Effect Of Pyroptosis And Oxidative Stress On Methylglyoxal-induced Injury In MC3T3-E1 Cells

Background Diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia which is contributed to various causes.It causes multi-system damage,e.g.cardiomyopathy,nephropathy,vascular disease and retinopathy.In skeletal system,it also causes damages including diabetic osteoporosis(DOP),delayed healing or nonunion of fracture.Studies show that both type 1(T1DM)and type 2 diabetes(T2DM)increase the risk of fracture.Accumulation of advanced glycation endproducts(Advanced glycation end-products,AGEs)has been proven to be an important pathogenic factor for multiple complications of diabetes,while its key precursor methylglyoxal(MG),has been extensively investigated.As the important precursor of AGEs,the accumulation of MG itself causes complications of diabetes,and it can also produces toxicity through formation of AGEs.The mechanism of abnormal bone metabolism induced by DM is complicated,of which osteoblast dysfunction is at the core.Therefore,investigation of the mechanism for MG-induced osteoblast injury provides important theoretical and clinical significance for prevention of diabetic osteoporosis.Objective 1.To explore the role of pyroptosis in MG-induced injury in MC3T3-E1 cells.2.To explore the role of oxidative stress in MG-induced injury in MC3T3-E1 cellsMethods 1.The viability of MC3T3-E1 cells was detected by Cell counting kit 8(CCK-8).2.The expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and cysteiny1 aspartate specific proteinase 1(Caspase-1)were analyzed by western blot assay.3.Caspase-1 small interfering RNA(si RNA)transfection was used for gene silencing.4.The intracellular level of reactive oxygen species(ROS)was detected by DCFHDA staining followed by photofluorography.5.Mitochondrial membrane potential(MMP)was measured by Rhodamine 123(Rh 123)staining followed by photofluorography.6.The alkaline phosphatase(ALP)activity was determined using the ALP kit.7.All data were represented as mean ± SEM and analyzed by SPSS21.0 version.Comparison among groups was conducted by one way analysis of variance(ANOVA)and pairwise comparison by LSD test.The results were considered significant if P < 0.05.Results 1 Pyroptosis mediated MG-induced injury in MC3T3-E1 cells.1.1 MG(0.6mmol/L)treatment for 24 h upregulated caspase-1 and NLRP3 expression in MC3T3-E1 cells.1.2 MG induced ROS generation in MC3T3-E1 cells.1.3 MG led to MMP loss in MC3T3-E1 cells.1.4 MG decreased cell viability in MC3T3-E1 cells.1.5 MG decreased ALP level in early osteoblast differentiation.1.6 Knockdown of caspase-1 with si RNA inhibited MG-induced ROS generation in MC3T3-E1 cells.1.7 Knockdown of caspase-1 with si RNA alleviated MG-induced MMP loss in MC3T3-E1 cells.1.8 Knockdown of caspase-1 with si RNA attenuated MG-induced cytotoxicity in MC3T3-E1 cells manifested by increasing cell viability.1.9 Knockdown of caspase-1 with si RNA inhibited MG-induced decrease of ALP activity.2 Oxidative stress mediated MG-induced injury in MC3T3-E1 cells.2.1 ROS scavenger N-Acetyl Cysteine(NAC)attenuated MG-induced cytotoxicity of MC3T3-E1 cells manifested by increasing cell viability.2.2 NAC inhibited MG-induced ROS generation in MC3T3-E1 cells.2.3 NAC alleviated MG-induced loss of MMP in MC3T3-E1 cells.2.4 NAC inhibited the elevated level of caspase-1 and NLRP3 by MG in MC3T3-E1 cells.2.5 NAC inhibited the MG-induced decrease of ALP activity in MC3T3-E1 cells.Conclusion: 1.MG induces injury in MC3T3-E1 cells.2.Pyroptosis mediates MG-induced injury in MC3T3-E1 cells.3.Oxidative stress mediates MG-induced injury in MC3T3-E1 cells.4.There is an interaction between pyroptosis and oxidative stress in MGinduced injury in MC3T3-E1 cells.