| Objective:1.To study the differences in miRNA expression in the serum of patients with phlegm-turbidity syndrome of unstable angina pectoris in coronary heart disease and healthy controls by high-throughput sequencing technology,to screen for miRNAs abnormally expressed in patients with unstable angina pectoris,and to explore the main target and mechanism of action of the warm bile broth in the fight against angina pectoris according to GO and KEGG enrichment analysis,in order to explore the biomarkers that are effective in predicting the development trend of coronary heart disease and traditional Chinese medicine(TCM)syndromes in the early stage of development.The biomarkers were analyzed according to GO and KEGG enrichment.2.To observe the effects of the standard intensive treatment and the combination of Wen-Dan Decoction on the symptoms of chest paralysis,the points of TCM syndromes and efficacy,ECG,CTnI,blood lipids,as well as the effects on the expression of miR-320c and COL4A1 in the patients with unstable angina pectoris in coronary atherosclerotic heart disease before and after the treatment.Preliminarily,to investigate the clinical effects of Wen-Gui-Tang in treating patients with phlegmatic syndrome of unstable angina pectoris of coronary heart disease and the effect of intervening miRNA expression.3.Through animal experiments,we investigated the effects of Wen-Dan Decoction on the expression of miR-320c and COL4A1 in different tissues of coronary heart disease model animals,and cell experiments verified the effects of abnormal expression of miR-320c on the proliferation of endothelial cells,cell migration,cell apoptosis,and COL4A1,so as to provide certain experimental bases for the promotion of the application of Wen-Dan Decoction in the clinical treatment of coronary heart disease and its therapeutic mechanism.Methods:1.5 patients with coronary heart disease unstable angina(phlegm-turbid evidence)were recruited,and on the basis of western medical treatment combined with the traditional Chinese medicine Wen-Dan Decoction was taken orally for 28 days as a course of treatment,and high throughput sequencing technology was applied to sequence microRNAs in peripheral serum samples before and after the treatment,and compared with 5 healthy volunteers,to preliminarily evaluate the sequencing characteristics of microRNAs in the patients with coronary heart disease unstable angina of phlegm-turbid evidence,and the effects of Wen-Dan Decoction on the microRNAs in these patients.and the effect of Wen-Dan Decoction on microRNA in this group of patients.The target genes of differentially expressed miRNAs were predicted using miRanda and TargetScan,and the intersection of the prediction results of these two software programs was taken to obtain the final miRNA target genes.From the perspective of biological pathways,GO and KEGG enrichment analysis was carried out with all differentially expressed miRNA target genes.2.Using a randomized,controlled,standard treatment-loaded clinical research method,80 patients with coronary artery disease unstable angina(phlegm-turbid evidence)were randomly divided into 2 groups,40 cases in each group.The control group was treated with standard intensive treatment of western medicine,and the Wen-Dan Decoction group was combined with oral administration of traditional Chinese medicine Wen-Dan Decoction on the basis of western medicine treatment,with 40 cases in each group and 28 days as a course of treatment.Observe the patients in the 2 groups:(1)Changes in TCM evidence points,reduction and stopping rate of nitroglycerin-containing tablets,blood cTnI.electrocardiogram,and blood lipids(TC,LDL-C.and TG)before treatment and on the 28th day after treatment were observed to assess their clinical efficacy.(2)The effects of blood.urine and feces routine,liver and kidney functions of patients before treatment and on the 28th day after treatment,and observe the safety of drugs.(3)The changes of serum miR-320c and COL4A1 mRNA expression were detected by qRT-PCR before and on the 28th day after treatment,and serum COL4A1 and MMP2 levels were detected by ELISA.3.30 SD rats were randomly divided into 3 groups,10 in the blank group were fed with basic feed,10 in the model group and 10 in the Wen-Dan Decoction group were fed with high-fat feed,and were given intraperitoneal injection of posterior pituitary hormone to establish the animal model of coronary artery atherosclerosis.After successful modeling,rats in the Wen-Dan Decoction group were gavaged daily with Wen-Dan Decoction,and the remaining two groups were gavaged with equal doses of saline for 28 consecutive days.At the end of the experiment,peripheral blood and heart and aortic arch tissues were collected from rats,and heart tissues were stained with HE.Masson stain and TUNEL stain.qRT PCR was used to detect the levels of miR-320c and COL4A1 mRNA in serum and myocardial tissues of rats,and plasma and cardiac tissues were tested for the content of COL4A1 and MMP2 by enzyme-linked immunosorbent assay(ELISA).4.Fluoresceinase reporter gene assay verified whether COL4A1 was a direct target gene of miR-320c.5.In cellular experiments,human umbilical vein endothelial cells HUVECs were selected and modeled with OX-LDL infiltration and then transfected with miR-320c mimics NC,miR-320c mimics,miR-320c inhibitor NC,miR-320c inhibitor,siRNA-NC,siRNA-COL4A1 transfection,observed the changes of cell proliferation,cell migration and apoptosis,qRT-PCR detected the expression of miR-320c and COL4A1 mRNA,and Western blot verified the expression of COL4A1 and MMP2.Results:1.5 miRNAs were down-regulated and 4 miRNAs were up-regulated in the serum of patients with phlegm-turbid syndrome of unstable angina pectoris compared with healthy controls.After treatment with Wen-Dan Decoction,9 miRNAs were up-regulated and 19 miRNAs were down-regulated in the serum of patients with phlegm-turbidity syndrome of unstable angina pectoris compared with those before treatment.Among them,hsa-miR-320c was significantly up-regulated in the serum of patients with phlegm-turbid syndrome of unstable angina compared with healthy controls,whereas hsa-miR-320c was significantly down-regulated after the treatment of Wen-Dan Decoction.By GO enrichment,the most significant biological functions enriched for differentially expressed miRNA target genes were macromolecular synthesis and denaturation processes,regulation of base compound metabolism,establishment of localization,and transcription;the most significant cellular components enriched were endosomes,nuclei,and organellar membranes;and the most significant molecular functions enriched were cation-binding,metal ion-binding,anion-binding,and DNA-binding.By KEGG enrichment,the most significant enrichment of differentially expressed miRNA target genes in patients with phlegmatic evidence of unstable angina was in the regulation of actin cytoskeleton,axon guidance,cGMP-PKG signaling pathway,autophagy,AMPK signaling pathway,synthesis,secretion,and action of growth hormone,thyroid hormone signaling pathway,cholinergic synapse,and cancer,AGE-RAGE signaling pathway in diabetic complications,longevity regulation pathway,circadian rhythm related pathway.The main pathways affected before and after treatment with warm bile broth were cancer,endocrine,calcium signaling pathway,Rapl signaling pathway,axon guidance,Hippo signaling pathway,mTOR signaling pathway,autophagy,dopaminergic synapses,sphingolipid signaling pathway,AMPK signaling pathway,cholinergic synapses,insulin resistance,glycerophospholipid metabolism,AGE-RAGE signaling pathway in diabetic complications,phospholipid regulation pathway,circadian rhythm-related pathways.RAGE signaling pathway,phosphatidylinositol signaling system,longevity regulation pathway,pancreatic cancer,inositol phosphate metabolic pathway,etc.2.Clinical research:Wen-Dan Decoction group is better than the control group in terms of improvement of Chinese medicine symptoms and efficacy of Chinese medicine symptoms for patients with unstable angina(phlegm-dampness syndrome),and the LDL-C level of the patients in the Wen-Dan Decoction group is significantly lower than the average value of the control group after treatment,which indicates that Wen-Dan Decoction can reduce the LDL-C level of patients to a certain extent.For blood TG,blood TC,blood HDL-C,chest paralysis symptom efficacy,electrocardiogram efficacy,and nitroglycerin administration,there was no statistical significance in the comparison between the control group and the Wen-Dan Decoction group.3.Morphological examination revealed that atherosclerosis aggravated myocardial injury and fibrosis in rats with coronary heart disease model,while myocardial injury and apoptosis were significantly improved after the intervention of warm bile broth.real-time PCR and ELISA results showed that atherosclerosis significantly up-regulated miR-320c and reduced COL4A1 in rats,and the effect was specific to both serum and aortic tissues.The effect was specific to serum and aortic tissues,and the treatment of warm bile broth inhibited this trend,altered the expression of miR-320c,and increased the content of COL4A1 in rats.4.Dual luciferase validation identified COL4A1 as a target gene of miR-320c.5.In cellular experiments,OX-LDL was found to not only affect endothelial cell survival and renewal,but also inhibit cellular functions including migration.High expression of miR-320c inhibited the proliferation of vascular endothelial cells on the basis of OX-LDL,promoted apoptosis and impaired their function,and inhibition of miR-320c reduced the endothelial cell damage caused by OX-LDL.miR-320c inhibited endothelial cell function by targeting the expression of COL4A1,resulting in endothelial cell impairment.Knockdown of COL4A1 in endothelial cells cultured in vitro showed similar effects as high expression of miR-320c.Conclusions:1.miR-320c expression in serum of patients with phlegm-turbid syndrome of unstable angina of coronary heart disease was significantly elevated,and COL4A1 expression was targeted and inhibited.2.Clinical studies have confirmed that Wen-Dan Decoction can improve the Chinese symptom score,Chinese symptom efficacy and serum LDL-C level in patients with phlegm syndrome of unstable angina in coronary heart disease.3.Animal experiments have confirmed that Wen-Dan Decoction improves miR-320c overexpression in serum and aorta of rats modeled for atherosclerosis and myocardial ischemia,and increases COL4A1 content,which can reduce myocardial injury and inhibit fibrosis.4.Cellular experiments revealed that inhibition of miR-320c had the effect of reducing endothelial cell injury and improving cell function.From the perspective of stabilizing coronary atherosclerotic plaques by type Ⅳ collagen(COL4A1),it provides a new idea for the mechanism of treating phlegm-turbidity evidence of unstable angina in coronary heart disease by Wen-Dan Decoction. |
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