| Cardiac repair and fibrosis after myocardial infarction are the key factors affecting the prognosis of myocardial infarction.The formation of collagen fiber in early myocardial infarction can prevent heart rupture,but excessive deposition of collagen can lead to myocardial interstitial fibrosis,affect myocardial contractile and diastolic function,and eventually lead to heart failure and even death.Therefore,searching for the regulatory mechanism of myocardial collagen deposition after infarction has become one of the hot topics.Recent researches found that the gut microbiota,as an endocrine organ,may be involved in the alteration of the myocardial structure and function in the process of cardiovascular disease.Gut microbiota may be one of the important driving factors for myocardial infarction.Destruction of gut microbiota structure could lead to poor prognosis in myocardial infarction.However,the communication mechanism that connects gut microbiota and collagen fiber formation after myocardial infarction has not been clarified.Gut microbiota transmit with to distant organs of the host through various biochemical signals and metabolites.Short chain fatty acids(such as butyric acid,propionic acid and acetic acid)are the main functional metabolites of gut microbiota via decomposing dietary fibers,which participate in certain biological function in the host.Butyric acid could affect body function by binding with receptors or inhibiting the activity of histone deacetylases(HDACs).Our previous studies found that histone deacetylase inhibitors can reduce infarction area in rats with myocardial infarction.We speculate that gut microbiota may inhibit HDACs activity through metabolites such as butyric acid and other metabolites,and affect collagen expression after myocardial infarction,which plays a key role in the process of myocardial infarction.Experimental objective:The current study is designed to investigate the effect of gut microbiota on collagen expression in myocardial infarction rats.Subsequently,the effect of butyric acid,a metabolite of gut microbiota,on the expression of collagen in rats with myocardial infarction will be clarified.Moreover,the target of butyric acid via HDACs mechanisms will be screened out and verified.Experimental Methods:1.To clarify the effect of gut microbiota on collagen expression after myocardial infarction.A myocardial infarction rat model was established by ligating the left anterior descending branch of coronary artery.The gut microbiota was inhibited by antibiotic cocktail(ABX).Fecal samples of rats were collected on the 7th,14 th,21st and 28 th days after myocardial infarction.The diversity,composition and abundance of gut microbiota were detected by 16 S r RNA sequencing.The injury of myocardial tissue after myocardial infarction was evaluated by HE staining analysis.The expression of collagen was detected by Sirius red staining.The expression of collagen related proteins was analyzed by Western blot and immunohistochemistry.2.To investigate the effect of gut microbiota generated butyric acid on collagen expression after myocardial infarction.The content of short chain fatty acids(butyric acid,propionic acid and acetic acid)in serum and myocardial tissue of rats was determined by GC-MS,and the relationship between butyric acid and spindle was determined.The injury of myocardial tissue after myocardial infarction was evaluated by HE staining.collagen expression was detected by Sirius red staining.The protein expression of collagen related protein was analyzed by Western blot and immunohistochemistry.3.To explore the target and mechanism of gut microbiota generated butyric acid involved in regulating collagen deposition after myocardial infarction based on HDACs.The dynamic changes of HDACs activity in serum and myocardial tissue of rats were detected by HDACs active kit.The effect of butyric acid concentration on HDACs activity was determined by inhibiting butyric acid producing bacteria and other gut microbiota;meanwhile,TSA was administrated as a positive control drug to determine the inhibitory effect of butyric acid by inhibiting HDACs activity.Transcriptomics was used to screen the target genes and pathways regulated by butyric acid,a metabolite of gut microbiota,through HDACs.Differential genes were verified in rat myocardial infarction model and NIH3T3 cell oxygen-glucose deprivation model.Experimental results1.Gut microbiota affects the expression of collagen after myocardial infarction The gut microbiota structure was significantly changed on the 7th,14 th,21st and 28 th day after myocardial infarction in rats.The results showed that the ratio of the bacteria group to the Firmicutes/Bacteroides(F/B)of the gut microbiota increased significantly(p < 0.05)on the 14 th day after myocardial infarction.The dynamic changes of the gut microbiota at the phylum level showed that the abundance of Bacteroidia,Peptostreptococcaceae and Turicibacteraceae increased significantly after myocardial infarction(p <0.05)The abundance of the Alcaligenaceae and Prevotellaceae was significantly reduced(p <0.05).The results of LEf Se analysis showed that the abundance of Peptostreptococcaceae,Alcaligenaceae and Prevotellaceae was significantly reduced on the14 th day after myocardial infarction,while the Proteobacteria,Erysipelotrichaceae and Streptococcaceae changed on the 21 st day;the 14 th and 21 st days were the key time points for the change of gut microbiota.In the altered gut microbiota,Prevotellaceae and Lachnostpiraceae were indirectly involved in the production of butyric acid.The HE,Sirius red staining,Western blot and immunohistochemistry analysis results showed that myocardial tissue injury was gradually aggravated,collagen deposition increased,granulation tissue and scar tissue gradually formed,and cardiac function decreased from 0 to 28 days after infarction.After inhibition of gut microbiota,increased expression levels of Collagen III,Collagen I and alpha-SMA were observed,meanwhile,collagen deposition,granulation tissue and scar formation increased,which further reduced cardiac function at 14,21 and 28 days after myocardial infarction.The most significant time point was 14 and 21 days after myocardial infarction.It is suggested that inhibition of gut microbiota including butyric acid producing bacteria could increase collagen expression after myocardial infarction,and increase granulation tissue and scar tissue formation.2.Gut microbiota generated butyric acid affect collagen expression via the gutheart axis after myocardial infarction The levels of butyric acid and propionic acid in serum and myocardial tissue were significantly decreased on the 14 th and 21 st days after myocardial infarction in rats.After inhibiting gut microbiota including butyric acid producing bacteria,the concentration of butyric acid in serum and myocardial tissue was further reduced(p <0.05).While,the content of butyric acid in serum and myocardial tissue samples was significantly increased when rats were administrated with sodium butyrate in drinking water before the operation(p <0.05).The results showed that butyric acid produced by gut microbiota was related to serum and myocardial content of butyric acid in myocardial infarction rats.It suggested that gut microbiota generated butyric acid mediated the gut-heart axis.Based on HE,Sirius red staining,Western blot and immunohistochemistry analysis,it is showed that sodium butyrate could decrease the expression of Collagen III,Collagen I and alpha SMA,inflammatory cell infiltration,collagen deposition and granulation tissue and scar tissue formation in myocardial infarction rats.When gut microbiota was inhibited and sodium butyrate was supplied,the expression levels of Collagen III,Collagen I and alpha SMA were significantly increased,the formation of granulation and scar tissue reduced in myocardial infarction rats.Therefore,gut microbiota generated butyric acid influenced collagen expression after myocardial infarction by mediating the gut-heart axis.3.The mechanism of gut microbiota generated butyric acid on the inhibition of HDACs activity and collagen expression The activity of HDACs in serum and myocardial tissue increased on the 14 th and 21 st days after myocardial infarction in rats.After inhibition of gut microbiota,the activity of HDACs in serum and myocardial tissue increased further(p <0.05).When inhibition of gut microbiota and butyric acid supplementation,the activity of class I HDACs significantly decreased.The HE,Sirius red staining,Western blot and immunohistochemistry analysis results showed that,sodium butyrate and histone deacetylase inhibitor TSA showed the similar regulatory effects on regulating the expression of Collagen III,Collagen I and alpha SMA,collagen deposition,granulation tissue and scar tissue formation,and consequently improve cardiac function.It indicated that gut microbiota generated butyric acid may improve cardiac function by inhibiting HDACs activity.Moreover,the results of gut microbiota generated butyric acid analysis showed that the differential expression genes affected by sodium butyrate after myocardial infarction were mainly includes col3a1,c3ar1,cola2 and tgfb3.Sodium butyrate related differential expression genes were critically enrichment in ECM receptor interaction,dilated cardiomyopathy,hypertrophic cardiomyopathy signal pathway,complement syste which were all closely related to collagen expression.It is suggested that butyric acid could inhibit HDACs activity,and subsequently regulating collagen expression after myocardial infarction.Moreover,it is validated that C3ar1 and Tgf beta were the significantly differentially expressed that increased in myocardial after myocardial infarction(p <0.001).In myocardial infarction rats,the protein expression of C3AR1 and TGF beta was significantly decreased by administration of sodium butyrate or TSA when gut microbiota was inhibited(p <0.001).The current results revealed that butyrate or TSA might inhibit the expression of collagen in OGD condition induced NIH3T3 fibroblasts by inhibiting the expression of C3AR1.Experimental conclusion:1.The gut microbiota was involved in the process of myocardial repair,and the abundance of butyrate-producing bacteria decreased significantly after myocardial infarction.2.Gut microbiota generated butyric acid could affect collagen expression and cardiac function after myocardial infarction by mediating the gut-heart axis.3.Butyric acid,a metabolite of gut microbiota could inhibit the expression of collagen after myocardial infarction and improve cardiac function by regulating HDACs activity.4.Gut microbiota generated butyric acid could regulate the expression of C3AR1 and inhibit the secretion of collagen in NIH3T3 based on the inhibitory effect of HDACs. |
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