CHBP and Caspase-3 Sirna Protected Mouse TCMK-1 Cells Treated by Cyclosporine A Via Suppressing Caspase-3 and Apoptosis.

Introduction Cyclosporine A (CsA) nephrotoxicity is one of main causes of chronic allograft dysfunction, which was characterized by tubular cell apoptosis and interstitial fibrosis. In this study, the effects of cyclic helix-B peptide (CHBP, a novel peptide derived from erythropoietin) and/or synthetic small interfering RNA (siRNA) targeting caspase-3 (an executing enzyme of apoptosis) on mouse epithelial TCMK-1 cells treated by CsA were investigated. Methods The dose and time responses of CsA or CHBP on TCMK1 cells were observed. Three sequences of caspase-3 siRNAs (s201121, s63385 and s63386, Ambion) were then transfected to TCMK-1 cells with or without CsA treatment. The changes in caspase-3 mRNA and apoptosis were detected by real-time qPCR and flow cytometry. Results There are gradual increases in both caspase-3 mRNA and apoptosis induced by 2.5, 5, 10, 20 and 40 ug/ml CsA at 24 h; 20 ug/ml CsA was chosen for further investigation with CHBP or caspase-3 siRNA. The CsA raised caspase-3 mRNA was gradually decreased by 2.5-40 ng/ml CHBP, with 27.2% significant difference at 20 ng/ml; and apoptotic cells were decreased by 53.7%. In addition, the CsA increased caspase-3 mRNA was also significantly down-regulated by three caspase-3 siRNAs at 30 nM after 24 h compared with the negative siRNA control, with 46.9% maximal silencing, while the level of apoptosis was also reduced 46.6%. Simultaneous administration of 20 ng/ml CHBP and the best performed siRNA even better reduced CsA induced apoptosis by 67.5%. Conclusion The renoprotection of CHSP against CsA nephrotoxicity might be partially through suppressing caspase-3 and apoptosis. CHBP and caspase-3 siRNA might have synergetic benefits in the treatment of CsA nephrotoxicity.