt/mTOR/STAT3 pathway.