自噬在低氧诱导人肾小管上皮细胞凋亡中的作用

Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis

摘要:

目的：探讨自噬在低氧诱导人肾小管上皮细胞株（HK-2）凋亡中的作用。方法：培养HK-2细胞，根据培养环境氧浓度不同分为对照组（21%O2）和低氧组（1%O2）；根据低氧培养时间不同，低氧处理细胞进一步分为：低氧6 h亚组、低氧12 h亚组、低氧18 h亚组和低氧24 h亚组。Western blot检测HK-2细胞自噬相关蛋白Beclin1、LC3Ⅱ/LC3Ⅰ、P62和凋亡相关蛋白活化的天冬氨酸特异性半胱氨酸蛋白酶（cleaved-caspase3）表达水平，流式细胞仪检测HK-2细胞凋亡情况；采用低氧诱导稳定表达GFP-LC3的HK-2细胞18 h，在荧光显微镜下观察GFP-LC3自噬小体的变化；进一步采用低氧联合自噬诱导剂雷帕霉素（ra－pamycin，RP）或自噬抑制剂巴弗洛霉素A1（bafilomycin A1，BAfA1）处理HK-2细胞，细胞分为常氧对照组、常氧+雷帕霉素/或巴弗洛霉素组、低氧对照组、低氧+雷帕霉素/或巴弗洛霉素组。Western blot检测Beclin1、LC3Ⅱ/LC3Ⅰ、P62和cleaved-caspase3表达水平，流式细胞仪检测细胞凋亡情况。结果：与对照组（0.161±0.043）相比较，低氧12 h[（0.315±0.060），P=0.019]、18 h[（0.586±0.063），P=0.000]、24 h[（0.698±0.085），P=0.000]亚组HK-2细胞cleaved-caspase3蛋白表达明显增多；低氧12 h、18 h、24 h亚组HK-2细胞凋亡率分别为（23.633±5.346）%、（25.200±4.782）%、（38.567±8.046）%，较对照组（11.800±3.966）%明显增多（P=0.036，P=0.021，P=0.000）。同时，低氧18 h、24 h亚组HK-2细胞表达Beclin1蛋白[（0.557±0.071），（0.897±0.074）[较对照组（0.249±0.040）明显上调（P=0.001，P=0.000）；低氧6 h、12 h、18 h、24 h亚组细胞表达LC3Ⅱ/LC3Ⅰ蛋白[（1.795±0.143），（2.873±0.078），（3.029±0.089），（3.735±0.059）]较对照组（1.322±0.191）明显上调（P=0.036，P=0.000，P=0.000，P=0.000）；细胞表达自噬底物P62蛋白[（0.776±0.061），（0.602±0.119），（0.419±0.117），（0.384±0.068）]较对照组（0.957±0.035）明显下调（P=0.028，P=0.001，P=0.000，P=0.000）；低氧组细胞GFP-LC3斑点计数相对值为（40.667±6.028），较对照组（18.667±5.508）明显增加（P=0.010）。雷帕霉素增加自噬后，低氧诱导的HK-2细胞凋亡（26.433±3.402）及cleaved-caspase3蛋白表达（0.265±0.066）较低氧对照组[（36.133±5.856），（0.358±0.060）]明显减少（P=0.012，P=0.000）；巴弗洛霉素A1抑制自噬后，低氧诱导的HK-2细胞凋亡增多（49.233±3.412）及cleaved-caspase3蛋白表达（1.242±0.110）较低氧对照组[（25.933±3.650），（0.659 ± 0.060）]明显增加（P=0.000，P=0.000）。结论：自噬在低氧诱导的HK-2细胞凋亡中起保护作用。

Abstract:

Objective：To investigate the effect of autophagy on the hypoxia-induced apoptosis in human renal proximal tubular cells（HK-2）. Methods：Human proximal tubular epithelial cells cultured in vitro were divided into the control group（21%O2） and hypoxia group（1%O2），based on the oxygen concentration. HK-2 cells cultured in hypoxia condition were then divided into 6 h hypoxia sub－group，12 h hypoxia subgroup，18 h hypoxia subgroup and 24 h hypoxia subgroup. The expression of autophagy-related proteins，Beclin1，LC3Ⅱ/LC3Ⅰ，P62 and apoptosis-related protein，activated cleaved-caspase 3 were detected by Western blot analysis，and the apoptosis was analyzed by flow cytometry assay. HK-2 cells stably expressing GFP-LC3 were induced by hypoxia for 18 hours，and the alteration of LC3 autophagosomes was examined by immunofluorescence（IF） assay. Then HK-2 cells were co-treated with hypoxia and autophagy inducer rapamycin or autophagy inhibitor Bafi-lomycin A1，and they were divided into normoxia control group，nor－moxia plus rapamycin/ Bafilomycin A1 group，hypoxia control group，hypoxia plus rapamycin/Bafilomycin A1 group. The expression of Beclin1，LC3Ⅱ/LC3Ⅰ，P62 and cleaved-caspase3 were detected by Western blot analysis，while the apoptosis was analyzed by flow cytometry assay. Results：Compared with the control group（0.161±0.043），the cleaved-caspase3 expression in 12 h[（0.315±0.060），P=0.019]，18 h[（0.586±0.063），P=0.000] and 24 h hypoxia subgroup[（0.698±0.085），P=0.000] increased significantly. The apopto－sis of HK-2 cells in 12 h[（23.633±5.346）%]，18 h[（25.200±4.782）%] and 24 h hypoxia subgroup[（38.567±8.046）%] increased significantly as compared with the control group[（11.800±3.966）%]（P=0.036，P=0.021，P=0.000）. The Beclin1 expression in 18 h[（0.557±0.071），P=0.001] and 24 h hypoxia subgroup[（0.897±0.074），P=0.000] were higher than that in the control group（0.249±0.04）. The LC3Ⅱ/LC3Ⅰ expression in 6 h[（1.795±0.143），P=0.036]，12 h[（2.873±0.078），P=0.000]，18 h[（3.029±0.089），P=0.000] and 24 h hypoxia subgroup[（3.735±0.059），P=0.000] were higher than that in the control group（1.322±0.191）. The P62 ex－pression in 6 h[（0.776±0.061），P=0.028]，12 h[（0.602±0.119），P=0.001]，18 h[（0.419± 0.117），P=0.000] and 24 h hypoxia subgroup[（0.384±0.068），P=0.000] were lower than that in the control group（0.957±0.035）. The counting value of GFP-LC3 puncta in HK-2 cells in hypoxia group（40.667±6.028） was obviously more than that in the control group（18.667±5.508）（P=0.010）. We also found that the treatment of the autophagy inducer rapamycin significantly reduced the hypoxia-induced apoptosis[（26.433±3.402），P=0.012] and cleaved-caspase3 expression[（0.265±0.066），P=0.000] of HK-2 cells compared with that in the hypoxia control group[（36.133±5.856），（0.358±0.060）]，while the treatment of the autophagy inhibitor Bafilomycin A1 significantly increased the hypox－ia-induced apoptosis[（49.233±3.412），P=0.000] and cleaved-caspase3 expression[（1.242±0.110），P=0.000] of HK-2 cells as com－pared to the hypoxia control group[（25.933±3.650），（0.659±0.060）]. Conclusion：Autophagy protects against the hypoxia- induced apoptosis of HK-2 cells.