| Background:Atrial fibrillation(AF)is a typically common,high-morbidity tachyarrhythmia.Atrial fibrillation is closely related to the occurrence and development of heart failure and is a high-risk factor for fatal diseases such as cardiogenic shock,stroke,and pulmonary embolism.Currently,atrial fibrillation’s onset and persistent causes are not fully understood.Myocardial fibrosis is one of the main pathological mechanisms of atrial fibrillation,and the inflammatory response is closely related to the formation of myocardial fibrosis.Epigenetics has been in an essential position in biology in recent years and includes three main mechanisms:methylation,histone modification,and including long and non-coding RNAs.DNA methylation is a chemical modification process where the cytosine-5 carbon position of genomic CpG dinucleotides binds to a methyl group by covalent bonding,which is used S-adenosine(SAM)as the methyl donor and under the catalysis of DNA methyltransferase(DNMT).LncRNA(long non-coding RNA)is a class of RNA molecules with a length of more than 200 nucleotides and has almost no ability to encode proteins but is involved in regulating various physiological and pathological processes.Studies have shown that a variety of lncRNAs can be modified by DNA methylation,among which lncRNA NEAT1(Nuclear Enriched Abundant Transcript1)is a long non-coding RNA transcribed by multiple endocrine tumor loci.This lncRNA is retained in the nucleus,participates in the core structure of the paracellular plaque,and can act as a transcriptional regulator of a variety of genes.Rgl is one of the 13 components of ginsenosides and has important roles in the cardiovascular system,such as anti-oxidative stress,anti-inflammatory,and anti-apoptosis.Objective:The study aims to clarify the specific expression of lncRNA NEAT1,DNMT3A,and NLRP3 inflammasome and their downstream genes in human myocardial fibrosis specimens and animal models and to explore whether lncRNA NEAT 1 and ginsenoside Rg1 can intervene cardiac fibrosis by inhibiting the NLRP3 inflammasome mediated cell pyroptosis.Furthermore,DNMT3 A and ginsenoside Rg1 may affect the expression of lncRNA NEAT 1,thereby producing a persistent effect in the continuous progression of myocardial fibrosis.Methods:Ninety SD(Sprague-Dawley)rats were selected for animal models of myocardial fibrosis and subcutaneously injected with ISO(Isoprenaline).The 90 male SD rats without specific pathogens(Specific pathogen Free,SPF)were randomly divided into control groups,ISO and ISO+Rg1,30 in each group.The ISO group was given abdominal subcutaneous ISO 5mg/kg/d,the control group received equivalent doses of saline,the ISO+Rg1 group was gavaged with Rg1 50mg/kg under ISO injection basis,once a day,and heart samples were obtained after 14 days.In addition,surgically obtained cardiac specimens from patients with atrial fibrillation and sinus rhythm were validated.Heart samples were washed with PBS(Phosphate buffered saline solution),fixed in general tissue fixed,and specimens were sliced for hematoxylin-eosin staining(HE staining),Sirius red staining,and Masson staining.Primary cardiac fibroblasts from neonatal SD mice were extracted using trypsin mixed with type II collagenase and cultured with medium containing 10%fetal calf serum at 37 ℃,5%CO2.Pyroptosis was induced in rat primary cardiac fibroblasts using LPS(Lipopolysaccharide)and transiently transfected with lncRNA NEAT 1,DNMT3 A small interfering RNA(siRNA),overexpressing DNMT3 A and lncRNA NEAT1 plasmids,and addition of different concentrations of ginsenoside Rg1.Immunofluorescence detected the expression of DNMT3A and NLRP3 in various groups of primary myocardial fibroblasts.In addition,apoptosis was detected by TUNEL staining and Hoechst staining,and the RNA expression levels of lncRNA NEAT1,DNMT3A,NLRP3,caspase1,IL-18,and type Ⅰ collagen(Collagen Ⅰ,COL1A1)were determined by quantitative reverse transcript polymerase chain reaction(qRT-PCR).The Western blot method detected the expression of DNMT3A,NLRP3,Cl-caspase1,Collagen Ⅰ,and IL-18.Results:Compared with patients with sinus rhythm,the expression of lncRNA NEAT1 was significantly reduced in the heart tissue from atrial fibrillation patients,but DNMT3A,NLRP3,Cl-caspase1,IL-18,and Collagen Ⅰ increased significantly.HE staining,Sirius Red staining,and Masson staining also indicate chaotic myocardial tissue and significantly collagen formation in atrial fibrillation patients.After ISO-induced myocardial fibrosis in SD rats.DNMT3A,NLRP3,Cl-caspase1,IL-18,and Collagen Ⅰ were expressed significantly higher than in control SD rats.HE staining,Sirius red staining,and Masson staining also showed chaotic myocardial tissue in the experimental group,showing extremely significant collagen fiber hyperplasia in the stroma.The expression of collagen fraction and DNMT3A,NLRP3,Cl-caspase1,IL-18,Collagen Ⅰ was decreased but lncRNANEAT1 increased in the ISO+Rg1 gavage group compared with the ISO group.Compared with commonly cultured primary cardiac fibroblasts and primary cardiac fibroblasts after LPS treatment,lncRNANEAT1 decreased,and DNMT3A,NLRP3,caspase1,IL-18,and Collagen I increased.In LPS-treated SD rat primary cardiac fibroblasts,compared with the empty plasmid group,the expression of NLRP3,Cl-caspase1,and IL-18 decreased significantly after transfection with NEAT1 overexpression plasmid.Compared with the negative control group,the expression levels of NLRP3,Cl-caspase1 and IL-18 were significantly increased after transfection with NEAT1 small interfering RNA.Compared with the empty plasmid group,the expression of NEAT1 decreased considerably after transfection with DNMT3A overexpression plasmid,while the expression of NLRP3 and Cl-caspasel increased significantly.Compared with the negative control group,the expression of NEAT 1 was significantly increased after transfection with DNMT3A small interfering RNA,and the expression of NLRP3,Cl-caspase1,and IL-18 was decreased considerably.In the group of 5-AzadC(5-nitrogen-2-deoxycytidine,methyltransferase inhibitor)and LPS into cardiac fibroblasts,NEAT1 expression was significantly increased compared with the LPS group.The expression of NEAT 1 was particularly in lower heart tissues in patients with atrial fibrillation than in sinus rhythm,and DNMT3A,NLRP3,Caspase1,IL-18,and Collagen Ⅰ was significantly higher than those with sinus rhythm.NEATl expression was observed considerably in lower cardiac tissue in SD rats in ISO models than in control SD rats,and DNMT3A,NLRP3,Cl-caspase1,IL-18,and Collagen Ⅰ was significantly higher than in standard control SD rats.The expression of NEAT 1 was markedly lower in primary cardiac fibroblasts treated with LPS than in the control cells,while DNMT3A,NLRP3,Cl-caspase1,and IL-18 were significantly higher than in the control group.Compared with LPS-stimulated primary cardiac fibroblasts,adding 10μM ginsenoside Rg1 to LPS-treated primary cardiac fibroblasts could promote the expression of NEAT1 and partially rescue LPS-induced high NLPR3,Cl-caspase1 and inhibit Collagen Ⅰ.Conclusion:In conclusion,this study suggests that DNMT3A methylation mediates lncRNA NEAT1 to regulate cell pyroptosis and thus affects the progression of myocardial fibrosis.In addition,gavage of ginsenoside Rg1 in the ISO rats can promote the expression of lncRNA NEAT 1,inhibit the NLRP3 inflammasome-mediated pyroptosis,reduce the degree of fibrosis,and offers new ideas for the regulatory mechanism of myocardial fibrosis.LncRNA NEAT1 can be used as an essential target to regulate the occurrence and development of myocardial fibrosis.Its epigenetic regulation mechanism in pyroptosis may be a potential treatment for myocardial fibrosis. |
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