| Backgrounds:Cardiac fibrosis is a common pathophysiological change in many cardiac remodeling-related diseases such as hypertension,coronary heart disease and cardiomyopathy.Its basic cellular biological mechanism is mainly excessive proliferation,migration and phenotypic transformation of cardiac fibroblasts(CFs),accompanied by excessive accumulation of extracellular matrix(ECM)proteins,which eventually leads to changes in cardiac structure.Its clinical manifestations are mainly decreased cardiac compliance and decreased diastolic and systolic functions.Due to the lack of obvious clinical symptoms and effective intervention and treatment methods in the early stage,it is of great significance to study the pathogenesis of cardiac fibrosis and find molecular markers for early diagnosis and potential targets for clinical treatment for improving cardiac diseases related to cardiac fibrosis,preventing sudden cardiac death and prognosis.The existing studies believe that the formation of cardiac fibrosis is closely related to the abnormal proliferation,migration and release of extracellular matrix of cardiac fibroblasts caused by the stimulation of various cell growth factors and inflammatory factors.Therefore,CFs are the direct effector cells of cardiac fibrosis.A large number of studies have found that transforming growth factor-(TGF-?1)is closely related to abnormal biological functions such as proliferation and migration of CFs.TGF-?1 can improve the phosphorylation level of SMADs protein and complete the activation of TGF-?1/SMADs signaling pathway,and finally complete its corresponding biological effects in nucleus.Because there are many factors involved in the occurrence and development of cardiac fibrosis,and its mechanism is relatively complex,the specific molecular mechanism of TGF-?1/SMADs signaling pathway in the occurrence and development of cardiac fibrosis,especially how to regulate downstream target genes,is not very clear.Epigenetics is one of the main regulatory methods in which eukaryotic cells play different biological functional phenotypes in the same genome.It involves reversible changes in chromatin conformation,DNA transcription,post-transcriptional processing and splicing to regulate gene expression.Especially in recent years,histone modification on nucleosome,which is closely related to reversible changes in chromatin conformation,has made important progress in regulating gene expression,and has put forward the hypothesis of "histone code" : whether a gene is expressed or not is related to histone modification on DNA sequence-chromatin remodeling.Experiments have proved that the chemical modification of lysine at the Nterminal of histone is an important mechanism for reversibility of chromatin conformation changes.Histones can regulate gene expression and silencing through histone acetylase(HAT),histone deacetylase(HDAC)and histone methyltransferases(HMT),which lead to the diversification of histone N-terminal modifications in the process of DNA sequence formation of nucleosomes,and make nucleosomes have tight and loose changes in the process of further chromatin formation,thus regulating gene expression and silencing.Therefore,epigenetic regulatory abnormalities lead to cell transformation of different phenotypic lineages under the same genome,and abnormal biological functions are closely related to theoccurrence of diseases.In recent years,histone modification has attracted much attention in the research field of pathogenesis of various diseases.Most of these studies focus on the occurrence of tumors and the outcome of drug therapy.Histone deacetylase inhibitors(HDACi),as a new type of anticancer drugs,have the functions of inhibiting cell proliferation,promoting cell differentiation or promoting cell apoptosis in tumor clinical research.However,in many heart disease models such as myocardial ischemia,studies have also confirmed that the deletion of class ? histone acetylase(such as Sir3)can lead to hypertrophy of myocardial cells and decrease the ability to remove accumulated extracellular matrix.In addition,HDACi histone deacetylation small molecule inhibitor can up-regulate the acetylation level of myocardial cells,inhibit the activity of angiotensin ?,and further reduce the degree of cardiac fibrosis.It is suggested that HDACi may play an important role in the pathophysiological process of cardiac fibrosis,which makes HDACi a new starting point for clinical research on the treatment of cardiac diseases such as pathological changes based on cardiac fibrosis and the prevention of sudden cardiac death.Basic on the previous animal experiments,we propose the hypothesis that sodium butyrate(NaBu),a small molecule histone deacetylase inhibitor(HDACi),can inhibit cardiac fibrosis by regulating the activation of TGF-?1/SMADs,a key signaling pathway in cardiac fibrosis through histone modification.In order to confirm our hypothesis,this study was designed to clarify the effect of sodium butyrate(NaBu)on molecular expression in TGF-?1/SMADs signaling pathway in cardiac fibroblasts,a direct effector cell in the occurrence and development of cardiac fibrosis,through histone modification apparent regulation mode,and its potential inhibitory effect and mechanism on cardiac fibrosis.In order to study and clarify themolecular mechanism of NaBu in the occurrence and development of cardiac fibrosis from the perspective of epigenetics,and to seek new therapeutic strategies and potential therapeutic targets.These research results may be helpful to develop new effective methods to antagonize cardiac fibrosis and lay an experimental and theoretical foundation.Methods:1.Angiotensin ? was used to establish rat cardiac fibrosis model.Forty-eight SD(Sprague Dawley,SD)rats were randomly divided into four groups: saline control group(sham operation group),angiotensin ? group,sodium butyrate group,angiotensin ? + sodium butyrate group.First of all,we established SD rat cardiac fibrosis model by subcutaneous implantation of microosmotic pump containing angiotensin ?(500ng/kg/min).Three days after the pump was implanted subcutaneously,blood pressure was detected by non-invasive eight-channel blood pressure detector in rat tail,and sodium butyrate(500mg/kg)was injected intraperitoneally daily.The modeling time is 28 days.2.On the 28 th day,SD rats in each group were examined by echocardiography under anesthesia,and the changes of cardiac fibrosis were observed in living animal models.3.On the 28 th day,6 of the 12 rats in each group were cut into abdominal cavity under anesthesia.After blood was taken from abdominal aorta,4% paraformaldehyde was perfusion and internal fixation was used.Heart tissue was taken out,4% paraformaldehyde was fixed,paraffin was embedded and sectioned.Cardiac fibrosis was detected by Masson staining and immunohistochemistry.The other 6 abdominal aorta puncture blood was taken after anesthesia,and the serum was separated after centrifugation and stored at-80 degrees for replacement.Heart tissue was taken out at low temperature for liquid nitrogen preservation,and tissue protein and RNAwere extracted.The expression of SMA,Collagen I,TGF-?1 and SMADs was detected by qPCR and Western blot.4.RAN-seq method was used to screen the differential gene expression of SD rats treated with sodium butyrate,without sodium butyrate and sham operation group induced by Ang ?,and qPCR was used to verify the differential gene expression of SD rats treated with sodium butyrate and sham operation group.5.Primary isolated and cultured neonatal rat cardiac fibroblasts were treated with Ang ? and NaBu for 48 hours respectively,and the cells were harvested and protein and RNA were extracted.The expression of related proteins and mRNA was detected in parallel with animal experiments.6.The proliferation and migration of CFs were detected by CCK8 method,cell counting method and cell scratch method,and the dose-effect concentration curves of Ang ? and NaBu on cells were established.7.SiRNA interference and luciferase reporter gene were used to detect the regulation of NaBu on gene expression and transcription of potential target proteins.8.Standard CO-IP,WB,ChIP and RT-PCR confirmed the effect of sodium butyrate on transcription and binding of potential target protein genes.Results:1.Sodium butyrate can significantly improve Ang ?-induced cardiac fibrosis in rats.Echocardiographic results showed that sodium butyrate could reduce the increase of blood pressure induced by Ang ? in SD rats,increase the left ventricular diameter(LVIDd)after diastole of Ang ?,reduce the thickness of left ventricular posterior wall(LVPWd)after diastole,and also have certain effects on ejection fraction(EF)and shortening fraction(FS).Masson staining showed that the area of cardiac fibrosis in NaBu + Ang ? group was significantly lower than that in Ang ? group,and the area of SMA and Collagen I positive staining in NaBu + Ang ? group was also significantly lower than that in Ang ? group.The above results suggest that NaBu can significantly antagonize Ang ?-induced cardiac fibrosis in SD rats.2.In vitro experiments show that NaBu's antagonism to Ang ? and inhibition of cardiac fibrosis are closely related to negative regulation of SMAD3.It is found that NaBu's down-regulation of SMAD3 does not depend on TGF-?1,but is not related to pSMAD2.Western blot and qPCR were used to detect the protein expression level of TGF-?1/SMADs signaling pathway,which is crucial in the occurrence and development of cardiac fibrosis.The results showed that there was no significant difference in TGF-?1 expression between Ang ? group and NaBu + Ang ? group(p > 0.05).Furthermore,the expression of SMAD2 and SMAD3 was not significantly different between SMAD2 and pSMAD2 groups(p > 0.05),but the histone expression of SMAD3 and pSMAD3 was significantly decreased in NaBu + Ang ?(p < 0.01),suggesting that NaBu antagonized Ang ? to improve cardiac fibrosis in SD rats,inhibit the protein expression of SMAD3 and reduce the phosphorylation level of pSMAD3,which is not dependent on TGF-?1regulation and is not related to pSMAD2.3.The antagonistic effect of NaBu on Ang ? on cardiac fibrosis in rats is closely related to the up-regulation of histone methyltransferase SETD4.The results of RNA-seq screening showed that there were 401 mRNA differentially expressed between Ang ? group and NaBu + Ang ? group,among which 208 mRNA were up-regulated and 121 mRNA were downregulated.Further GO enrichment and KEGG enrichment analysis werecarried out for the 401 differentially expressed mRNAs.Ten mRNAs with the most significant enrichment(p-value level,P < 0.001)were selected from BP,CC and MF respectively for GO enrichment analysis.The results suggest that the GO enrichment function includes R-SMAD binding,cell adhesion molecule binding,cell adhesion molecule binding,cell adhesion molecule binding,cell adhesion molecule binding,cell adhesion molecule binding,and other differentially expressed mRNAs.KEGG enrichment analysis revealed that there were three signaling pathways related to cell migration and proliferation,including regulation of cytoskeleton actin,Apelin signaling pathway,and cardiomyocyte adrenergic signaling in cardiomyocytes.Futhermore,the results showed that the expression of Acta2,YY1,SMAD3 and SETD4 in NaBu + Ang ? group had the strongest correlation with the expression of SETD4.We analyzed the GO enrichment function of 21 mRNA with the most significant differences,such as Acta2,YY1,SMAD3 and SETD4,through online database,and found that there was a certain interaction between these 21 differentially expressed mRNA.According to the results of GO enrichment function analysis,four up-regulated mRNA related to epigenetic modification and cell proliferation(Asmt,Setd4,Yy1)and four down-regulated mRNA related to fibrosis(Smad3,Acta2,TGF-?2)were selected.Quantitative PCR was used to further analyze their expression in heart tissues of Ang ? group and Ang ? + NaBu group.These genes have been proved to have significant expression changes after NaBu treatment.The results of quantitative PCR were consistent with those of RNA-seq,and the mRNA of histone methyltransferase 4(SETD4)was significantly higher than that of Ang ? and sham operation group(p < 0.001).The quantitative PCR method was used to further detect the expression of SETD4 in cardiac tissue,and the quantitative results were consistent with those of RNA-seq,which enhanced the credibility of RNA-seq results.4.In vivo and in vitro experiments confirmed that NaBu up-regulates SETD4 and negatively regulates SMAD3 to inhibit Ang ?-induced rat cardiac fibrosis.In vitro CFs were used to verify the significantly differentially expressed mRNA screened by RNA-seq in SD rat heart tissue.The results showed that the expression level of SETD4 protein and mRNA in NaBu treated group was significantly higher than that in Ang ? group.Furthermore,the expression levels of SMAD3,pSMAD3,SMA and Collagen I genes were detected in CFs by immunofluorescence and Western blot.The results showed that the expression level of SETD4 was negatively correlated with SMAD3,pSMAD3,SMA and Collagen I.5.NaBu significantly inhibited the proliferation and migration of CFs induced by Ang ? by up-regulating SETD4.The levels of SETD4 protein and mRNA in Ang ? treatment group transfected si-SETD4 with NaBu intervention were lower than those in Ang ? control group transfected si-NC(p < 0.001).The results of EdU and scratch tests showed that the ability of NaBu to antagonize Ang? to inhibit CFs proliferation and migration decreased significantly in the Ang ? treatment group transfected with si-SETD4 compared with the Ang ? control group transfected with si-NC(p < 0.001).The above results further prove that NaBu negatively regulates SMAD3 to antagonize Ang ? and is closely related to SETD4.It is further suggested that NaBu antagonizes the negative regulation of CFs proliferation and migration by Ang ?,and the epigenetic participation of histone methylation modification is involved in improving cardiac fibrosis.6.NaBu upregulates SETD4 and inhibits SMAD3 expression at epigenetic level by binding to YY1 and H3K9me3.RNA FISH results showed that SETD4 was abundant in the nucleus and cytoplasm of CFs.According to the biological function analysis of online SETD and literature reports,WB was used to detect histone modification sites that SETD4 may act on in CFs treated with NaBu and Ang ?,including H3K9 me,H3K4me3,H3K27me3 and H3K36me3.The results suggest that NaBu up-regulates SETD4 negative regulation of SMAD3 and may inhibit SMAD3 transcription activity through H3K9me3.According to our GO enrichment analysis and UCSC on-line analysis of differentially expressed mRNA,we found that YY1 binding site exists in SMAD3 promoter region.YY1 is a trans-acting factor with chromatin remodeling enzyme activity.After interfering with SETD4 and YY1,the transcription activity in SMAD3 promoter region is significantly higher and SMAD3 expression is significantly increased.CHIP experiment demonstrated that the promoter region of SMAD3 has YY1 binding and abundant H3K9me3 signal,and SETD4 and YY1 can interact with each other in the promoter region of SMAD3.The above results show that NaBu up-regulates the expression of SMAD3 through H3K9me3 by binding YY1 to SETD4.Luciferase reporter gene experiment proved that the activity of SMAD3 promoter increased after interfering with SETD4 and YY1.WB test showed that SETD4 did not affect the expression of YY1.CO-IP assay demonstrated that SETD4 could bind to YY1 protein.ChIP assay demonstrated that interfering with SETD4 decreased the binding of YY1 protein and the level of H3K9me3 in SMAD3 promoter region.The above results show that SETD4 up-regulates histone methylation level in SMAD3 promoter region by binding with YY1,which makes chromatin structure in SMAD3 promoter region compact and eventually leads to transcription shutdown of SMAD3,thus realizing gene expression regulation atepigenetic level.Conclusion:To sum up,this study further explored the function and mechanism of NaBu up-regulating SETD4 and negatively regulating SMAD3 on the basis of Ang ?-induced rat cardiac fibrosis model in vivo and in vitro.It was found that NaBu significantly up-regulated the expression of Ang ?induced cardiac fibrosis in SD rats and antagonized the cardiac fibrosis induced by Ang ?.In vitro and in vivo functional experiments confirmed that SETD4 can bind YY1 to inhibit transcription activity of SMAD3 promoter region via H3K9me3.NaBu inhibits cardiac fibrosis by upregulating SETD4 and negatively regulating SMAD3 expression.SETD4 increases histone methylation in SMAD3 promoter region by binding to YY1 in nucleus,and inhibits transcription of SMAD3 gene by changing its chromatin structure.Our research proves that NaBu up-regulates the biological function of SETD4 in inhibiting cardiac fibrosis.Moreover,the apparent regulatory molecular mechanism of NaBu negative regulation of SMAD3 gene expression by SETD is clarified for the first time,which provides new evidence for understanding the biological function and mechanism of histone methylation modification in cardiac fibrosis,and also provides a potential molecular marker for the treatment and prognosis of cardiac fibrosis. |
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