| OBJECTIVEClinical screening of miRNAs differentially expressed in serum exosomes of patients with chronic heart failure(CHF)combined with hyperuricemia(HUA)of interior accumulation of dampness and turbidity,evaluating their value as potential novel molecular markers for precise diagnosis of CHF combined with HUA of interior accumulation of dampness and turbidity,and further analyzing their potential therapeutic targets.Moreover,observe the effects of Huashi Jiangzhuo Formula on differentially expressed miRNAs and related signaling pathways obtained through clinical screening and explore the intervention effect of Huashi Jiangzhuo Formula,which has achieved sound therapeutic effects in the early clinical application,on myocardial cell autophagy in CHF combined with HUA model rats.METHODS1.Clinical study:This controlled study included 30 patients with CHF combined with HUA of interior accumulation of dampness and turbidity admitted to the Cardiovascular Department of Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine were selected as the observation group(group T),and 30 healthy volunteers were selected as the control group(group C)during the same period.6 samples were selected from each group,and highthroughput sequencing was used to analyze the differential expression of exosomal miRNAs in the serum of the two groups.Then,RT-PCR was used to verify the high-throughput sequencing results on the remaining 24 samples from group T and 24 from group C.R software was used to perform GO and KEGG enrichment analysis on the different expression of exosomal miRNAs.2.Experimental research:50 SD male rats were randomly selected,of which 8 were selected as the blank control group(Control),and the remaining 42 rats were subjected to CHF combined with HUA modeling.Firstly,CHF model rats were prepared by intraperitoneal injection of doxorubicin(2.5mg·kg-1)once a week for 6 consecutive weeks.Then,the CHF model rats were administered by gavage of ethambutol(250mg·kg-1)combined with subcutaneous injection of potassium oxonate(200mg·kg-1)once a day for 6 consecutive weeks to prepare the CHF combined with HUA model rats.To observe the effects of Huashi Jiangzhuo Formula on serum uric acid(SUA),cardiac function,blood expression of miR-27a-5p and miR-139-3p,and myocardial pathological structure in CHF combined with HUA model rats,and further observe the impact of Huashi Jiangzhuo Formula on cardiomyocyte autophagy and adenosine monophosphate-activated protein kinase(AMPK)-mammalian target of rapamycin(mTOR)signaling pathway in CHF combined with HUA model rats.42 SD rats were subjected to CHF combined with HUA modeling,and 6 died during the CHF modeling process,the remaining 36 rats successfully prepared CHF combined with the HUA pathological model.They were randomly divided into 3 groups:model group(Model),Huashi Jiangzhuo Formula group(HSJZF),and Huashi Jiangzhuo Formula+Acadesine(AMPK agonist)group(HSJZF+A),with 12 rats in each group.HSJZF administered a suspension of Huashi Jiangzhuo Formula(10g·kg-1·d-1)by gavage,followed by intramuscular injection of an equal amount of physiological saline.HSJZF+A was given a suspension of Huashi Jiangzhuo Formula(10g·kg-1·d-1)by gavage,followed by intramuscular injection of Acadesine(50mg·kg-1·d-1).The Control and Model rats were given equal amounts of distilled water by gavage,followed by intramuscular injection of an equal amount of physiological saline,with a volume of 10mL·kg-1.The rats in each group were weighed once a week,and then the dosage of drugs and distilled water was adjusted until the material was taken.After the successful preparation of the CHF combined with the HUA pathological model,the general mental state,water consumption,food intake,haire condition,and activity of each group of experimental rats were recorded daily.One hour after the last administration,each experimental rat’s left ventricular ejection fraction(LVEF)was measured by echocardiography and blood samples were collected from the carotid artery.After anesthesia,the rats were euthanized,and their blood B-type natriuretic peptide(BNP),SUA,blood urea nitrogen(BUN),and serum creatinine(SCr)were measured by ELISA.RT-PCR measured the relative expression levels of blood miR-27a-5p and miR-139-3p.Myocardial tissues were taken from rats,and the expression of Beclin 1,LC3-II,p62,and AMPK-mTOR proteins by Western Bolt method in one section,while HE staining,Masson staining,and TUNEL staining were used in the other section for light microscopy examination.We also observed the mitochondrial structure of cardiomyocytes by using transmission electron microscopy.RESULTS1.Clinical study:(1)A total of 42 differentially expressed miRNAs were detected in group T by high-throughput sequencing;among them,miR-27a-5p was significantly upregulated(p=0.000179),and miR-139-3p was significantly downregulated(p=0.000058).(2)RT-PCR validated the differential expression of miRNAs,and the results showed that the expression of miR-27a-5p was significantly upregulated(p=0.004)and miR-139-3p was significantly downregulated(p=0.005)in the serum exosomes of CHF patients combined with HUA of interior accumulation of dampness and turbidity.The area under the ROC curve(AUC)was used further to predict the diagnostic value of differential expression of miRNAs.It revealed that the expression of miR-27a-5p and miR-139-3p predicted the AUC of CHF combined with HUA of interior accumulation of dampness and turbidity,which were 0.708(95%CI:0.562-0.855)and 0.734(95%CI:0.593-0.876),respectively.The maximum AUC predicted by the combination of miR-27a-5p and miR-139-3p for CHF and HUA of interior accumulation of dampness and turbidity was 0.899(95%CI:0.812-0.987).(3)GO enrichment analysis identified autophagy is the most highly enriched target,and KEGG enrichment analysis revealed that activating the AMPK-mTOR signaling pathway may be one of the differentially expressed targets of miR-27a-5p and miR-1393p.2.Experimental research:(1)The successful preparation of the CHF combined with HUA model was verified by measuring LVEF through cardiac ultrasound and measuring SUA and BNP through orbital blood sampling.(2)General life condition observation:Compared with Model,both HSJZF+A and HSJZF could significantly improve the general life condition of CHF combined with HUA model rats,but the improvement effect of HSJZF was significantly better than that of HSJZF+A.(3)Comparison of SUA:Compared with Model,both HSJZF+A and HSJZF could significantly reduce SUA levels in CHF combined with HUA model rats,and the effect of HSJZF was better than HSJZF+A.(4)Comparison of BNP and LVEF:Compared with Model,HSJZF+A and HSJZF could significantly reduce BNP and increase LVEF levels in CHF combined with HUA model rats,and HSJZF has a significantly better effect than HSJZF+A.(5)Comparison of renal function:HSJZF+A and HSJZF could significantly improve the renal function in CHF combined with HUA model rats when compared with Model,and HSJZF has a more significant effect.(6)Pathological and ultrastructural changes in myocardial tissue:Compared with Model,the myocardial tissue structure and pathological changes in HSJZF and HSJZF+A rats were significantly improved.The collagen fiber content,myocardial cell apoptosis,and autophagy degree in myocardial tissue were reduced,and the improvement effect of HSJZF was significantly better than that of HSJZF+A.(7)Comparison of relative expression levels of blood miR-27a-5p and miR-139-3p:Compared with Model and HSJZF+A,HSJZF can significantly reduce the expression level of blood miR-27a-5p and significantly increase the expression level of miR-1393p in CHF combined with HUA model rats.(8)Expression of myocardial autophagy-related proteins Beclin 1,LC3-Ⅱ,and p62:Compared with Model,HSJZF+A and HSJZF could significantly reduce the expression of Beclin 1 and LC3-Ⅱ,and increase the expression of p62 in the myocardium of CHF combined with HUA model rats,and the improvement effect of HSJZF was significantly better than that of HSJZF+A.(9)Expression of myocardial AMPK-mTOR:Compared with Model,both HSJZF+A and HSJZF could significantly reduce the expression of AMPK and increase the expression of mTOR in the myocardium of CHF combined with HUA model rats,and the effect of HSJZF was significantly better than that of HSJZF+A,indicating that AMPK agonists partially reverse the intervention effect of Huashi Jiangzhuo Formula.CONCLUSIONThe upregulation of miR-27a-5p and downregulation of miR-139-3p in serum exosomes may have significant value in accurately diagnosing novel molecular markers in patients with CHF combined with HUA of interior accumulation of dampness and turbidity.Activating the AMPK mTOR signaling pathway to promote autophagy response in cardiomyocytes may be one of the targets of differentially expressed miR-27a-5p and miR-13 9-3p.Huashi Jiangzhuo Formula has an intervention effect on the expression of miR-27a-5p and miR-139-3p in the blood of CHF combined with HUA pathological model rats,thereby inhibiting the myocardial AMPK-mTOR signaling pathway and exerting an inhibitory effect on the autophagy response of myocardial cells."Huashi Jiangzhuo Fang," which is the clinical protocol party,has a targeted intervention effect on the expression of miR-27a-5p and miR-139-3p in the blood of CHF combined with HUA pathological model rats,thereby inhibiting the myocardial AMPK mTOR signaling pathway and exerting an inhibitory effect on the autophagy response of cardiomyocytes. |
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