穿心莲内酯及水溶性衍生物致HK-2细胞毒性筛选及亚硫酸氢钠穿心莲内酯相对毒性机制

Toxicity screening of andrographolide and its watersoluble derivatives on HK-2 cells and relative toxicity mechanism induced by andrographolide sodium bisulfite

目的观察和比较穿心莲内酯及其水溶性衍生物〔亚硫酸氢钠穿心莲内酯(ASB)、穿琥宁和炎琥宁〕的原料药对人肾小管上皮细胞HK-2的毒性作用,探讨ASB所致内质网应激作用机制。方法 MTT法检测分别给予4种药物作用后HK-2细胞存活率,计算半数抑制浓度(IC50)。ASB给药组采用Hoechst33342染色和流式细胞术检测细胞凋亡;检测细胞内超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;Western蛋白质印迹法检测免疫球蛋白重链结合蛋白(Bip)、增强子结合蛋白同源蛋白(CHOP)和胱天蛋白酶4的表达。结果 4种药物在一定浓度下均能抑制HK-2细胞增殖,呈时间和浓度依赖性。药物作用24 h,穿心莲内酯IC50为30.6μmol·L-1,均比其他衍生物小,而穿琥宁和炎琥宁的IC50(分别为16.2和15.6 mmol·L-1)相差不大,ASB的IC50为29.4 mmol·L-1。在ASB(0,15,30和60 mmol·L-1)处理24 h后,细胞凋亡率上升,SOD活性下降,MDA含量上升,均呈现浓度依赖性。与对照组比较,8 h时,ASB(30和60 mmol·L-1)组CHOP表达水平增加(P<0.01),Bip和胱天蛋白酶4蛋白无显著变化。另外,24 h时,ASB(60 mmol·L-1)组Bip蛋白水平减少(P<0.05),ASB(30和60 mmol·L-1)组CHOP蛋白表达增加(P<0.01);活化的胱天蛋白酶4随ASB浓度升高表达增加(P<0.01)。结论穿心莲内酯及其水溶性衍生物对HK-2细胞具有一定毒性作用,内质网应激相关的CHOP和胱天蛋白酶4通路参与了ASB诱导的细胞凋亡。

OBJECTIVE To observe and compare the cytotoxicity induced by andrographolide（AD）and its water soluble derivatives：andrographolide sodium bisulfite（ASB）,active pharmaceutical ingredients of Chuanhuning and Yanhuning on human renal tubular epithelial cells（HK- 2）,and to explore the ASB-induced endoplasmic reticulum stress（ERS）mechanism. METHODS HK-2 cells were treated with the above four drugs respectively. The survival rate was examined by methyl thiazolyltetrazolium（MTT） assay and 50% inhibitory concentration（IC50） was calculated. In ASB treated group,Hoechst33342 staining and flow cytometry analysis were used to determine cell apoptosis,intracellular superoxide dismutase（SOD）activity and malondialdehyde（MDA）content were examined,and the protein expressions of binding immunoglobulin protein（Bip）,C/EBP- homologous protein（CHOP）and cysteine-containing aspartate-specific protease 4（caspase 4）were detected by Western blotting. RESULTS The four drugs inhibited HK-2 cell growth in a time-dependent and concentrationdependent manner. At 24 h,the IC50 of AD（30.6 μmol · L^- 1）was lower than that of others. Active pharmaceutical ingredients of Chuanhuning and Yanhuning（16.2 and 15.6 mmol · L^- 1） were very close,ASB was 29.4 mmol·L^- 1. ASB（0,15,30 and 60 mmol·L^- 1）increased the apoptotic rate and caused the decrease in SOD activity and the increase in MDA content in a dose-dependent manner.Compared with control group,the protein expression of CHOP increased（P〈0.01）at 8 h with ASB（30 and 60 mmol·L^- 1）treatment,Bip and caspase 4 had no significant change. In addition,at 24 h,ASB（60 mmol·L^-1） decreased the expression of Bip（P〈0.05）,ASB（30 and 60 mmol·L^-1）promoted the expression of CHOP（P〈0.01）,and the protein expression of activated caspase 4 increased in a concentration-dependent manner（P〈0.01）. CONCLUSION AD and its water soluble derivatives have a toxic effect on HK-2 cells. CHOP and caspase 4 pathway related to ERS is involved in ASB-induced apoptosis.