环孢菌素A对肾小管上皮细胞自噬-溶酶体通路的影响

摘要

Objective To investigate the effect of cyclosporine A (CsA) on autophagylysosomal pathway in tubular epithelial cells.Methods Human renal tubular epithelial cell line (HK-2 cell) was treated with different concentrations (3,5 and 10 μmol/L) of CsA for 24 h.Then the viability and apoptosis of cells were measured by MTT assay or AnnexinV-PI staining followed by flow cytometry analysis,respectively.Autophagy-related protein LC3-Ⅱ and p62 were detected by immunofluorescence assay.Autophagic flux was analyzed in HK-2 cells transfected with a tandem mRFP-GFP fluorescent-tagged LC3 (ffLC3) plasmid by laser confocal microscope.The lysosomal degradation was evaluated by DQ-ovalbumin staining followed by flow cytometry analysis.Results The viability of HK-2 cells was significantly decreased with CsA stimulation when compared with control group (P ＜ 0.01),but the number of apoptotic cells was markedly increased by CsA treatment (P ＜ 0.05).Compared with the control group,different doses of CsA dramatically increased the expressions of LC3-Ⅱ (P ＜ 0.01) and p62 (P ＜ 0.05) in HK-2 cells.Moreover,HK-2 cells treated with CsA displayed a significant increase in autophagosomes but a marked decrease in autolysosomes.In HK-2 cells,exposured to CsA caused a decrease in lysosomal degradation by DQ-ovalbumin staining when compared with control group (P ＜ 0.01).Conclusion Blockade of autophagy via disrupting lysosome degradation may represent a novel mechanism of CsA-induced tubular epithelial cells injury.%目的 探讨环孢菌素A(CsA)对肾小管上皮细胞(TEC)自噬-溶酶体通路的影响.方法 不同浓度(3、5、10 μmol/L)CsA刺激人近端肾小管上皮(HK-2)细胞24h.用MTT法检测细胞活力;AnnexinV-PI双染流式细胞术检测细胞凋亡;免疫荧光法检测细胞自噬相关蛋白微管相关蛋白1轻链3(LC3)Ⅱ和p62表达;共聚焦显微镜下观察mRFP-GFP串联荧光标记LC3(tfLC3)质粒转染HK-2细胞后自噬体与溶酶体融合及降解情况;用流式细胞术检测HK-2细胞溶酶体对DQ-卵清蛋白的消化能力.结果 与对照组相比,5、10 μmol/L CsA组细胞活力下降(均P＜ 0.01);细胞凋亡数增多(均P＜0.05).与对照组相比,3、5、10 μmol/L CsA组细胞LC3-Ⅱ和p62表达增加(均P＜0.05);自噬体降解受阻;细胞溶酶体消化DQ-卵清蛋白能力下降(均P＜ 0.01).结论 CsA通过损害溶酶体功能而阻滞肾小管上皮细胞自噬通路,可能是CsA肾病的发生机制之一.