柴胡皂苷b2对肝癌细胞增殖的影响及机制

Effects and mechanism of saikosaponin b2 on hepatocellular carcinoma cell proliferation

目的研究柴胡皂苷b2(SSb2)对肝癌细胞增殖的影响,并进一步探讨柴胡皂苷b2抑制肝细胞癌发生发展的分子机制。方法将细胞分为空白对照组(NC)、SSb2组、沉默信息调节因子6(SIRT6)si-RNA组(si-SIRT6)、si-SIRT6+SSb2组,NC组与SSb2组分别加入含阴性质粒的转染溶液,si-SIRT6组和si-SIRT6+SSb2组加入含si-SIRT6质粒的转染溶液,转染24 h后,SSb2组和si-SIRT6+SSb2组加入80 mg·L^(-1)的SSb2,各组继续转染24 h。检测各组HepG2细胞中腺苷三磷酸(ATP)含量、细胞上清液中葡萄糖以及乳酸的含量,用蛋白质印迹法检测HepG2细胞中SIRT6、缺氧诱导因子1α(HIF1α)、葡萄糖转运蛋白1(GLUT1)的表达水平变化。结果NC组、SSb2组、si-SIRT6组和si-SIRT6+SSb2组细胞的ATP含量分别为(36.81±2.42)、(26.22±2.09)、(74.16±2.65)和(33.95±2.00)μmol·gprot^(-1),葡萄糖摄取量分别为(1.41±0.07)、(0.49±0.05)、(1.86±0.10)和(1.06±0.12)mmol·L^(-1),乳酸生成量分别为(7.09±0.30)、(4.13±0.25)、(11.49±0.68)和(5.76±0.27)mmol·L^(-1),SIRT6蛋白相对表达水平分别为0.29±0.06、0.70±0.06、0.06±0.03和0.17±0.04,HIF1α蛋白相对表达水平分别为0.18±0.03、0.06±0.03、0.44±0.04和0.22±0.04,GLUT1蛋白相对表达水平分别为0.63±0.05、0.30±0.09、1.34±0.12和0.51±0.09。上述指标,SSb2组与NC比较,差异均有统计学意义(P<0.05,P<0.01);si-SIRT6组与NC比较,差异均有统计学意义(均P<0.01);si-SIRT6+SSb2组与SSb2组比较,差异均有统计学意义(P<0.05,P<0.01)。结论SSb2在体外可以抑制肝癌细胞的增殖,其作用可能是通过调控SIRT6介导的糖代谢通路实现的。

Objective To investigate the effect of Saikosaponin b2(SSb2)on proliferation of hepatocellular carcinoma(HCC)cells,and to further explore the molecular mechanism of SSb2 inhibiting the occurrence and development of HCC.Methods The cells were divided into negative control(NC)group,SSb2 group,silent information regulator 6(SIRT6)si-RNA(si-SIRT6)group and si-SIRT6+SSb2 group.Negative plasmids were added to NC group and SSb2 group,si-SIRT6 and si-SIRT6+SSb2 groups were added with si-SIRT6 plasmids.HepG2 cells were transfected for 24 h firstly and then 80 mg·L^(-1)SSb2 was added in si-SIRT6 group and si-SIRT6+SSb2 group for another 24 h for subsequent experiments.Adenosine triphosphate(ATP),glucose and lactic acid level of HepG2 cells in each group were detected by kits.The expressions of SIRT6,hypoxia inducible factor 1α(HIF1α),glucose transporter 1(GLUT1)in HepG2 were analyzed using Western blot.Results The amount of ATP produced in NC group,SSb2group,si-SIRT6 group and si-SIRT6+SSb2 group was(36.81±2.42),(26.22±2.09),(74.16±2.65)and(33.95±2.00)μmol·gprot^(-1),respectively;glucose intakes were(1.41±0.07),(0.49±0.05),(1.86±0.10)and(1.06±0.12)mmol·L^(-1),respectively;lactic acid productions were(7.09±0.30),(4.13±0.25),(11.49±0.68)and(5.76±0.27)mmol·L^(-1),respectively;the relative expression levels of SIRT6 protein were0.29±0.06,0.70±0.06,0.06±0.03 and 0.17±0.04,respectively;the HIF1αprotein levels were 0.18±0.03,0.06±0.03,0.44±0.04 and 0.22±0.04;the GLUT1 protein were 0.63±0.05,0.30±0.09,1.34±0.12 and 0.51±0.09.Compared with NC group,the differences in SSb2 group were statistically significant(P<0.05,P<0.01),and the difference between si-SIRT6 group and NC group was statistically significant(P<0.01).In addition,the difference between si-SIRT6+SSb2 group and SSb2 group was statistically significant(P<0.05,P<0.01).Conclusion SSb2 exerts anti-tumor effects in vitro by inhibiting the proliferation of hepatocellular carcinoma cells,and its effect may be related to the regulation of glucose metabolism pathway mediated by SIRT6.