Shenkang Injection Antagonizes The Effects Of Aristolochic Acid On Human Proximal Tubular Epithelial Cells In Vitro

ObjectiveChronic aristolochic acid nephropathy (CAAN) has been known to bea progressive renal tubulointerstitial nephropathy that is caused byChinese herbs containing aristolochic acid (AA). So far, the potentialpathogenic mechanism of CAAN has not been completely clear, and thetherapeutic measures for CAAN are still poor. Shen kang injection (SKI)is an extractive of Chinese herbs complex that containing Danshen (RadixSalviae Miltiorrhizae), Honghua (Flos Carthami), Dahuang (Radix EtRhizoma Rhei) and Huangqi (Radix Scutellariae). It had been found SKIcould delay the progression of chronic renal fibrosis in some kinds ofCKD animal models and could be clinically used to treat chronic renalfailure. This project is designed to study whether SKI could antagonizethe fibrogenic effects induced by AA on human proximal tubularepithelial cells (HKC) in vitro.MethodsCultured HKC were divided into the following four groups: control group, AA-Na group, AA-Na + SKI group and SKI group. The effects ofAA-Na or/and SKI on HKC proliferation and cytotoxicity weredetermined by MTT assay and LDH release assay, respectively. Aftertreatment with AA-Na or/and SKI for 12h, the mRNA expression oftransforming growth factor-β1 (TGF-β1), connective tissue growth factor(CTGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) andplasminogen activator inhibitor-1 (PAI-1) was measured by RT-PCR.After treatment for 24h, the protein expression of TGF-β1, TIMP-1 andPAI-1 was measured by ELISA. After treatment for 36h, the proteinexpression of CTGF was also measured by western blotting (WB).ResultsTen mg/L AA-Na or/and 8mg/ml SKI had no any effects of cellproliferation and cytotoxicity on HKC. mRNA expression of TGF-β1,CTGF, TIMP-1 and PAI-1 was significantly up-regulated by 10 mg/LAA-Na. Compared with the control group, their mRNA expression wasup-regulated to 1.84, 1.58, 1.62 and 1.29 times, respectively (P＜0.05).The AA-induced up-regulated mRNA expression of TGF-β1, CTGF,TIMP-1 and PAI-1 was significantly inhibited by 8mg/ml SKI. Comparedwith the AA-Na group, their inhibition rates were 41.6%, 43.7%, 43.8%and 24.4%, respectively (P＜0.05). About the protein expression, quitesimilar to mRNA expression, 10mg/L AA-Na also significantly increased the expression ofTGF-β1, TIMP-1, PAI-1 (by ELISA) and CTGF protein(by WB). Compared with the control group, their expression wasup-regulated to 1.12, 1.63, 1.42 and 1.29 times, respectively (P＜0.05).Eight mg/ml SKI also significantly inhibited the AA-inducedup-regulated expression of TGF-β1, TIMP-1, PAI-1 and CTGF protein.Compared with the AA-Na group, their inhibition rates were 34.3%,43.3%, 31.1% and 21.9%, respectively (P＜0.05). Eight mg/ml SKI alonehad no any effects on both mRNA and protein expression of TGF-β1,CTGF, TIMP- 1 and PAI-1.ConclusionAA-Na can up-regulate the mRNA and protein expression ofpromoting extra cellular matrix (ECM) synthesis factors (TGF-β1, CTGF)and inhibiting ECM degradation factors (TIMP-1, PAI-1), and theabove-mentioned AA-induced fibrogenic effects can be antagonized bySKI.