百令提取液对环孢素A导致肾小管损伤的防治作用

Cordyceps Sinensis extractant protect against cyclosporine induced injury of tubular epithelium cell

目的:从细胞水平观察百令提取液对环孢素A(CsA)肾毒性的影响。 方法:体外培养肾小管上皮细胞,通过3H-TdR掺入、MTT比色法、PI染色法和细胞LDH释放等观察百令提取液对CsA引起的肾小管上皮细胞毒性的防治作用。 结果:3H-TdR掺入和MTT比色法显示在一定的剂量范围百令提取液可明显促进培养的肾小管上皮细胞增生, 5mg/L以上的CsA抑制细胞增殖;细胞周期的研究显示15mg/L以上的CsA使小管上皮细胞细胞周期阻滞在G2期;此外,更大剂量的CsA(25mg/L)对细胞有直接的毒性作用,使培养上清LDH值升高。百令提取液能部分逆转CsA引起的细胞增殖受抑和细胞周期阻滞,但是对CsA导致 的细胞培养上清LDH的升高没有明显的影响。 结论:本研究证实了百令提取液可以促进体外培养的肾小管上皮细胞增生,对CsA的肾小管细胞毒性具有一定的拮抗作用。

Objective: Cyclosporine (CsA) has been widely applied in the field of organ transplantation with improved allograft survival. However, its unsolved nephrotoxicity remains to be the major limiting factor in the use of CsA. Our previous study has shown Cordyceps Sinensis attenuate the structural and functional changes of CsA nephrotoxicity in rats. In this study, LLC-PK1 and HKC cells were used as proximal tubular cell models to investigate the protective effects of Cordyceps Sinensis on CsA cytotoxity in vitro. Methodology:LLC-PK1 and HKC cells were treated with different dose of Cordyceps Sinensis for 24hours. 3H-TdR incorporation and MTT cell proliferation assay were applied to analyze cell proliferation. CsA was applied with or without Cordyceps Sinensis to cultured cells for 24 hours. Flow cytometry was used to investigate alterations in the DNA content and cell cycle. Culture medium LDH level was determined to evaluate cell injury induced by cyclosporine and the protective effect of Cordyceps Sinensis on CsA cytotoxicity. Results: Both 3H-TdR incorporation and MTT cell proliferation assay showed that Cordyceps Sinensis stimulated tubular epithelium cell proliferation. CsA significantly reduced DNA synthesis and induced cell cycle arrest. MTT incorporation was inhibited by CsA at concentration over 5mg/L in a dose dependent manner. In flow cytometric analysis, CsA at 15mg/L was found to cause a block of cell cycle at G2/M phase, as characterized by depletion of S-phase cells and increased percentage of cells in G2 phase. Percentage of S phase cells was reduced from 33.72.41 to 10.8 1.96 compared with control. G2/M phase cells were increased from 21.8?.53 to 34.42.01. Sub G0/G1 peak indicative of apoptosis was not found in any of the CsA dosage groups in this study. Cellular injury was evident at concentration of 25mg/L, characterized by elevation of LDH activity in culture medium. CsA induced inhibition of cell proliferation and cell cycle arrest were partially reversed by Cordyceps Sinensis, while not for the elevated LDH levels in culture medium. Conclusions: Cordyceps Sinensis can stimulate proliferation of tubuloe- pithelium cells and ameliorate CsA induced inhibition of cell proliferation and cell cycle arrest. The underlying mechanism requires further research.