Effects And Mechanism Of N-acetylcysteine On The Injury Of Human Renal Tubular Epithelial Cells Induced By Methylglyoxal

Objective: To investigate the effect and mechanism of N-acetylcysteine on the injury of human proximal tubular epithelial cel s(HK-2 cells)induced by methylglyoxal(MG).Methods: HK-2 cells were treated with MG to establish a model of tubular injury of diabetic nephropathy(DN)in vitro,and then treated with different concentrations of Nacetylcysteine(NAC).Cell viability was detected by CCK-8 method,nuclear morphology was observed by DAPI staining,mitochondrial transembrane potential(??m)was measured by rhodamine 123(Rh123),and intracellular reactive oxygen species(ROS)were measured by DCFH-DA.The expression of Bax,Bcl-2,C leaved-caspase-3,pERK1/2,K lotho,TLR4,N LRP3,ASC,Caspase-1,IL-1?,IL-18,TNF-? and NF-?B protein was measured by Western blot.Inaddition,TLR4 inhibitor(TAK-242)was added to observe its effect on the cell viability and the expression of TLR4-NLRP3 inflammatory signaling pathway-associated protein.Results: 1.Compared with control group,the cell viability,??m and the expression of Bcl-2 of HK-2 cells decreased significantly,while intracellular ROS levels,the number of apoptotic cells and the expression of proapoptotic protein Bax and apoptosis effector protein cleaved caspase-3 increased in MG group.However,the cell viability,??m,the expression of Bcl-2 in HK-2 cells increased while the intracellular ROS leve ls,the number of apoptotic cells and the expression of Cleaved-caspase-3 and Bax decreased after treated with NAC.2.Compared with control group,the expression of p-ERK1/2 protein increased,while the expression of K lotho protein decreased in MG group.However,the above effect could be reversed by NAC.3.Compared with control group,the expression of TLR4,NLRP3,ASC,Caspase-1,IL-1?,IL-18,TNF-?,NF-?B in HK-2 cells of MG group was increased significantly,while the cell viability was decreased.Compared with MG group,the expression of the above protein was decreased and the cell viability of HK-2 cells was increased after treated with NAC.4.TLR4 inhibitor(TAK-242)showed the protective effect similar to NAC on HK-2 cells against MG-induced injuries,including improving the cell viability and decreasing the expression of TLR4,NF-?B,NLRP3,ASC,Caspase-1,IL-1?,IL-18,TNF-? protein.Although the expression of TLR4,NLRP3,ASC,Caspase-1,IL-1?,IL-18 and TNF-? protein could be further reduced by combined use of NAC and TAK-242,but it didn't exert more protective effect than either drug as a single agent.Conclusion: 1.NAC could resist the apoptosis of HK-2 cells induced by MG,the mechanism could be though the reduction of oxidative stress,stabilization of mitochondrial membrane potential,inhibition of ERK1/2 signaling pathway and promotion of Klotho protein.2.NAC may attenuate MG-induced damage of HK-2 cells by inhibiting the activation TLR4-NLRP3 inflammatory signaling pathway.