白花丹醌对大鼠肝细胞肝癌自噬活性的影响并机制初探

Effect of plumbagin on autophagy activity in rat hepatocellular carcinoma and underlying mechanism

背景自噬是一个支持营养循环和代谢适应的多步骤溶酶体降解途径,被认为是一个调节癌症进展的过程.本研究使用白花丹醌处理大鼠肝细胞癌(hepatocellular carcinoma,HCC)细胞,探究其对HCC自噬活性影响,为HCC治疗提供新的思路.目的探讨白花丹醌对黄曲霉毒素B1(aflatoxin B1,AFB1)诱导型大鼠HCC细胞自噬活性的影响,分析其发生的可能机制.方法AFB1制作大鼠HCC模型并用白花丹醌干预,透射电镜观察肝组织和自噬细胞的超微结构.RT-PCR和免疫组织化学染色技术测AKT1 mRNA和蛋白的表达.Western Blot测肝组织中LC3BⅠ、LC3BⅡ蛋白表达.结果AFB1诱癌模型组和白花丹醌处理组大鼠肝组织中多见细胞自噬现象.与对照组相比,AFB1诱癌模型组大鼠肝组织中AKT1 mRNA和蛋白表达水平明显增加(t值分别为17.013、9.986,均P<0.001);2 mg/kg、3 mg/kg白花丹醌处理组大鼠肝组织中AKT1 mRNA表达明显低于AFB1诱癌模型组(前者t=-2.378、P=0.030;后者t=-17.980,P<0.001).与AFB1诱癌模型组相比,2 mg/kg白花丹醌、3 mg/kg白花丹醌处理组大鼠肝组织中LC3B-II/I比值均明显升高(前者t=2.420,P=0.028;后者t=35.136,P<0.001),大鼠肝组织中LC3B-II/I比值在2 mg/kg白花丹醌和3 mg/kg白花丹醌处理组间的差异有统计学意义(t=21.316,P<0.001).3 mg/kg白花丹醌处理组大鼠肝组织中AKT1 mRNA表达与LC3B-II/I比值间存在明显相关性(r=-0.611,P=0.035).结论白花丹醌可能通过抑制ATK1表达,增强大鼠HCC细胞自噬活性.

BACKGROUND Autophagy is a multi-step lysosomal degradation pathway supporting nutritional cycle and metabolic adaptation,which is considered to be a process of regulating cancer progression.In this study,rats with hepatocellular carcinoma(HCC)were treated with plumbagin to explore its effect on the autophagy activityin HCC,in order to provide a new idea for the treatment of this malignancy.AIM To investigate the effect of plumbagin on autophagy activity in aflatoxin B1(AFB1)-induced rat hepatocellular carcinoma and to explore the possible mechanism involved.METHODS AFB1 was used to develop a rat hepatocellular carcinoma model.The rats were then treated with plumbagin.The ultrastructure of liver tissues and autophagic cells was observed by electron microscopy.The expression of AKT1 mRNA and protein was measured by RT-PCR and immunohistochemical staining,respectively.Western blot was used to detect the expression of LC3BⅠand LC3BⅡprotein in liver tissue.RESULTS Autophagy was common in liver tissues of the AFB1-induced cancer model group and plumbagin treated group.Compared with the control group,the expression levels of AKT1 mRNA and protein in the liver tissues of the AFB1-induced cancer model group were significantly increased(t=17.013 and 9.986,respectively,P<0.001).The expression of AKT1 mRNA in liver tissue of rats treated with 2 mg/kg and 3 mg/kg plumbagin was significantly lower than that of the AFB1-induced cancer model group(t=-2.378,P=0.030;t=-17.980,P<0.001).Compsared with the AFB1-induced cancer model group,the LC3B-II/I ratio in the liver tissue of rats treated with 2 mg/kg and 3 mg/kg plumbagin was significantly increased(t=2.420,P=0.028;t=35.136,P<0.001).The LC3B-II/I ratio in rat liver tissue was significantly different between the 2 mg/kg and 3 mg/kg plumbagin treatment groups(t=21.316,P<0.001).There was a significant correlation between the expression of AKT1 mRNA and the LC3BII/I ratio in the liver tissue of rats treated with 3 mg/kg plumbagin(r=-0.611,P=0.035).CONCLUSION Plumbagin may enhance the autophagy activity of rat HCC cells by inhibiting the expression of ATK1.