Effects Of Pyroptosis,necroptosis And COX-2 Pathway On High Glucose-induced Inflammation And Injury In MC3T3-E1 Osteoblasts

BackgroundOsteoporosis(OP),a chronic metabolic bone disorder,is characterized by decreased bone strength and increased fracture risk.Diabetes mellitus(DM)-associated osteoporosis(DOP)is a common cause of secondary osteoporosis.It is well known that both type 1 diabetes mellitus(T1DM)and T2DM have higher bone fragility and worse fracture outcomes.However,the mechanisms underlying increased skeletal fragility in patients with DM are unclear.Importantly,osteoblast dysfunction has been revealed to play a central role in the pathogenesis of OP.Hence,it is crucial to explore the pathophysiological mechanisms of high glucose(HG)-induced injury and provide a fundamental and an experimental basis for the clinical application in DOP.Objective(1)To explore the role of pyroptosis in HG-induced inflammation and injury in MC3T3-E1 cells;(2)To explore the effects of necroptosis and COX-2 pathway on HG-induced inflammation and injury in MC3T3-E1 cells.Methods1.The cell counting kit 8(CCK-8)was used to determinate the cell viability of MC3T3-E1 cells.2.The expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),cysteinyl aspartate specific proteinase 1(Caspase-1),receptor-interacting protein 1(RIP1),receptor-interacting protein 3(RIP3)and cyclooxygenase-2(COX-2)were analyzed by western blot assay.3.Casepase-1 small interfering RNA(siRNA)transfection was used for gene silencing.4.The secretion levels of interleukin-18(IL-18)and IL-1β were detected by ELISA.5.The intracellular level of reactive oxygen species(ROS)was measured by DCFH-DA staining followed by photofluorography.6.Mitochondrial membrane potential(MMP)was examined by Rhodamine 123(Rh 123)staining followed by photofluorography.7.The alkaline phosphatase(ALP)activity was determined using the ALP kit.8.The number of mineralized nodules was detected by alizarin red S staining.9.All data was represented as mean ± SEM and analyzed by SPSS21.0 version.Comparison between groups was conducted by one way analysis of variance(ANOVA)and pairwise comparison using LSD-t test.The results were considered significant if P<0.05.Results1.Pyroptosis contributed to the HG-induced inflammation and injury in MC3T3-E1 osteoblasts1.1 High glucose(45 mmol/L glucose,HG)induced the up-regulation of NLRP3 and Caspase-1 expression in MC3T3-E1 cells;1.2 Knockdown of Casepase-1 gene inhibited the HG-induced the secretion of IL-18 and IL-1β in MC3T3-E1 cells;1.3 Knockdown of Casepase-1 gene inhibited the HG-induced cytotoxicity in MC3T3-E1 cells;1.4 Knockdown of Casepase-1 gene inhibited the HG-induced accumulation of intracellular reactive oxygen species(ROS)in MC3T3-E1 cells;1.5 Knockdown of Casepase-1 gene inhibited the HG-induced a loss of mitochondrial membrane potential(MMP)in MC3T3-E1 cells;1.6 Knockdown of Casepase-1 gene inhibited the HG-induced a decrease of ALP activity in MC3T3-E1 cells;1.7 Knockdown of Casepase-1 gene inhibited the decline in mineralization capability induced by HG in MC3T3-E1 cells;2.The effects of necroptosis and COX-2 pathway on the HG-induced inflammation and injury in MC3T3-E1 cells2.1 HG enhanced the expression levels of RIP1 and RIP3 in MC3T3-E1 cells;2.2 HG upregulated the expression level of COX-2 in MC3T3-E1 cells;2.3 Nec-1(the inhibitor of necroptosis)and NS-398(the inhibitor of COX-2)attenuated the HG-induced cytotoxicity in MC3T3-E1 cells;2.4 Nec-1 and NS-398 inhibited the HG-induced the secretion of IL-18 and IL-1βin MC3T3-E1 cells;2.5 Nec-1 and NS-398 ameliorated the HG-induced ROS generation in MC3T3-E1 cells;2.6 Nec-1 and NS-398 alleviated HG-induced a loss of MMP in MC3T3-E1 cells;2.7 Nec-1 and NS-398 inhibited the HG-induced a decrease of ALP activity in MC3T3-E1 cells;2.8 Nec-1 and NS-398 inhibited the decline in mineralization capability induced by HG in MC3T3-E1 cells;2.9 There is a positive interaction between necroptosis and COX-2 pathway in HG-treated MC3T3-E1 cells2.9.1 Nec-1 inhibited the HG-induced an increase in the expression level of COX-2 in MCT3-E1 cells;2.9.2 NS-398 inhibited the HG-induced an increase in the expression levels of RIP1 and RIP3 in MC3T3-E1 cells.Conclusion1.High glucose induced inflammation and injury in MC3T3-E1 cells;2.Pyroptosis contributes to the HG-induced inflammation and injury in MC3T3-E1 cells;3.Interaction between necroptosis and COX-2 pathway contribute to the HG-induced inflammation and injury in MC3T3-E1 cells.