Mechanism Of DNMT1 Silencing On LncRNA GAS5 Regulation Of Myocardial Fibroblast Pyroptosis Through NLRP3

Objective Myocardial fibroblasts are the main effector cells for fibrosis in the heart.Influencing the growth of myocardial fibroblasts can effectively inhibit the development of myocardial fibrosis.Through the preliminary research of our research group,we determined that LncRNA GAS5 in long-chain non-coding RNA plays an important role in the pyrolysis of cardiac fibroblasts and cardiac fibrosis.Further studies have shown that the down-regulation of DNMT1 leads to increased GAS5 expression by reversing promoter hypermethylation in cardiac fibroblasts.In this experiment,we aimed to provide a new therapeutic target for cardiac fibrosis by studying the mechanism of LncRNA GAS5 regulating myocardial fibroblast pyrolysis.Methods Forty SD rats were randomly divided into an experimental group and a control group,each with 20 rats.Rats in the experimental group were injected subcutaneously with ISO,and rats in the control group were injected with the same amount of normal saline for two weeks.HE and Masson,Sirius staining were used to observe the pathological changes of the heart tissue and collagen changes in rats.Immunohistochemical analysis of the expression of caspase1 and NLRP3 in tissues.Cardiac fibroblasts from SD rats were extracted,transiently transfected with LncRNA GAS5 overexpression plasmid and its negative control,small interfering GAS5 si RNA and its negative control,cells were collected 48 hours later,and GAS5,DNMT1,and type 1 collagen were detected by qRT-PCR(Col 1),mRNA expression of NLRP3.Western blot was used to analyze the protein expression of caspase 1,NLRP3 and DNMT1.pyroptosis was detected by flow cytometry and Hoechst 33258 staining.Transient transfection of siDNMT1,qRT-PCR was used to detect its effect on LncRNA GAS5 expression and LPS-induced myocardial fibroblast pyroptosis,and qRT-PCR and Western blot were used to analyze the effect of siDNMT1 on NLRP3 expression.Results Compared with the normal control group,the myocardial fibrosis group treated with ISO,HE,Masson,and Sirius staining showed that the cardiac fibrotic tissue was significantly proliferated and distributed more densely.Immunohistochemical results showed that the expressions of caspase1 and NLRP3 were increased.The qRT-PCR results showed that compared with the control group,the expression of GAS5 in myocardial tissues induced by ISO decreased significantly,and the mRNA expression of DNMT1 and Col1A1 increased significantly.In vitro,myocardial fibroblasts were stimulated with LPS.QRT-PCR results showed that the expression of DNMT1 and Co l1A1 in myocardial fibroblasts induced by LPS was significantly increased,and the expression of LncRNA GAS5 was significantly reduced.Western blot results showed that DNMT1 and Col1A1 expression increased significantly after LPS induction.Hoechst 33258 staining showed a significant increase in CFs focal death after LPS induction.qRT-PCR results showed that the expression of GAS5 in CFs transfected with siDNMT1 was significantly increased,and the expression of DNMT1 and caspase 1was significantly reduced.qRT-PCR showed that in CFs transfected with pc DNA3.1-GAS5,GAS5 expression increased significantly,while caspase1 expression level decreased significantly.The expression of GAS5 in CFs transfected with si GAS5 was significantly reduced,and the expression level of caspase 1 was significantly increased.Flow cytometry analysis showed that increased pyroptosis after transfection si GAS5,and decreased si GAS5 after transfection with pc DNA3.1-GAS5.In CFs transfected with siDNMT1,the expression of NLRP3 was significantly reduced.The expression of NLRP3 in CFs transfected with pc DNA3.1-GAS5 was significantly reduced,while the expression of NLRP3 in CFs transfected with si GAS5 wassignificantly increased.Conclusion In the model of myocardial fibrosis in SD rats induced by ISO,the expression of GAS5 decreased significantly,the expression of DNMT1 increased significantly,and the expression of pyroptosis-related proteins increased.In vitro,in the CFs after LPS induction,the expression of GAS5 decreased significantly while the expression of DNMT1 and NLRP3 increased significantly.Conversely,high expression of GAS5 will cause reduced pyroptosis,while low expression of DNMT1 can lead to increased expression of GAS5 and decreased pyroptosis.Therefore,we can conclude that DNMT1 silenced LncRNA GAS5 can reduce the occurrence and development of pyroptosis by inhibiting NLRP3.