Drp-1调节LPS脓毒血症诱导的肾小管上皮细胞自噬的研究

Drp-1 Regulates Autophagy in Renal Tubular Epithelial Cells Induced by LPS Sepsis

目的:探讨发动蛋白-1(Dynamin-1,Drp-1)在脓毒血症诱导肾小管自噬中的影响。方法:用LPS诱导体外培养的肾小管上皮细胞,CK-8法检测细胞存活率。分别采用雷帕霉素、3-MA、氯喹或NAC处理NRK-52E细胞后,进行免疫荧光、Drp-1、LC3-Ⅱ转录和蛋白水平检测。构建过表达Drp-1或Drp-1-shRNA的慢病毒,感染NRK-52E细胞,检测Drp-1、LC3-Ⅱ转录和蛋白表达量。结果:确定1μg/ml的LPS浓度干预24 h作为建立NRK-52E细胞损伤模型的干预条件。与对照组相比,LPS浓度依赖性降低NRK-52E细胞的存活率,增加Drp-1的表达(P<0.05)。干扰或过表达Drp-1能够的表达后可观察到自噬增强或减,Drp-1可调节由LPS诱导的自噬。结论:Drp-1能够调节脓毒血症诱导肾小管上皮细胞自噬。

Objective:To investigate the role of dynamin-1(Dynamin-1,Drp-1)in sepsis-induced renal tubular autophagy and further explore its possible mechanism.Methods:Renal tubular epithelial cells were cultured in vitro and stimulated with LPS.The cell proliferation was detected by cell counting Kit-8(CCK-8)assay.NRK-52E cells were incubated with rapamycin,3-MA,CL or NAC respectively.Immunofluorescence staining was employed to detect the autophagy.Drp-1,LC3-II transcription and protein level detection were performed.The lentiviral vector expressing the Drp-1 was constructed and transfected into NRK-52E cells.Western blot was used to detect the expressions of Drp-1 and LC3B.Results:NRK-52E cells were treated with 1μg/ml of LPS for 24 hours.Compared with the control group,the cell proliferation and the ratio of LC3Ⅱ/LC3Ⅰin NRK-52E cells in the LPS groups were significantly decreased(P<0.05),and the expression of Drp-1 was significantly increased(P<0.05).Autophagy is enhanced or decreased after interference or overexpression of Drp-1 capable of expression,and Drp-1 regulates autophagy induced by LPS.Conclusion:Drp-1 is involved in regulating sepsis-induced renal tubular epithelial cells autophagy.