Effects Of MiR-590-3p On Pyroptosis Of Cardiac Fibroblasts In High Glucose Cultured And It’s Mechanism

Objective: To investigate the effect of miR-590-3p on cardiac fibroblast pyroptosis under high glucose(HG)culture,the relationship with JAK2/STAT3 pathway,and to explore the molecular mechanism initially.Methods:1.Neonatal rat cardiac fibroblasts(NRCFs)were cultμred in primary cultures of neonatal SD rats(1-3 days),and intracytoplasmic vimentin(Vimentin)was detected by immμnofluorescence to identify the purity of cardiac fibroblasts in primary cultures.2.50 mmol/L glucose concentration in complete medium was cultured for 48 hours to establish a high glucose model,and CCK-8 assay,immunofluorescence double-staining technique,ELISA,Western blot and fluorescence qμantitative PCR(q RT-PCR)were used to probe the effect of high glucose on cardiac fibroblast activity,fibrosis,type I collagenase,MMP-9,α-SMA,FSP-1 NLRP3,GSDMD,cleaved caspase-1,IL-1β,IL-18 protein,and miR-590-3p expression.3.miR-590-3p mimic(mimic)and its control(mimic NC)were transfected into NRCFs by transient transfection and transfection efficiency was examined by q RT-PCR.ELISA,immunofluorescence double-staining and Western blot were used to probe the effect of up-regulation of miR-590-3p on the focal death rate of NRCFs,fibrosis,type I collagenase MMP-9,α-SMA,FSP-1,NLRP3,GSDMD,cleaved caspase-1,IL-1β,IL-18 protein expression.4.The target genes of miR-590-3p were predicted by miRNA target gene prediction website,and JAK2/STAT3,a pathway closely related to myocardial metabolism and inflammatory response,was selected.5.AG490,a specific inhibitor of JAK2/STAT3 pathway,was selected and added in cardiac fibroblasts transfected with miR-590-3pmimic,and its effect on miR-590-3pmimic action was verified by double-stained immμnofluorescence and Western blot.Result:1.Primary cultured cardiomyocytes were observed by fluorescence microscopy for purity identification.Green fluorescence(Vimentin)was located in the cytoplasm of cardiac fibroblasts,and blue fluorescence(DAPI)was located in the nucleus of all cells.The purity of cultured cardiac fibroblasts was higher than 95%.2.Compared with the control group,cell activity was increased in the high glucose group(P<0.05),fibrosis and scorching rate increased with increasing culture time(P<0.001),and expression of MMP-9,α-SMA,type I collagenase,NLRP3,GSDMD,cleaved caspase-1,IL-1β,IL-18 increased(P<0.001),FSP-1 expression was reduced(P<0.001).4.QRT-PCR results showed that the expression of miR-590-3p was significantly reduced in the high glucose group compared with the control group(P<0.05),and the expression of NLRP3 and IL-18 was reduced after transfection with miR-590-3pmimic compared with the high glucose group(P<0.001).5.increased expression of type I collagenase,NLRP3,GSDMD,cleaved caspase-1,IL-1β,IL-18 in HG+mimic+AG490 group compared to HG+mimic group(P<0.05).Conclusion:1.miR-590-3p redμces fibrosis and scorching of cardiac fibroblasts under high glucose culture.2.The protective effect of miR-590-3p on cardiac fibroblasts under high glucose culture is related to the JAK2/STAT3 pathway.