| Background and Object Sepsis is a systemic inflammatory response syndrome caused by infection,acute kidney injury(AKI) is one of the most common serious complication of sepsis.Septic acute kidney injury is a common critical disease with high incidence rate,high case fatality rate and poor prognosis,and up to now there is still lack of effective treatments,so it is urgent to find effective treatment strategies.Some studies have shown that inflammatory response is one of important mechanisms in the occurrence and development of septic acute kidney injury,and antiinflammatory treatment is of great significance for the treatment of septic acute kidney injury.In recent years,LPS/TLR4 signaling pathways are widely researched in the studies for prevention and control of inflammatory response.Autophagic flux is a highly dynamic process,including four steps proceed in succession.They are autophagy bubble formation,autophagosome formation,autolysosome fusion and autolysosome degradation.Autophagic flux can illustrate the process of autophagy better.In recent years,the researches of autophagy process are more and more widely used in the field of kidney diseases which indicated that the occurrence of autophagy plays an important role in the development of many kidney diseases,but so far the mechanism of specific is not yet clear.The purpose of this study is to investigate the role of autophagic flux in the inflammatory response in renal tubular epithelial cells(HK-2 cells)induced by lipopolysaccharide(LPS),and whether toll-like receptor 4(TLR4)was involved the procedure.Finally,it is expexted to provide a new approach for the clinical prevention and treatment of septic acute kidney injury.Methods1.To stimulate HK-2 cells using LPS,detected the m RNA expression of IL-1β.HK-2 cells in logarithmic phase were stimulated by LPS: using different concentrations(0,50,100,200 μg/m L)of LPS to stimulate HK-2 cells,the m RNA expressions of IL-1β were detected by fluorescence quantitative PCR(qPCR)at different time points(0,4,12,24 h),so that the optimal concentration and time piont of LPS stimulation were determined for subsequent experiments.2.To detect the m RNA expression of IL-1β,TLR4,autophagy-related genes LC3 and p62 after intervention of autophagy and blockade of TLR4.HK-2 cells were divided into five groups for the intervention experiment,including the negative control group,LPS group,rapamycin(RAP)combined with LPS group,chloroquine(CQ)combined with LPS group,TLR4 inhibitor(TAK242)combined with LPS group.RAP,CQ and TAK242 was respectively used to pretreat HK-2 cells for 2 h,and then 100 μg/m L of LPS was added to every experimental group,distilled water was added to the negative control group.The m RNA expressions of IL-1β,TLR4,LC3 and p62 were detected by qPCR after culturing for 12 h.3.To detect the protein expression of IL-1β,TLR4,autophagy-related proteins LC3-II and p62 after intervention of autophagy and blockade of TLR4.HK-2cells were divided into five groups for the intervention experiment: the negative control group,LPS group,RAP combined with LPS group,CQ combined with LPS group and TAK242 combined with LPS group.RAP,CQ and TAK242 was respectively used to pretreat HK-2 cells for 2 h,after that 100 μg/m L of LPS was added to every experimental group,and distilled water was for the negative control group.The protein expression of IL-1β,TLR4,LC3-II and p62 were detected by western blot(WB)after culturing for 12 h.4.To detect autophagy flux changes using autophagy double-tagged adenovirus to transfect HK-2 cells.HK-2 cells were transfected using autophagy double-tagged adenovirus for 24 h.After transfection,transfected HK-2 cells were divided into five groups including the negative control group,LPS group,RAP combined with LPS group,CQ combined with LPS group and TAK242 combined with LPS group for the intervention experiment.RAP,CQ and TAK242 was respectively used to pretreat HK-2 cells for 2 hours,and then 100μg/m L of LPS was added to every experimental group,distilled water was added to the negative control group.The number of autolysosomes are observed under confocal microscope to judge the patency degree of autophagic flux.Results1.LPS induced the expression of IL-1β in renal tubular epithelial cells.HK-2cells were stimulated with LPS of 50,100,200 μg/m L for 4,12,24 h respectively,the m RNA expression of IL-1β in HK-2 cells are up-regulated(P<0.05).LPS concentration of 100 μg/m L stimulated HK-2 cells for 12 h,the expression of IL-1β is the most significant(P<0.01).2.Autophagic flux and TLR4 are involved in the regulation of IL-1β expression in renal tubular epithelial cells.qPCR and WB results: compared with the negative control group,the m RNA and protein expressions of IL-1β are up-regulated in the LPS group(P<0.05);Compared with the LPS group,the m RNA expression of IL-1β in RAP combined with LPS group is significantly down-regulated(P<0.01),and the protein expression of IL-1β is down-regulated(P<0.05).Compared with the LPS group,the m RNA and protein expressions of IL-1β are up-regulated in CQ combined with LPS group(P<0.05).Compared with the LPS group,the m RNA expression of IL-1β in TAK242 combined with LPS group is significantly down-regulated(P<0.01),and the protein expression of IL-1β is down-regulated(P<0.05).3.Autophagic flux is involved in the regulation of TLR4 expression in renal tubular epithelial cells.qPCR and WB results: compared with the negative control group,the m RNA expression of TLR4 is up-regulated in the LPS group(P<0.05),and the protein expression of TLR4 is significantly up-regulated(P<0.01).Compared with the LPS group,the m RNA and protein expressions of TLR4 in RAP combined with LPS group are significantly down-regulated(P<0.01).Compared with the LPS group,the m RNA and protein expressions of TLR4 in CQ combined with LPS group are significantly up-regulated(P<0.01).Compared with the LPS group,the m RNA and protein expressions of TLR4 in TAK242 combined with LPS group are significantly down-regulated(P<0.01).4.TLR4 mediates autophagic flux retardation in renal tubular epithelial cells.4.1 Detection of LC3 and p62.qPCR and WB results: compared with the negative control group,the m RNA expression of LC3 is up-regulated in the LPS group(P<0.05),and the protein expression of LC3-II is significantly up-regulated(P<0.01),and the m RNA and protein expressions of p62 are significantly up-regulated(P<0.01).Compared with LPS group,the m RNA expression of LC3 and the protein expression of LC3-II in RAP combined with LPS group are up-regulated(P<0.05),the m RNA expression of p62 has no significant down-regulation(P>0.05),but the protein expression of p62 is significantly down-regulated(P<0.01).Compared with LPS group,the m RNA expression of LC3 and the protein expression of LC3-II in CQ combined with LPS group have no significant change(P>0.05),but the m RNA and protein expression of p62 are significantly up-regulated(P<0.01).Compared with LPS group,the m RNA expression of LC3 and the protein expression of LC3-II in TAK242 combined with LPS group are up-regulated(P<0.05),and the m RNA expression of p62 has no down-regulation(P>0.05),but the protein expression of p62 is significantly down-regulated(P<0.01).4.2 Autophagic flux was detected by autophagy double-tagged adenovirus.Compared with the negative control group,autophagosomes are significantly increased while autolysosomes are few in the LPS group.Compared with LPS group,autolysosomes are significantly increased in the RAP combined LPS group,autolysosomes are particularly scarce in the CQ combined with LPS group,while autolysosomes are significantly increased in the TAK242 combined with LPS group.Conclusions1.Autophagic flux negatively regulates LPS-induced inflammatory response in renal tubular epithelial cells.2.TLR4 may mediate LPS-induced inflammatory response in renal tubular epithelial cells through autophagic flux retardation. |
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