The Effect Of Wenzhongyiqi Decoction To Exosomal MiR-320a Mediated Myocardial Fibrosis Via Regulation Of PIK3CA In Chronic Heart Failure

Objective:Chronic heart failure（CHF）is a cardiovascular diseases with high diseases incidence and mortality.On the orther hand,myocardial fibrosis（MF）have an important influence on disease progression of CHF,it also was pathological basis of many cardiovascular diseases,such as coronary heart disease,hypertensive heart disease,dilated cardiomyopathy and so on.So,take some measures effectively to regulate and prevent progress of MF is becoming a new means for CHF treatment.MicroRNAs（miRNAs）have been reported to be involved in cardiovascular diseases by way of exosomes,it was impotent factor of myocardial fibrosis.CHF was defined as"palpitations","edema"or"dyspnea"in Chinese medicine.Wenzhong Yiqi decoction has good curative effect on CHF,it can improves the function of the spleen and stomach on the basis of traditional Chinese medicine treatment.This study in order to explore a new idea to control and cure CHF.We plan to find and verify the miRNA which was differentially expressed in CHF.secondly,discover the relationship between miRNA and exosomes.finally,the effect of miRNA on myocardial fibrosis and the potential treatment mechanism of Wenzhong Yiqi decoction will be investigated.Methods:Part Ⅰ:Find differentially expressed miRNAs in chronic heart failure by genetic database,and clear out it’s target gene information.Part Ⅱ:The differential genes were verified by RT-PCR,the expression of ST₂in CHF patients and healthy person’s serum will be compare in the meantime.Collected clinical and laboratory data to explore the association between miRNA,ST2 and NT-pro BNP,LEVF and NYHA classificationPart Ⅲ:Exosomes was extracted by kit.Identification of exosomes by weston blot,NTA and electron microscopy.RT-PCR was used to detected the level of miR-320a in exosomes and the remainder that after extraction of exosomes.Exosomes are co-cultured with cardiac fibroblasts,Observe cells uptake of exosomes,explored the effects of exosomes on cells.Part Ⅳ:miR-320a mimics and inhibitor transfected cardiac fibroblasts,prepared drug-containing serum of Wenzhong yiqi decoction.cell viability and apoptosis was detected by MTT and flow cytometry.ELISA,RT-PCR were used to assessed the level of ST₂and PIK3CA.Evaluated ofα-SMA,collagen I,collagen III and PIK3CA protein expression by Weston blot.investigated the effects of Wenzhong yiqi decoction on the expression levels of PIK3CA,α-SMA,type I,III collagen and ST2.Results:Part Ⅰ:Bioinformatics analysis showed that miR-320a is differentially up-regulated in chronic heart failure patients,PIK3CA maybe is potential target gene of it.Part Ⅱ:Expression of miR-320a and ST₂were significantly up-regulated in the serum of patients with chronic heart failure（p<0.05）/（p<0.01）.There was a positive correlation between miR-320a and ST₂,NT-pro BNP and NYHA cardiac function classification,and a significant negative correlation with LEVF in patients with chronic heart failure.Part Ⅲ:Weston blot showed positive expression of exosome surface markers such as CD63/CD9/TSG101.NTA diameter analysis showed that diameter size of the exosomes sample we extracted were accord to exosomes.Transmission electron microscopy successfully captured the specific structure of the exosomes.Expression of miR-320a in exosomes,HF group>NOR group（P<0.05）.miR-320a was mainly derived from exosomes（p<0.05）.Exosomes can be taken up by cardiac fibroblasts,and promote miR-320a up-regulation.Part Ⅳ:miR-320a can stimulate the proliferation of cardiac fibroblasts via PIK3CA,and increase the expression levels of ST₂、α-SMA,type I and III collagen（p<0.01）.Wenzhong Yiqi decoction not only can down-regulate the expression of miR-320a and PIK3CA in cardiac fibroblasts（p<0.05）,but also inhibits proliferation of cardiac fibroblasts and promotes early apoptosis of cells（p<0.01）,and decrease the levels ofα-SMA,type I,type III collagen,and ST2 in the meantime（p<0.01）.Conclusion（s）:1、miR-320A is highly expressed in serum and exosomes of patients with chronic heart failure.miR-320a of serum was mainly derived from eoxsomes.2、There was a positive correlation between miR-320a and ST₂,NT-pro BNP and NYHA cardiac function classification,and a significant negative correlation with LEVF in patients with chronic heart failure.3、Exosomes can be taken up by cardiac fibroblasts,and promote miR-320a up-regulation after enter the cell.4、miR-320a can Promote myocardial fibrosis via PIK3CA.5、Wenzhong Yiqi decoction can improve myocardial fibrosis by inhibits proliferation of cardiac fibroblasts and promotes early apoptosis of cells,and down-regulate the expression of miR-320a and PIK3CA,decrease accumulation of ECM.