ABSTRACT
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 concentration-dependent 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.