| The chymase secreted by cardiac mast cells is a prion-like serine protease discovered in recent years and is widely distributed in the myocardial interstitium.Clinical studies have shown that chymase promotes the production of angiotensin 2 in arteries in patients with atherosclerosis and aneurysms,thereby aggravating the condition of acute myocardial infarction.Animal experiments have shown that cardiac chymase is directly involved in the pathogenesis of myocardial infarction in experimental animals.Oral chymase inhibitor (CHI) can significantly improve hemodynamic parameters such as left ventricular systolic pressure,end-diastolic pressure and mean arterial pressure.Reducing the mortality of rats with myocardial infarction;at the same time,it was found that transgenic mice carrying human cardiac chymase gene catalyze the formation of Angl and have obvious tendency of cardiac hypertrophy,but the effect of chymase on CFs aggravating cardiac hypertrophy has not been reported..Studies have shown that the PI3K-Akt pathway is an important endogenous cardiac hypertrophy regulatory pathway that plays an important role in promoting cell growth,inhibiting apoptosis,and maintaining cell survival.A variety of studies have confirmed that activation of myocardial PI3K-Akt pathway signaling pathway can improve systolic and diastolic function and inhibit cardiomyocyte apoptosis,while endogenous GSK3? factor can prevent myocardial hypertrophy.Therefore,this study will initially investigate the effects of chymase on CFs proliferation and collagen synthesis at the cellular level;and establish a rat myocardial infarction model to investigate the effect of chymase on ventricular remodeling after myocardial infarction in rats,and study related clinical drugs.The effects of chymase on the chymase and PI3K-AKt-GSK3? pathway are expected to further elucidate the specific mechanism of action of chymase on myocardial remodeling.Methods1.Cell experiment1.1 The preparation,culture and grouping of rat CFsRat fibroblasts were isolated by differential adherence method,and the required CFs were identified and detected by SABC immunohistochemical staining and trypan blue staining.Cells in the log phase growth phase were divided into control group(added serum-free DMEM medium),chymase induction group (30 ?g/L chymase was added to serum-free DMEM medium) and chymase inhibitor SBTI intervention group(Add 30 ?g/L SBTI to serum-free DMEM medium).The cells were cultured in serum-free DMEM for 48 h, and then culture medium,chymase or SBTI were added separately.After 24 h of culture,cells were harvested for subsequent experiments.1.2 MTT colorimetric assay for CFs proliferation rateThe effect of chymase on the proliferation of CFs was compared by MTT colorimetric assay.The optical density values (OD values,n=5) of the above respective cells after the culture were measured at 490 nm using an enzyme-linked immunosorbent assay.Cell proliferation rate=OD test well/OD control* 100%.1.3 flow cytometry analysis of each group of cell cycleThe effect of chymase on the proliferation cycle of CFs was compared by flow cytometry.The cycle of the cells was analyzed using Multicycle (MCYCLE) software and the proliferation index (PI) was calculated.PI=(S+G2/M)/(G0/G1+S+G2/M)*100%.1.4 3H-proline incorporation assay for total collagen synthesis in each group of cellsThe effect of chymase on total collagen synthesis of CFs was analyzed by 3H-proline incorporation method.The radioactivity of each group was measured using a liquid scintillation counter,and the obtained data was expressed in cpm/well.1.5 RT-PCR method for determination of CO?-? and CO?-? collagen mRNA in each groupThe effect of chymase on the expression of CO?-? and CO?-? collagen mRNA in CFs was analyzed by RT-PCR.The agarose gel electrophoresis bands of each group were observed under UV projector,and the gray values of the bands of target genes CO?-?,CO?-? and reference GAPDH were analyzed systematically.2 Animal experiments2.1 Preparation and grouping of AMI rat modelsFifty male SPF SD rats were randomly divided into sham operation normal control group,AMI model group,chymase inhibitor SBTI intervention group,ACEI drug ramipril intervention group and Chinese patent medicine Tongluo drug.Qiqiang capsule intervention group (n=10).Except for the normal control group of sham operation,the other four groups were established by ligating the left coronary artery of the rat.After 24 hours of myocardial infarction,for 4 weeks,each group was given daily saline,SBTI (10 mg/kg,ip),ramipril (10 mg/kg,po),and sputum powder (0.5).g/kg,po).All the above groups were treated with penicillin anti-inductive treatment within 3 days after operation.After 4 weeks of myocardial infarction,various indexes were detected.The rats were sacrificed by cervical dislocation and the heart was taken out for subsequent experimental analysis.2.2 Determination of cardiac function and left ventricular mass index (LVWI) in each groupCardiac morphology and functional tests were performed before the 4th week of myocardial infarction.Measurements included left ventricular end diastolic diameter(LVEDD),left ventricular end systolic diameter (LVESD),left ventricular internal diameter shortening (LVFS),left ventricular ejection fraction (LVEF),and left ventricular posterior wall thickness (PWT).The body weight of each group was weighed (Body Weight,BW),the rats were sacrificed,the heart was quickly removed,the left ventricular mass (LVW) was weighed and the left ventricular mass index(LVWI=LVW/BW*100%) was calculated..2.3 Myocardial pathological analysis of each group of ratsThe myocardial pathological results of each group were determined by HE and Masson staining.The myocardial pathology scores of each group of rats were calculated according to the staining results,and the collagen fiber volume integral(CV,myocardial collagen area/measured field of view area) was calculated.2.4 Immunohistochemical method for determination of CO?-?,CO?-? collagen and p-PI3K,p-Akt protein expression in myocardial tissueThe content of CO?-?,CO?-? collagen and p-PI3K and p-Akt protein in myocardial tissue of each group were detected by immunohistochemistry.Quantitative gray value determination was performed on the positive area of the field of view image selected for each slice using the Image-Pro Plus 6.0 image analysis system.2.5 RT-PCR method for determination of CO?-? and CO?-? collagen mRNA in myocardial tissueReal-time quantitative PCR was used to detect the expression of CO?-? and CO?-? collagen mRNA in myocardial tissue of each group.The agarose gel electrophoresis bands of each group were observed under UV projector,and the gray values of the bands of target genes CO?-?,CO?-? and reference GAPDH were analyzed systematically.2.6 Immunohistochemical method for determination of p-PI3K and p-Akt protein expression in myocardial tissueThe p-PI3K and p-Akt proteins in the myocardial tissue of each group were detected by immunohistochemistry.Quantitative gray value determination was performed on the positive area of the field of view image selected for each slice using the Image-Pro Plus 6.0 image analysis system.2.7 Western blotting detection of GSK3? protein expression in rat myocardial tissueThe expression of GSK3? protein in rat myocardial tissue was detected by Western blot.The optical density was measured by Image-QuaNT software,and the relative optical density values of each group of proteins were calculated by using?-actin as an internal reference.StatisticsStatistical analysis was performed using SPSS 21.0.The measurement data were expressed as meanąstandard deviation.One-way ANOVA test was used to compare the mean of multiple groups,and LSD-t test was used for comparison.P<0.05 There are statistical differences.Results1.Cell experiment results1.1 The effect of chymase on the proliferation of CFsCompared with the control group,the OD value and the value-added rate of the chymase-induced group were significantly increased (P<0.05),but the intervention of the chymase inhibitor SBTI significantly decreased the OD value and the rate of increase of CFs (P<0.05).The above results indicated that chymase significantly promoted the proliferation of rat CFs cells,while chymase inhibitor SBTI significantly inhibited the proliferation of rat CFs cells.1.2 Effect of chymase on the proliferation cycle of CFsCompared with the control group,the proportion of G0/G1 phase cells in the chymase-induced group was significantly decreased,and the proportion of cells in the S phase and the cell proliferation index were significantly increased (P<0.05).The proportion of cells in the G0/G1 phase of the SBTI intervention group was significantly increased.The proportion of cells in S phase and the cell proliferation index were significantly lower (P<0.05).Compared with the chymase-induced group,the cells in the SBTI intervention group also increased significantly in the G0/G1 phase,and the proportion of cells in the S phase and the cell proliferation index also decreased significantly (P<0.05).The above results indicated that chymase significantly decreased the number of CFs cells in G0/G1 phase,increased the proportion of cells in S phase,and significantly increased the proliferation index of CFs cells.1.3 Effect of chymase on total collagen synthesis of CFsCompared with the control group,the amount of 3H-valine incorporation in the chymase-induced group was significantly increased (P<0.01),and the amount of 3H-proline in the SBTI-treated group was significantly decreased (P<0.01).Compared with the chymase-induced group,the amount of 3H-valine incorporation in the SBTI intervention group was also significantly lower (P<0.01).The above results indicate that chymase significantly increases the expression of total collagen synthesis in CFs cells.1.4 Effect of chymase on the expression of CFs CO?-? and CO?-? collagen mRNACompared with the control group,the relative expressions of CO?-? and CO?-? collagen mRNA in CFs of chymase-induced group were significantly increased (P<0.05);the relative expression of CO?-? collagen mRNA in SBTI-induced group was significantly decreased (P<0.05).There was no significant difference in the relative expression of CO?-? collagen mRNA (P>0.05).Compared with the chymase-induced group,the relative expression levels of CO?-? and CO?-? collagen mRNA in CFs of SBTI-suppressed group were significantly lower(P<0.05).The above results indicated that chymase significantly increased the expression of CO?-? and CO?-? collagen mRNA and promoted the synthesis of collagen.2 animal experiment results2.1 The effect of chymase on cardiac ultrasound parameters of rats in each groupCompared with the sham-operated control group,LVEDD,LVESD and PWT were significantly increased in the AMI model group,SBTI intervention group,ramipril group and sputum group (P<0.05),and LVEF and LVFS were significantly decreased (P<0.01),Compared with the AMI model group,LVEDd,LVESD and PWT were significantly lower in the SBTI intervention group,ramipril group and sputum group (P<0.05),and LVEF and LVFS were significantly increased (P<0.01).)·Compared with the sputum inhibitor SBTI intervention group,there was no significant difference in LVEDd,LVESD,PWT,LVEF and LVFS between the ramipril group (P>0.05);LVEDd,LVESD and PWT in the sputum group There was no statistical difference,but LVEF and LVFS were significantly lower (P<0.01).The above results showed that the cardiac function indexes of rats after acute myocardial infarction decreased significantly,while chymase inhibitors and ramipril and sputum cardiac intervention significantly improved the heart after acute myocardial infarction in rats.Function,while chymase inhibitors have no significant difference in the improvement of cardiac function in rats with myocardial infarction and drugs such as ramipril and sputum.2.2 Effect of chymase on left ventricular mass index in each groupCompared with the sham-operated control group,LVWI was significantly increased in the AMI model group and the sputum group (P<0.05),and there was no statistically significant difference in the LVWI between the sputum inhibitor SBTI intervention group and the ramipril group (P>0.05).Compared with the AMI model group,the LVWI of the sputum inhibitor SBTI intervention group and the ramipril and sputum cardiac intervention group were significantly lower (P<0.05).There was no significant difference in LVWI between the sputum inhibitor SBTI intervention group and the ramipril and sputum cardiac intervention group (P>0.05).The above results showed that the left ventricular mass index increased significantly in rats 4 weeks after acute myocardial infarction,while the chymase inhibitor SBTI and ramipril and sputum stress significantly reduced the left ventricular mass index,and SBTI.There was no significant difference between drugs such as ramipril and sputum in reducing the left ventricular mass index of rats.Therefore,chymase inhibitors and ramipril and sputum strong drugs have significantly improved ventricular remodeling induced by acute myocardial infarction,and chymase has a significant role in promoting myocardial infarction-induced ventricular remodeling.2.3 Effect of chymase on pathological results of HE staining in ratsCompared with the normal sham operation group,the HE pathological scores of the AMI model group,the SBTI intervention group,the ramipril group and the sputum group were significantly increased (P<0.05).Compared with the AMI model group,the HE pathological scores of the SBTI intervention group,the ramipril group and the sputum group were significantly lower (P<0.05).Compared with the SBTI intervention group,the HE pathological score of the Qiangxinxin group was significantly increased (P<0.05),but there was no significant difference in the HE pathological score between the SBTI intervention group and the ramipril group(P>0.05).).The above results showed that myocardial injury was more obvious 4 weeks after acute myocardial infarction in rats,while sputum inhibitors SBTI and ramipril and sputum and other drugs significantly reduced the degree of myocardial damage,and SBTI in protecting the myocardium There is no significant difference between ramipril.Therefore,chymase inhibitors and ramipril and sputum strong drugs have significantly improved acute myocardial infarction-induced myocardial injury,and chymase has a significant role in promoting myocardial infarction-induced myocardial injury.2.4 Effect of chymase on pathological results of myocardial Masson staining in ratsCompared with the normal sham-operated group,the percentage of Masson staining positive in the AMI model group,SBTI intervention group,ramipril group and sputum group was significantly increased (P<0.05).Compared with the AMI model group,the percentage of Masson staining positive in the SBTI intervention group,the ramipril group,and the sputum group was significantly lower (P<0.05).Compared with the ramipril group,the percentage of Masson staining positive area in the SBTI intervention group and the sputum group was significantly increased(P<0.05).There was no significant difference in the percentage of Masson staining positive area between the SBTI intervention group and the Qiangxin group (P>0.05).The above results indicate that myocardial fibrosis induced by ventricular remodeling in rats after acute myocardial infarction is obvious,chymase inhibitors significantly improve fibrosis induced by acute myocardial infarction,and chymase is significant in myocardial infarction-induced collagen fiber deposition.enhancement.2.5 Effects of chymase on the contents of CO?-? and CO?-? collagen in myocardial tissue of rats in each groupCompared with the sham-operated control group,the collagen expression of the rats in the AMI model group,the SBTI intervention group,and the ramipril and sputum-strength drug intervention groups increased significantly (P<0.05), and the CO?-?/? collagen ratio also decreased.Significant increase (P <0.05).Compared with the AMI model group,the collagen expression of the SBTI intervention group and the ramipril and sputum cardiac intervention group were significantly decreased(P<0.05),and the CO?-?/? collagen ratio was also significantly decreased (P<0.05).There was no significant difference in the expression of collagen and CO?-?/?collagen between the SBTI intervention group and the ramipril and sputum drug intervention groups (P>0.05).The above results showed that the expression of collagen induced by ventricular remodeling was significantly increased after acute myocardial infarction in rats.-The chymase inhibitor significantly inhibited the expression of type ? and ? collagen induced by acute myocardial infarction.There is a significant promotion in the expression of type ? and ? collagen.2.6 Effects of chymase on the expression of CO?-? and CO?-? collagen mRNA in myocardial tissue of ratsCompared with the sham-operated control group,the expression levels of CO?-?and CO?-? collagen mRNA in the AMI model group,SBTI intervention group and ramipril and sputum-strength drug intervention group were significantly increased(P<0.05).Compared with the AMI model group,the expression levels of CO?-? and CO?-? collagen mRNA in the SBTI intervention group and the ramipril and sputum cardiac intervention group were significantly lower (P<0.05).Compared with the ramipril drug intervention group,the expression levels of CO?-? and CO?-?collagen mRNA in the SBTI intervention group and the Qiangxin drug intervention group were significantly increased (P<0.05),but the SBTI intervention group and ?There was no significant difference in the expression of CO?-? and CO?-? collagen mRNA between the two groups (P>0.05).The above results indicate that the expression of collagen mRNA induced by ventricular remodeling in rats after acute myocardial infarction is significantly increased.The chymase inhibitor significantly inhibits the expression of type ? and ?,collagen mRNA induced by acute myocardial infarction.There was a significant promotion in the process of infarct-induced expression of type ? and ? collagen mRNA.2.7 Effect of chymase on the relative expression of p-PI3K and p-Akt in myocardial tissue of ratsThe expression of p-PI3K and p-Akt protein in the AMI model group was significantly higher than that in the control group (P<0.01).The SBT1 intervention group and the ramipril and sputum cardiac intervention group were more p-PI3K,p-than the AMI model group.Akt protein expression was significantly reduced (P <0.01).Compared with the Qiangxin drug intervention group,the expression of p-PI3K protein in the SBTI intervention group and the ramipril intervention group was significantly lower (P<0.05).There was no statistically significant difference in the expression of p-Akt protein between the three groups (P>0.05).These results indicate that ventricular remodeling significantly induces the expression of p-PI3K and p-Akt protein after acute myocardial infarction in rats.The chymase inhibitor significantly inhibited the phosphorylation of PI3K and Akt in the PI3K-AKt-GSK3? pathway.2.8 Effect of chymase on the relative expression of GSK3? protein in myocardial tissue of rats in each groupThe statistical results showed that compared with the sham-operated control group,the relative expression of GSK3? in the AMI model group,the SBTI intervention group,and the ramipril and sputum cardiac intervention group were significantly lower (P<0.05).Compared with the AMI model group,the relative expression of GSK3? in the SBTI intervention group and the ramipril and sputum cardiac intervention group were significantly increased (P<0.05),but the SBTI intervention group and ramipril and sputum cardiac intervention There was no significant difference in the relative expression of GSK3P protein between the three groups (P>0.05).The above results indicated that ventricular remodeling significantly inhibited the expression of GSK3? protein after acute myocardial infarction in rats,and chymase inhibitor significantly induced the expression level of GSK3? protein in PI3K-AKt-GSK3? pathway.Conclusions1.The chymase significantly promoted the proliferation of rat CFs cells,decreased the cells in G0/G1 phase,increased the proportion of cells in S phase,and significantly increased the proliferation index of CFs cells.2.The chymase significantly increased the total collagen synthesis and the expression of CO?-? and CO?-? collagen mRNA in CFs cells,and promoted the synthesis of CFs collagen.3.The chymase inhibitor SBTI and ramipril and sputum intensive intervention significantly improved the cardiac function after acute myocardial infarction in rats,decreased the left ventricular mass index,and improved myocardial damage and fiber induced by acute myocardial infarction.Hyperplasia.4.The chymase inhibitor SBTI and ramipril and sputum cardiac intervention significantly inhibited the expression of type ? and ? collagen and mRNA:induced by acute myocardial infarction,and significantly inhibited the GSK3? protein in PI3K-AKt-GSK3? pathway.Expression and phosphorylation of PI3K and Akt proteins. |
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