| ObjectivesTo investigate the effects of tanshinoneⅡA（Tan ⅡA）on ventricular remodeling（VR）in rats with myocardial infarction（MI）,the effects of interleukin 33（IL-33）/growth stimulation expressedgene 2 protein（ST2）signaling pathway on VR,and whether the role of epicardial adipose tissue（EAT）is also through the IL-33/ST2 axis.Methods1.The animal model of myocardial infarction was established by ligating the left anterior descending branch of coronary artery in 80 SPF female SD rats with a body mass of（200±20）g.The elevation of ST segment in postoperative ECG was used as one of the bases for the success of myocardial infarction model.60 successful rats were randomly divided into MI group,low,middle and high dose Tan ⅡA groups and positive control group,with 12 rats in each group.In addition,12 rats with left anterior descending coronary artery were randomly selected as sham operation group（Sham group）.Tan ⅡA low,middle and high dose groups(7.5,15,30mg kg-1·d-1),positive control group（XST group）,valsartan 30mg kg-1·d-1group,MI group and sham operation group were injected intraperitoneally with saline for 28 days.During the administration period,the general condition and body weight of the mice were observed,and some rats died of heart failure.The death of rats in each group was counted,the survival rate of each group was calculated,and the survival curve was made.Before the rats were sacrificed,they were fasted and water deprived for 12 hours,body weight was measured,and heart mass index（HW/BW）and epicardial fat mass index（FW/HW）were calculated.2.The levels of serum N-terminal pro-brain natriuretic peptide（NT-pro BNP）and high-sensitivity cardiac troponinⅠ（hs-c Tn-Ⅰ）were detected by enzyme-linked immunosorbent assay（ELISA）.3.The cardiac function of rats was measured by electrocardiogram and ultrasound.The electrocardiogram of rats in each group was measured before operation,20min after operation and before death,and the changes of ST segment in leadⅡwere observed.The changes of left ventricular end-systolic diameter（LVDs）,left ventricular end-diastolic diameter（LVDd）,decreased left ventricular ejection fraction（LVEF）and left ventricular shortening fraction（LVFS）were measured after28 days of animal B-ultrasound.4.The contents of malondialdehyde（MDA）,superoxide dismutase（SOD）,glutathione（GSH）and catalase（CAT）in serum were detected by Bio Tek test method.5.2,3,5Triphenyltetrazolium chloride staining（TTC）was used to observe myocardial infarction and quantify the infarct size.6.Pathological changes and myocardial fibrosis were observed by HE and Masson staining.7.Masson staining was used to observe the morphological changes of epicardial adipose tissue.8.ELISA was used to detect the levels of serum inflammatory factors:interleukin-10（IL-10）and tumor necrosis factor-α（TNF-α）.9.The serum levels of IL-33 and ST2 were detected by ELISA.10.The protein expression and location of IL-33,ST2 and my D88 in myocardial tissue and epicardial adipose tissue were detected by immunohistochemistry.11.The expression levels of IL-33/ST2 signaling pathway and related proteins NF-κB（p65）and TGF-β1 in myocardial tissue and epicardial adipose tissue were detected by Western blotting.Results1.Effects of Tan ⅡA on general state and body weight of rats with myocardial infarction（MI）:rats in MI group were in poor spirit,sparse hair and dark color,decreased water intake and activity,and abdominal swelling.The mental state of rats in the low,middle and high dose groups of Tan ⅡA and XST group was better,the hair was smooth and bright,and the amount of water intake and activity increased.After 2weeks,there was no significant difference in body weight between the administration group and the MI group（P>0.05）.After 4 weeks,the body weight of the administration group was heavier than that of the MI group,and there was significant difference between the middle and high dose group of Tan ⅡA,the XST group and the MI group（P<0.05）.2.Effect of Tan ⅡA on the survival rate of MI rats:53 rats survived after intervention,12 rats in Sham group,6 rats in MI group,7 rats in Tan ⅡA-L group,8rats in Tan ⅡA-M group,10 rats in Tan ⅡA-H group and 10 rats in XST group.By log-rank（Mantel-Cox）test,there was significant difference between MI group and Sham,Tan ⅡA-H,XST（P<0.05）.The cause of death in rats is related to myocardial injury or heart failure after myocardial infarction.The survival rate of the administration group was improved and the time of death was delayed,suggesting that the administration group could improve the cardiac function.3.Effects of Tan ⅡA on cardiac index and fat index of MI rats:cardiac hypertrophy,cardiac index,epicardial fat thickening and fat index in MI group were significantly higher than those in Sham group（P<0.01）.The cardiac index and fat index in low,middle and high dose groups of Tan ⅡA and XST group decreased,and there were significant differences in cardiac index and fat index between low and middle dose groups of Tan ⅡA and MI group（P<0.05）.There were significant differences in cardiac index and fat index between Tan ⅡA high dose group and XST group and MI group（P<0.01）.4.The effect of Tan ⅡA on the contents of serum NT-pro BNP and hs-c Tn-Ⅰin MI rats:NT-pro BNP and hs-c Tn-Ⅰin MI group were significantly higher than those in Sham group（P<0.01）.NT-pro BNP and hs-c Tn-Ⅰdecreased in the treatment group,especially in the middle and high dose Tan ⅡA groups and XST group,which were significantly lower than those in the MI group（P<0.01）.5.Effect of Tan ⅡA on ECG and ultrasound of MI rats:after 28 days of administration,the ST segment of each group decreased compared with the MI group,and the ST segment of the middle and high dose Tan ⅡA groups and XST group decreased significantly,close to the baseline level.28 days after administration,echocardiography was performed to evaluate the cardiac function of rats.The cardiac function of rats in MI group was significantly impaired and the index of cardiac function decreased,including the increase of left ventricular end-systolic diameter（LVIDs）and left ventricular end-diastolic diameter（LVIDd）,the decrease of left ventricular ejection fraction（LVEF）and left ventricular shortening fraction（LVFS）.After 28 days of administration,LVIDs and LVIDd in low,middle and high dose groups of Tan ⅡA and XST group decreased,while LVEF and LVFS increased.There was significant difference in LVEF and LVFS between middle and high dose groups and MI group（P<0.01）.6.Effect of Tan ⅡA on serum oxidation index of MI rats:the content of serum MDA in MI group was significantly higher than that in Sham group（P<0.01）,and the activities of SOD,GSH-Px and CAT were significantly lower than those in Sham group（P<0.05）.MDA decreased and SOD,GSH-Px and CAT activities increased in low,middle and high dose groups of Tan ⅡA and XST group.There was significant difference between middle and high dose groups of Tan ⅡA and XST group and MI group（P<0.01）,but there was no significant difference in MDA content and CAT activity between low dose Tan ⅡA group and MI group（P>0.05）.7.Effect of Tan ⅡA on infarct size of MI rats:the infarcted myocardium was pale white and normal myocardium was brick red in TTC staining.The results of TTC staining showed that there was no obvious myocardial infarction in Sham group,and myocardial infarction in left ventricular free wall and anterior wall in MI and administration group（not stained,pale）.The pale infarct area in MI group was significantly lower than that in Sham group（P<0.01）.The infarct size in low,middle and high dose groups of Tan ⅡA and XST group were all decreased,and there were significant differences between middle and high dose groups of Tan ⅡA and XST group and MI group（P<0.01）.8.Effect of Tan ⅡA on myocardial histopathology and myocardial fibrosis in MI rats:HE staining showed that cardiomyocytes arranged regularly and there were a few fibers in myocardial interstitium in Sham group.In MI group,the arrangement of cardiomyocytes was irregular,with a large number of fibrous tissue formation and inflammatory cell infiltration.The number of fibrous cells in the low,middle and high dose groups of Tan ⅡA and XST group was less,most of the cardiomyocytes were arranged orderly,and there was a small amount of neutrophil infiltration.After Masson staining,the cardiomyocytes were red and the interstitial collagen fibers were blue.There were very few collagen fibers in the myocardial interstitium in the Sham group,and obvious collagen fibers were found in the myocardial tissue in the MI group.The degree of fibrosis in the low,middle and high dose groups of Tan ⅡA and XST group was significantly less than that in the MI group.9.Effect of Tan ⅡA on pathomorphology of adipose tissue of MI rats:the mirror results showed that compared with the Sham group,the adipocytes in the MI group were uneven in size,disordered in arrangement and larger in size.Compared with MI group,the low,middle and high dose groups of Tan ⅡA and XST group were neatly arranged,uniform in size,and the number of large adipocytes was significantly reduced.10.The effect of Tan ⅡA on the content of IL-10 and TNF-αin serum of MI rats:the content of IL-10 in MI group was significantly lower than that in Sham group（P<0.05）,and the content of TNF-αin MI group was significantly higher than that in Sham group（P<0.01）.IL-10 increased in all treatment groups,and the levels in middle and high dose Tan ⅡA groups and XST group were significantly higher than those in MI group（P<0.01）.The content of TNF-αdecreased in all treatment groups,especially in the middle and high dose groups of Tan ⅡA and XST group,which was significantly lower than that in MI group（P<0.01）.11.The effect of Tan ⅡA on the contents of IL-33 and ST2 in serum of MI rats,the expression of IL-33/ST2 signal pathway and its related proteins NF-κB（p65）and TGF-β1 in myocardial tissue and epicardial adipose tissue:The contents of serum IL-33 and ST2 in MI group were significantly higher than those in Sham group（P<0.01）,and the contents of IL-33 and ST2 in treatment group were lower than those in MI group（P<0.01）.The contents of IL-33 in middle and high dose groups of Tan ⅡA and XST group were significantly lower than those in MI group（P<0.01）.The immunohistochemical results of myocardial tissue and epicardial adipose tissue were consistent.The results showed that the expression of IL-33,ST2 and my D88 in MI group was significantly higher than that in Sham group（P<0.01）,and the expression was mainly in the infarct area and peri-infarct area.The brown expression in low,middle and high dose groups of Tan ⅡA and XST group was lower than that in MI group,and there was significant difference between middle and high dose groups,XST group and MI group（P<0.01）.The Westernblotting results of myocardial tissue and epicardial adipose tissue were consistent,and the trend was basically consistent with that of IHC.The results showed that the expression of IL-33 and ST2 protein in MI group was significantly higher than that in Sham group（P<0.01）.The expression of TGF-β1 protein in myocardial tissue in MI group was higher than that in Sham group（P>0.05）,and the expression of NF-kappa B（p65）in myocardial tissue and TGF-β1 and NF-kappa B（p65）in epicardial adipose tissue in MI group was significantly higher than that in Sham group（P<0.01）.The expression of IL-33,ST2,TGF-β1 and NF-kappa B（p65）decreased in low,middle and high dose groups of Tan ⅡA and XST.The expression of IL-33 and ST2 in myocardial tissue of high dose Tan ⅡA group and XST group was significantly lower than that of MI group（P<0.01）.The expression of TGF-β1 and NF-kappa B（p65）in myocardial tissue of middle dose Tan ⅡA group was significantly lower than that of MI group（P<0.01）.The expression of IL-33 and ST2 in epicardial adipose tissue in middle dose Tan ⅡA group and XST group was significantly lower than that in MI group（P<0.01）,and the expression of TGF-β1 and NF-kappa B（p65）in epicardial adipose tissue in middle and high dose Tan ⅡA group and XST group was significantly lower than that in MI group（P<0.01）.ConclusionsTan ⅡA can reduce inflammatory reaction and oxidative stress and improve ventricular remodeling after myocardial infarction by inhibiting epicardial fat and myocardial tissue IL-33/ST2 signal pathway. |
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