Calreticulin Inhibits High-glucose Induced Pyroptosis,Inflammatory Response And Damages In Mouse Osteoblasts

Background:Diabetic osteoporosis(DOP)is a common cause of secondary osteoporosis,and its incidence increases with the progress of population aging.Studies have shown that the risk of fractures increases in patients with type 1 and type 2 diabetes,resulting in heavy economic burden for individuals and countries.Calreticulin(CRT)is a type of heat shock protein that plays a role in physiological and pathological processes such as protein folding,tumor immunity,and cellular calcium balance.Dysfunction of osteoblasts is a key link in osteoporosis,and calreticulin can maintain the functional homeostasis of cells and tissues.Therefore,exploring whether calreticulin has a protective effect on osteoblasts against high glucose damage and its related mechanisms is of great significance for the prevention and treatment of DOP.Objective:To investigate the effect of calreticulin expression on pyroptosis in a high glucose(HG)-induced embryonic osteoblast cell(MC3T3-E1)model of mice and to observe the effects of calreticulin on the inflammatory level and cell damage of osteoblasts.Method:1.Cell Counting Kit-8(CCK-8)was used to detect the cell viability of MC3T3E1 cells.2.Western blot was used to detect the expression levels of calreticulin(CRT)and nucleotide-binding oligomerization domain-like receptor protein-3(NLRP3),an indicator of pyroptosis.3.siRNA was used to interfere with the expression of CRT in MC3T3-E1 cells.4.Enzyme-linked immunosorbent assay(ELISA)was used to determine the secretion levels of inflammatory factors such as interleukin-1β(IL-1β)and interleukin-18(IL-18)in the cell supernatant.5.DCFH-DA staining was used to detect the level of intracellular reactive oxygen species(ROS).6.Rhodamine 123(Rh123)staining was used to detect the mitochondrial membrane potential(MMP)of MC3T3-E1 cells.7.SPSS 25.0 software was used for statistical analysis of experimental data.Data were expressed as mean ± SEM for quantitative data.One-way analysis of variance(ANOVA)was used for inter-group comparisons,and least significant difference(LSD)-t test was used for pairwise comparisons.P<0.05 was considered statistically significant.Results:1.High glucose(60 mmol/L)increased the expression levels of CRT and NLRP3 in MC3T3-E1 cells.2.Interfering with CRT expression further increased the expression level of NLRP3 in HG-induced MC3T3-E1 cells.3.Interfering with CRT expression further increased the secretion of IL-1β and IL-18 in HG-induced MC3T3-E1 cells.4.Interfering with CRT expression further enhanced the cytotoxicity(decreased cell viability)of HG-induced MC3T3-E1 cells.5.Interfering with CRT expression further increased the generation of ROS in HG-induced MC3T3-E1 cells.6.Interfering with CRT expression further increased the loss of MMP in HGinduced MC3T3-E1 cells.Conclusion:Inhibition of calreticulin alleviates high glucose-induced pyroptosis,inflammatory response,and cellular injury in mouse embryonic osteoblasts.