| Myocardial fibrosis(MF)can increase myocardial stiffness,impair heart contraction and diastolic function,and seriously threaten the quality of life of patients.Actively carrying out research on the prevention and treatment of MF is of great significance to improve patient prognosis.miRNA is a non-coding single-stranded small RNA composed of about 20-22 nucleotides,which can play a regulatory role by degrading the target mRNA or inhibiting the post-transcriptional translation process.More and more studies have shown that miRNAs play a pivotal role in cardiovascular disease.Previous studies by the research group have shown that FZHY can reduce the area of myocardial infarction,improve cardiac function,and prevent and treat myocardial fibrosis,but its mechanism is still unclear.Therefore,in this study,rat models of acute myocardial infarction were prepared to observe the intervention effect of FZHY on the differential miRNAs in MF after myocardial infarction,with a view to revealing the mechanism of action of FZHY against myocardial fibrosis after MI from the perspective of miRNA.Objective:To reveal the changes of differential miRNAs expression profile in myocardial fibrosis after myocardial infarction.Using miRNA as the entry point,the possible mechanism of FZHY against myocardial fibrosis after myocardial infarction was explored.Methods:Rat models of acute myocardial infarction were prepared by ligation of anterior descending branch of left coronary artery,which were randomly divided into model group and Fuzheng Huayu Capsule Group(FZHY).Each group was then randomized to four weeks and eight weeks after surgery.In the sham operation group,only thread was inserted without ligation.Five in each group.The FZHY group was administrated with Fuzheng Huayu Capsule 0.4 g/(kg·d),and the sham operation group and model group were given iso-volume deionized water.Once a day for 4 weeks and 8 weeks respectively Histopathological changes of myocardium were detected by HE and Masson staining.The gene microarray was used to detect difference of miRNAs expression between the sham operation group and the model group at 4 weeks and 8 weeks,respectively.The TargetScan and microRNAorg databases jointly predicted target genes of differential miRNAs.GO and KEGG enrichment analysis of common target genes was performed by DAVID online tool.Quantitative Real-time PCR was used to verify the detection results of gene chip and the effect of FZHY on the expression of differential miRNAs.miRNAs that FZHY might target for regulation were screened and further predicted by DAVID online tool.Protein interaction network(PPI)analysis of proteins corresponding to target genes in the pathway was performed by STRING database and Cytoscape software.Results:(1)Compared with the sham operation group,the myocardial collagen volume fraction(CVF)increased(P<0.01),the myocardial tissue was destroyed,the myocardial cells were broken,collagen was largely aggregated,and fibrosis was more severe in the model group at 4 and 8 weeks after surgery.Compared with the model group,the CVF in the FZHY group was reduced(P<0.01),the myocardial tissues were arranged more neatly,the collagen was relatively reduced,and the degree of fibrosis was reduced(2)Compared with the sham operation group,the expressions of miR-13 2-3p,miR-140-3p,miR-140-5p,miR-155-5p,miR-199a-3p,miR-199a-5p,miR-21-5p,miR-214-3p,miR-221-3p,miR-222-3p,miR-31a-3p,miR-31a-5p,and miR-335 were up-regulated in the 4-week model rats after surgery(FC?2,P?0.05).A total of 2155 target genes were obtained from these 13 miRNAs.GO analysis showed that differential miRNAs can affect cellular components such as cytoplasm,endoplasmic reticulum,mitochondrion.Regulation of molecular functions such as protein binding,protein kinase binding,transcription factor binding.Involved in biological processes such as apoptotic process,aging,and cellular response to glucose starvation(P<0.01).KEGG analysis showed that differential miRNAs can target 63 related pathways including Insulin signaling pathway,Insulin resistance,MAPK signaling pathway,etc(P<0.05).(3)Compared with the sham operation group,9 miRNAs in the 8-week model rats after surgery were differentially expressed(FC?1.2,P?0.05).Among them,the expressions of miR-106b-5p and miR-10b-5p were up-regulated,and the expressions of miR-20b-5p,miR-30e-3p,miR-352,miR-378a-5p,miR-378b,miR-425-5p and miR-483-3p were down-regulated.A total of 990 target genes were obtained from these 9 miRNAs.GO analysis showed that differential miRNAs can affect cellular components such as cytoplasm,extracellular exosome,Golgi apparatus.Regulation of molecular functions such as protein binding,protein kinase binding,protein kinase activity.Involved in biological processes such as protein transport,aging,response to glucose,apoptotic processes(P<0.01).KEGG analysis showed that differential miRNAs can target 20 related pathways including TNF signal pathway,TGF-? signal pathway,MAPK signal pathway,etc(P<0.05).(4)The results of quantitative Real-time PCR showed that the expression trend of miR-140-3p,miR-140-5p,miR-199a-3p,miR-199a-5p,miR-21-5p,miR-214-3p,miR-221-3p,miR-222-3p,miR-31a-3p and miR-31a-5p in 4-week rats after surgery was consistent with the microarray results.In addition to miR-140-3p,the expressions of the other 9 miRNAs in the model group were significantly higher than those in the sham operation group(P<0.05 or P<0.01)(5)FZHY reduced the expression of miR-140-5p,miR-199a-3p,miR-199a-5p,miR-214-3p,miR-221-3p,miR-222-3p and miR-31a-3p in model rats 4 weeks after surgery,but the differences were not statistically significant(P>0.05).(6)The miR-199a family is most likely to be the miRNA directly targeted by FZHY.378 target genes of miR-199a-3p/5p were predicted.GO analysis showed that miR-199a can affect cellular components such as mitochondrial outer membrane,cytoplasm,Golgi apparatus.Regulation of molecular functions such as protein binding,protein kinase binding,and kinase activity.Involved in biological processes such as hypoxia,insulin,amino acid,fatty acid(P<0.01).KEGG analysis showed that miR-199a can target 10 related pathways including Insulin signaling pathway,Insulin resistance,PI3K-Akt signaling pathway,etc(P<0.05).Among them,the insulin signaling pathway is most closely related to miR-199a-3p/5p.PPI analysis showed that mTOR was the most important protein in the insulin signaling pathway as a central protein.Conclusion:(1)Different miRNAs were involved in different stages of myocardial fibrosis after myocardial infarction.Bioinformatics analysis showed that these differential miRNAs target multiple functions and signaling pathways,providing ideas for further exploration of the mechanism of myocardial fibrosis(2)Combining the effect of Fuzheng Huayu Capsules on the expression of differential miRNAs,it was speculated that the regulation of miR-199a3p/5p targeting the Insulin-PI3K/Akt-mTOR signaling pathway might be the mechanism of Fuzheng Huayu Capsules in improving MF after myocardial infarction at the gene level. |
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