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Objective To investigate whether myeloid-derived growth factor (Mydgf) can improve cardiomyocyte pyroptosis induced by hyperglycemia by regulating nucleotide-binding domain-like receptor protein 3 (NLRP3). Methods Cultured rat cardiomyocyte H9c2 cells were divided into 4 groups: normal group (Nor), high glucose group (HG, 50 mmol/L glucose for 24 h), Mydgf group (25 ng/mL recombinant Mydgf protein for 12 h, followed by 50 mmol/L glucose for 24 h), and necrosulfonamide group (NSA, 32 μmol/L necrosulfonamide for 0.5 h, followed by 50 mmol/L glucose for 24 h). Western blotting was used to detect the protein levels of cleaved Caspase-1, NLRP3, apoptosis-related speckle-like proteins (ACS) and gasdermin D (GSDMD). The secretions of IL-1β and IL-18 in cell culture medium were detected by ELISA. CCK-8 assay was used to detect the effect of Mydgf or NLRP3 on the viability of H9c2 cells. Lentiviral vector carrying NLRP3 and enhanced green fluorescent protein (EGFP) (NLRP3-EGFP) and empty viral vector (EGFP) were transfected into H9c2 cells respectively, and the changes of pyroptosis related indexes were detected by recovery experiments. Results Western blot results showed that the expression levels of pyroptosis related proteins, cleaved Caspase 1, ACS, NLRP3 and GSDMD were significantly higher in the HG group than the Nor group (P < 0.05). Mydgf treatment effectively inhibited the expression of above proteins (P < 0.05), but the inhibitory efficiencies were lower than that in the NSA group (P < 0.05). ELISA demonstrated that the secretions of IL-1β and IL-18 were increased in the HG group (P < 0.05), but decreased in the Mydgf group (P < 0.05), and more significantly reduced in the NSA group (P < 0.05). CCK-8 assay indicated that Mydgf at a concentration of below 25 ng/mL had no significant effect on the viability of H9c2 cells, while when the concentration above 25 ng/mL, the treatment significantly decreased the cell viability (P < 0.05). Compared with the control group, overexpression of NLRP3 significantly decreased H9c2 cells viability under the condition of HG. Transfection of lentiviral vector NLRP3-EGFP resulted in obvious increases in the mRNA and protein expression of NLRP3 (P < 0.05), but that of empty vector had no such effects (P>0.05). In the recovery experiment, Western blot results showed that in the high-glucose environment, the expression level of NLRP3, ASC, and cleaved Caspase-1 were significantly increased (P < 0.05), and the secretions of IL-1β and IL-18 were also significantly enhanced in the Mydgf +NLRP3-EGFP group compared with the Mydgf group (P < 0.05). Conclusion Mydgf may ameliorate cardiomyocyte pyroptosis induced by hyperglycemia by inhibiting NLRP3. |
