肾康注射液拮抗马兜铃酸对人近端肾小管上皮细胞作用的研究

Shenkang Injection Solution Antagonizes the Fibrogenic Effects of Aristolochic Acid on Human Proximal Tubular Epithelial Cells in Vitro

目的:探讨肾康注射液(SKI)能否拮抗马兜铃酸钠盐(AA-Na)诱发的人近端肾小管上皮细胞(HKC)的促纤维化效应。方法:AA-Na(10mg/L)加或不加SKI(8mg/ml)与HKC孵育,然后检测转化生长因子-β1(TGF-β1)、结缔组织生长因子(CTGF)、金属蛋白酶组织抑制物-1(TIMP-1)及纤溶酶原激活物抑制物-1(PAI-1)的mRNA表达(孵育12h,RT-PCR方法检测)和蛋白质表达(孵育36h用免疫印迹法检测胞内CTGF,孵育24h用ELISA法检测上清中其他因子)。结果:AA-Na能显著上调HKC对TGF-β1、CTGF、TIMP-1、PAI-1的表达,与对照组比较,mRNA表达分别上调1.84,1.58,1.62,1.29倍,蛋白质表达分别上调1.12,1.63,1.42,1.29倍,P均<0.05;加SKI后,上述因子的高表达均被显著抑制,与AA-Na组比较,mRNA表达的抑制率分别为41.6%,43.7%,43.8%及24.4%,蛋白质表达的抑制率分别为34.3%,43.3%,31.1%及21.9%,P均<0.05。结论:AA-Na能刺激HKC显著上调促细胞外基质(ECM)合成因子(TGF-β1、CT-GF)及抗ECM降解因子(TIMP-1、PAI-1)的mRNA及蛋白质表达,而SKI能拮抗AA-Na的上述作用。

Objective：To study whether Shenkang injection solution （SKI） can antagonize the fibrogenic effects of aristolochic acid （AA）on human proximal tubular epithelial cells （HKC） in vitro.Methods：HKC were incubated with medium alone （control group）, medium containing AA-Na 10 mg/L, medium containing SKI 8 mg/ml,or medium containing AA-Na 10 mg/L and SKI 8 mg/ml, respectively. The mRNA expression of transforming growth factor-β1（TGF-β1）, connective tissue growth factor（CTGF）, tissue inhibitor of metalloproteinase-1（TIMP-1）and plasminogen activator inhibitor-1 （PAI-1）in cell lysate was measured by RT-PCR after 12 hours; The protein expression of TGF-β1, TIMP-1 and PAI-1 in supernate was measured by ELISA after 24 hours, and the protein expression of CTGF in cell lysate was measured by western blotting after 36 h incubation hours.Results：The mRNA and protein expression of TGF-β1,CTGF, TIMP-1 and PAI-1 was significantly up-regulated by 10 mg/L AA-Na. Compared with the control group, their the mRNA expression was up-regulated to 1.84, 1.58,1.62 and 1.29 times, respectively（P〈0.05）, and the protein expression was up-regulated to 1.12,1.63,10.42and 1.29 times, respectively（P〈0.05）.The up-regulated mRNA and protein expression of TGF-β1,CTGF,TIMP-1 and PAI-1 by AA-Na was significantly inhibited by 8 mg/ml SKI. Compared with the AA-Na group,the inhibition rates of mRNA expression were 41.6%,43.7%,43.8% and 24.4%, respectively（P〈0.05）,and those of protein expression were 34.3%, 43.3%, 31.1% and 21.9%,respectively（P〈0.05）.8 mg/ml SKI alone had no effects on both mRNA and protein expression of TGF-β1,CTGF,TIMP-1 and PAI-1.Conclusion：AA-Na can up-regulate the mRNA and protein expression of extracellular matrix（ECM）promoting synthesis factors（TGF-β1,CTGF）and inhibiting inhibit ECM degradation factors（TIMP-1, PAI-1）, which can be antagonized by SKI.