虎蝎化积膏联合顺铂诱导肝癌H22细胞凋亡作用及其机制

Effect and mechanism of Huxie Huaji Ointment combined with cisplatin on apoptosis of H22 cells of liver cancer

目的探讨虎蝎化积膏联合顺铂诱导肝癌H22细胞凋亡作用及其机制。方法将SD大鼠随机分为空白对照组和虎蝎化积膏组,连续给药3 d后,腹主动脉取血,制备虎蝎化积膏含药血清。将对数期H22细胞随机分为5组:正常组、空白血清对照组、顺铂组、20%虎蝎化积膏含药血清组、20%虎蝎化积膏含药血清+顺铂组,采用CCK-8法检测虎蝎化积膏含药血清对H22细胞增殖的影响;Hoechst 33258染色法观察各组H22细胞凋亡形态变化;高内涵细胞成像分析技术检测H22细胞内B细胞淋巴瘤2基因(Bcl-2)、凋亡相关蛋白细胞色素C(Cyt C)、B细胞淋巴瘤2关联蛋白X(Bax)、天冬氨酸蛋白水解酶9(caspase-9)蛋白表达变化。结果与空白血清对照组比较,虎蝎化积膏含药血清组H22细胞增殖呈剂量-时间依赖关系的抑制作用,20%虎蝎化积膏含药血清的作用效果最佳。与空白血清对照组和单一用药组比较,20%虎蝎化积膏含药血清+顺铂组H22细胞呈现不同程度的核染色质聚集、核固缩,呈现出更典型的细胞凋亡特征;H22细胞内Cyt C、Bax和caspase-9平均荧光强度值明显增加(P<0.01、P<0.05),蛋白表达明显增多;Bcl-2平均荧光强度值明显降低(P<0.01、P<0.05),蛋白表达显著减少。结论 20%虎蝎化积膏含药血清联合顺铂可明显抑制肝癌细胞H22增殖,诱导其凋亡,作用机制可能与细胞内Bax和Bcl-2蛋白表达比例失调,胞质中Cyt C分泌增多,促使caspase-9蛋白表达增加有关。

Objective To investigate the effect and mechanism of Huxie Huaji Ointment combined with cisplatin on apoptosis of H22 cells of liver cancer.Methods SD rats were randomly divided into blank control group and Huxie Huaji Ointment,and the blood samples were collected from abdominal aorta for preparing medicated serum of Huxie Huaji Ointment after treatment continuously for 3 d.The logarithmic H22 cells were randomly divided into 5 groups:normal group,blank control group,cisplatin group,20%of medicated serum group and 20%of medicated serum+cisplatin group.The influence of the medicated serum on H22 apoptosis was detected by using CCK-8 method,and apoptotic morphologic changes of H22 cells was observed after Hoechst 33258 staining in all groups.The expressions of B-cell lymphoma-2(Bcl-2),cytochrome C(Cyt C),BCL2-associated X(Bax)and cysteinyl aspartate specific proteinase-9(caspase-9)in H22 cells were detected by using high-content cell imaging analysis technology.Results Compared with blank control group,the proliferation of H22 cells was inhibited in a dose-time-dependent manner in medicated serum group,and the efficacr was the best in 20%of medicated serum group.Compared with blank control group,cisplatin group and medicated serum group,H22 cells showed nuclear chromatin aggregation and karyopyknosis in varying degrees,and more typical apoptotic characteristics in medicated serum+cisplatin group.The mean fluorescence intensity values of Cyt C,Bax and caspase-9 increased significantly in H22 cells(P<0.01 of P<0.05),and their protein expressions increased significantly.The mean fluorescence intensity value of Bcl-2 decreased significantly in H22 cells(P<0.01 of P<0.05),and its protein expression decreased significantly.Conclusion The medicated serum of Huxie Huaji Ointment combined with cisplatin can significantly inhibit the proliferation and induce the apoptosis of H22 cells,and the mechanism may be related to the imbalance of Bax and Bcl-2 protein expressions,and increases of Cyt C secretion and caspase-9 protein expression.