Necroptosis And Inflammatory Factors Expression In Renal Tubular Epithelial Cells And The Molecule Mechanism

Objective: Chronic ?idney disease(CKD)is a ?ind of destructive disease.In recent years,CKD has gradually been regarded as a major public health problem.The course of CKD is slow,which leads to irreversible renal unit loss and end-stage renal disease.The continuous inflammatory response of ?idney is closely related to the occurrence and development of CKD.Necroptosis is an important type of programmed cell death in addition to apoptosis,which was first proposed by Degterev et al in 2005 and named "necroptosis".Necroptosis is a caspase-independent cell death mode that can be regulated by TNF-induced receptor integrated protein(RIP).Necroptosis plays an important role in the inflammatory response of many diseases.However,the relationship between necroptosis and the expression of inflammatory factors in human renal tubular epithelial cells is still unclear.In our previous studies,we found that Necrostatin-1(Nec-1),a necroptosis inhibitor,can reduce renal inflammatory response in unilateral ureteral obstruction(UUO)mice,but the specific regulatory mechanism has not been clarified.On the basis of previous experiments,this experiment ta?es HK2 as the research object,TNF-? and pan caspase inhibitor Z-VAD-FMK were used as stimulants to construct necroptosis cell model.Furthermore,administering Nec-1 and NF-?B specific inhibitor pyrrolidine dithiocarbamate(PDTC)to the cells separately or in combination,and transfecting the RIP1 overexpression plasmid.To further explore the mechanism of necroptosis and related inflammation in human renal tubular epithelial cells,so as to provide new ideas for elucidating the role of renal tubular injury in the progression of CKD.METHODS:Human renal tubular epithelial cells HK2 were cultured in vitro,and stimulated with TNF-? and Z-VAD-FMK(T/Z)for 24 h.Lactate dehydrogenase(LDH)cytotoxicity assay was used to detect the percentage of cell necrosis.The protein levels of receptor interacting protein 1(RIP1),IKK-? and NF-?B p65 were determined by Western blot.The levels of interleu?in-1?(IL-1?)and monocyte chemotactic protein 1(MCP-1)were detected by Western blot and ELISA.RT-PCR was used to detect the m RNA expression level of NF-?B p65.Furthermore,Nec-1 and PDTC were used separately and in combination for intervention,and RIP1 overexpressed plasmids were transfected to detect the above indicators.Co-immunoprecipitation was used to detect the expression of ubiquitin(Ubiquitin,Ub)protein,a ubiquitination-related indicator of RIP1.RESULTS:1.TNF-? / Z-VAD-FMK induces RIP1 expression in HK2 cellsWestern blot results showed that protein levels of RIP1 and cleaved caspase3 were increased when TNF-? stimulated HK2 cells alone,compared with the Control group.T/Z stimulated the protein level of RIP1 in HK2 cells to further increase(P<0.05),while the protein level of cleaved-caspase3 decreased(P<0.01).The results of LDH cytotoxicity test showed that compared with the Control group,the percentage of necrosis increased after T/Z stimulation of HK2 cells(P<0.01).When Nec-1 was administered under this stimulation condition,the percentage of necrosis of HK2 cells decreased(P<0.01),and the percentage of necrosis of transfected RIP1 overexpressing plasmid cells increased significantly(P<0.01).2.Necroptosis induces expression of inflammatory factors in HK2 cellsThe results of Western blot and ELISA showed that the levels of IL-1?(P<0.05)and MCP-1(P<0.01)protein in HK2 cells stimulated by T/Z were higher than those in the Control group.Under this stimulating condition,the protein level of HK2 cells was reduced by Nec-1 intervention(P<0.05 or P<0.01);on the contrary,the transfection of RIP1 overexpression plasmid significantly increased its protein level(P<0.05 or P<0.01).3.Necroptosis activates NF-?B pathway in HK2 cellsWestern blot showed that the protein levels of IKK-??NF-?B p65?and p-NF-?B p65 in HK2 cells stimulated by T/Z were higher than those in the Control group(P<0.01).Under this stimulating condition,the protein level of HK2 cells was dropped by Nec-1 intervention(P<0.05 or P<0.01);on the contrary,the transfection of RIP1 overexpression plasmid significantly raised its protein level(P<0.01).Real-time PCR results showed the same trend as Western blot results.4.Inhibition of NF-?B pathway reduces expression of inflammatory factors in HK2 cells induced by necroptosisWestern blot showed that the protein levels of IL-1? and MCP-1 in HK2 cells stimulated by T/Z were higher than those in Control group(P<0.01).Under the stimulation condition,the above protein level was significantly reduced after the PDTC intervention of HK2 cells alone(P<0.01),especially after the combined application of Nec-1(P<0.01).5.TNF-? / Z-VAD-FMK induces RIP1 ubiquitination in HK2 cellsThe results of co-immunoprecipitation showed that compared with the control group,T/Z stimulated HK2 cells to increase the ubiquitination level of RIP1 protein.CONCLUSION:1.RIP1 mediated necroptosis is involved in the expression of inflammatory factors in human renal tubular epithelial cells,which may be partially mediated by the activation of NF-?B signaling pathway.2.TNF-?/Z-VAD-FMK may induce the ubiquitination of RIP1,suggesting that it may be involved in the expression of inflammatory factors in human renal tubular epithelial cells.