N-acetyl-cysteine Improves Methylglyoxal-induced HaCaT Cells Injuries And Mechanisms Underlying

Background/Aim:Accumulation of advanced glycation end products(AGEs)is a major cause of diabetes mellitus(DM)skin complications.Methylglyoxal(MGO),a reactive dicarbonyl compound,is a crucial intermediate of AGEs generation.which can form AGEs in DM rapidly.N-acetyl-L-cysteine(NAC),an active ingredient of some medicines,can induce endogenous GSH and hydrogen sulfide generation,and set off a condensation reaction with MGO.However,there is rare evidence to show NAC can alleviate DM-induced skin cells injuries through inhibition of AGEs generation or toxicity.The present study aims to investigate the effects of NAC on MGO-induced skin cells damages and its molecular mechanism.Methods:The levels of AGEs in plasma of diabetic patients and healthy volunteers were measured by enzyme-linked immunosorbent assay(ELISA).AGEs were prepared by incubation with bovine serum albumin(BSA)and MGO at 37 ° C for 3 d.According to the characteristics of AGEs with spontaneous fluorescence and the formation of brown solution,the fluorescence intensity in 340/465 nm reaction system was observed by fluorescence microplate reader and the color of mixed solution of BSA and MGO was observed.The effect of NAC on MGO induced AGEs formation was further observed.Human adult low calcium kertinocytes(HaCaT)were administered to MGO to mimic the high MGO status in diabetic blood or tissue and establish an in vitro model of diabetic skin wound healing disorder.Cell viability,mitochondrial membrane potential,cell adhesion and migration function,secretion of inflammatory factors,nuclear factor kappa B p65 subunit translocation,expression of MMP-9 and the contents of extracellular matrix were evaluated.Before treatment of HaCaT cells with MGO,pretreatment with NAC for 1 h to observe the effects of NAC on MGO-induced changes in the above-mentioned indexes to clarify the protective effects of skin cells.Finally,by detecting the expression of receptor for advanced end products(RAGE)and the effects of neutralizing antibody(Neutralizing antibody against RAGE,N-ABR)pretreatment on MGO-induced HaCaT cell damage,MGO induction was finally revealed and whether the cytoprotective effect of NAC depends on the activation of RAGE.Results:1.The plasma levels of AGEs in diabetic patients were higher than those healthy subjects,the difference was statistically significant(P <0.05).2.MGO and BSA reaction,the solution color deepened,fluorescence enhanced,suggesting that MGO can induce the formation of AGEs.NAC treatment can reduce the color and fluorescence intensity of the solution(P <0.01),suggesting that it can inhibit the formation of MGO-induced AGEs.In addition,treatment of HaCaT cells with MGO increased the contents of AGEs in cell culture medium,and NAC pretreatment could significantly inhibit the production of AGEs induced by MGO,the difference was statistically significant(P <0.01).3.Treatment of HaCaT cells with MGO can dose-dependently reduce cell viability,impair mitochondrial membrane potential and cell adhesion and migration behavior,which can be attenuated by NAC pretreatment.Suggesting that NAC can attenuate MGO-induced cell damage.4.HaCaT cells treated with MGO,the secretion of inflammatory factors IL-6 and IL-8 increased,NF-?B subunit p65 nuclear translocation enhanced,NAC pretreatment can inhibit MGO-induced inflammatory factor secretion and NF-?B subunit p65 nuclear translation.It Suggests that NAC can inhibit MGO-induced inflammatory response.5.MGO treatment of HaCaT cells can lead to the up-regulation of MMP-9 expression and the reduction of type I collagen,which can be partially reversed by NAC pretreatment.It Suggests that NAC improves MGO-induced cell migration disorders that may be associated with improved levels of extracellular matrix.6.MGO treatment can upregulate the expression of RAGE in HaCaT cells,and NAC pretreatment can inhibit the overexpression of MGO-induced RAGE.Importantly,MGO-induced cell damage,inflammatory factor release,MMP-9 overexpression,and NF-?B subunit p65 nuclear translocation were partially improved after RAGE neutralizing antibody hindered its effect.It Suggests that MGO-induced skin cell inflammation is associated with activation of RAGE,whereas NAC antagonizes the damage of MGO by inhibiting RAGE.Conclusion:In this study,it was confirmed that NAC attenuated MGO-induced skin cell damage,and its mechanism was related to inhibition of AGEs production and activation of AGEs / RAGE / NF-?B signaling pathway.This study provides basic information and theoretical support for the clinical application of NAC-related drugs to prevent and treat diabetic skin complications.