| Part One Effect of PTX3 KD on myocardial fibrosis in patients with heart failure after myocardial infarctionObjective: Pentraxin 3(PTX3)is a Pentraxin produced by stroma and myeloid cells that activates complement and regulates inflammation.Animal and clinical trials have shown that PTX3 is pleiotropic in cardiovascular diseases,but the molecular mechanism of PTX3 in heart failure after myocardial infarction,especially in myocardial fibrosis,has been less reported.Based on this,this experiment aims to verify the effect of PTX3 on myocardial fibrosis through bioinformatics analysis and HF mouse model after MI.Methods:1.The GSE86569 dataset was downloaded from the Gene Expression Omnibus(GEO)database,and Kaplan-Meier survival curve analysis and logarithmic rank test were used to compare the difference in overall survival(OS)between patients with heart failure and normal volunteers.RNA-seq data of Sh PTx3-transfected cardiomyocytes were retrieved from GEO database to identify differentially expressed genes(DEGs)and to perform PTX3 interaction gene analysis.GO and KEGG pathways were used to analyze IL-6pathways in HF patients.2.PTX3 knockdown lentivirus vector was constructed,and overexpressed and interfered lentivirus was injected into the heart of experimental mice,and mice were divided into Sham group,model group and PTX3 KD group.Echocardiography and electrocardiogram were performed to evaluate myocardial function of mice in different groups.3.After obtaining mouse heart tissue,Masson trichromatic staining,TTC staining and WGA staining were used to detect myocardial infarction area and myocardial fibrosis degree of mice in each group.The expression of α-SMA in each group was detected by immunofluorescence staining.Western blot was used to detect the difference in the expression of collagen I(Col-I)and collagen III(Col-III).Results:1.Compared with the healthy control group,the expression of PTX3 in HF patients was significantly up-regulated;Survival analysis showed that high expression levels of PTX3 were associated with lower overall survival.GSEA results showed that the expression of PTX3 was related to the production pathway of inflammatory factor IL-6.GO and KEGG analysis showed that IL-6 production pathway,inflammatory response pathway,GTPase activity pathway and cell surface receptor pathway were enriched in HF patients.2.Compared with model group,LVAWd,LVAWs,ES and FS in PTX3-KD group were significantly decreased(P<0.05),but LVIDd and LVIDs were significantly increased(P<0.05);Compared with model group,the E and A peak rates,PVA,PVD and PVS in PTX3-KD group were imp-roved(P<0.05).3.TTC results showed that compared with model group,myocardial infarction area of mice in PTX3-KD group was significantly reduced(P<0.05).The cross-sectional area(CSA)of myocardial cells in PTX3-KD group was significantly lower than that in model group(P<0.05).4.The fluorescence intensity(α-SMA,red fluorescence)of cardiac fibroblasts after PTX3 KD was significantly lower than that in model group.The fibrosis rate of PTX3-KD group was significantly lower than that of model group(P<0.05).Compared with model group,the expressions of Col-I and Col-III in myocardial tissue of PTX3-KD group were significantly downregulated(P<0.05).Conclusion: Interference with PTX3 expression can improve myocardial hypertrophy and cardiac dysfunction.Part Two Mechanism of PTX3 KD inhibiting myocardial fibrosis in patients with heart failure after myocardial infarctionObjective: To investigate the molecular mechanism of PTX3 in myocardial fibrosis based on the effect of PTX3 on myocardial fibrosis in patients with heart failure after myocardial infarction.Methods: Cardiac fibroblasts were isolated and cultured and transfected with IL-6 overexpressing lentivirus and PTX3 knockdown lentivirus;PTX3-KD+IL-6 group and PTX3-NC+IL-6 group were cultured in DMEM containing 10 ng/m L TGF-β1 for 72 h,and the fibroblast activity was detected by CCK-8 method.The difference of PTX3 expression in different groups was detected by q RT-PCR.Results:1.After overexpression of IL-6,the expression of IL-6,p-STAT3,Col-I and Col-III increased significantly(P<0.05);However,compared with the model group,IL-6 and p-STAT3 in PTX3-KD group were significantly decreased(P<0.05).2.Compared with the PTX3-NC group,the expression of PTX3 in PTX3-KD constituent fiber cells was significantly down-regulated(P<0.001).The cell viability of cardiac fibroblasts in PTX3-KD group was significantly lower than that in NC group(P<0.05),on the contrary,the cell viability was significantly enhanced after IL-6 stimulation(P<0.05).Compared with PTX3-NC+TGF-β1 group,the expressions of Col-I and Col-III in PTX3-KD+TGF-β1 group were significantly decreased(P<0.05).However,compared with PTX3-KD +TGF-β1 group,the expressions of Col-I and Col-III in PTX3-KD +TGF-β1+IL-6 group were significantly increased(P<0.05).Conclusion: Interfering with the expression of PTX3 can antagonize myocardial fibrosis by inhibiting the IL-6/STAT3 pathway,suggesting that PTX3 may be a therapeutic target for heart failure after myocardial infarction.Part Three Effect of PTX3 on myocardial function in hypertensive heart failureObjective: Cardiac remodeling is a determinant of the clinical course of heart failure.However,the role of PTX3 in the progression of hypertensive cardiac remodeling remains unclear.In this study,the effect of PTX3 on Ang II-induced cardiac remodeling and dysfunction were explored through in vivo and in vitro experiments.Methods:1.Angiotensin Ang II was injected subcutaneously with an implantable osmotic pump to construct a hypertensive heart remodeling model.The model was divided into two groups: control group and Ang II group.After successful construction,PTX3 recombinant protein or PBS was injected intraperitoneally every week.Set groups: Vehicle group and PTX3 group;Echocardiography was used to measure myocardial function,and ejection fraction(EF)and shortening fraction(FS)were calculated.2.After obtaining the mouse heart,HE staining,Masson staining and wheat germ agglutinin(WGA)staining were used to detect the degree of myocardial hypertrophy and fibrosis.3.The overexpressed plasmid of PTX3 was constructed and transient transfected into cardiomyocytes,and divided into no-load group and overexpressed group.Myocardial cells were treated with Ang II or normal Saline and divided into saline group and Ang II group.The m RNA and protein expressions of myocardial fibrosis and myocardial hypertrophy were detected by q RT-PCR and Western blot.Results:1.Compared with Vehicle group,the m RNA and protein of PTX3 in Ang II group were significantly increased(P<0.05);Compared with Vehicle group,EF and FS values in PTX3 group were significantly increased(P< 0.05).2.Compared with the control group,the heart size,heart weight to body weight ratio(HW/BW)and cross-sectional area of cardiomyocytes in Ang II group were significantly increased(P<0.05),indicating that Ang II could promote myocardial hypertrophy.Compared with Vehicle group,HW/BW in PTX3 group was significantly decreased(P<0.001),as well as myocardial cell cross-sectional area and heart size(P<0.05).Compared with Vehicle group,hypertrophy markers ANP,BNP and MYH7 were significantly decreased in PTX3 group(P<0.05).In addition,compared with Vehicle group,the degree of cardiac fibrosis in PTX3 group was improved.3.Compared with the control group,the expressions of collagen I,collagen III and α-SMA in Ang II group were significantly increased;Compared with Vehicle group,collagen I,collagen III and α-SMA in PTX3 group were significantly decreased(P<0.05).4.Compared with the control group,the expressions of TGF-β1 and p-Smad2/3 in Ang II group were increased,and the expressions of TGF-β1and p-Smad2/3 in PTX3 group were decreased compared with the Vehicle group.Compared with the control group,the m RNA expressions of inflammatory factors IL-1β,IL-6 and TNF-α were significantly increased in Ang II group,but after Ang II induction,the m RNA expressions of inflammatory factors IL-1β,IL-6 and TNF-α were significantly decreased in PTX3 group compared with Vehicle group.5.Compared with the control group,the expression of p-IKKα and p-p65 protein in Ang II group was significantly increased;However,after Ang II,p-IKKα and p-p65 protein expression decreased in PTX3 group compared with Vehicle group.6.Compared with Saline group,myocardial hypertrophy markers ANP,BNP and MYH7 in Ang II group were significantly increased(P<0.05);Moreover,after induction of Ang II,ANP,BNP and MYH7 in PTX3 group were significantly decreased compared with no-load group(P<0.05).Compared with Saline group,Collagen I and III cells in Ang II group were significantly increased(P<0.05),and after induction of Ang II,compared with the no-load group,Collagen I and III m RNA levels in PTX3 group were significantly decreased(P<0.05).7.Compared with Saline group,TGF-β1 and p-Smad2/3 were increased in Ang II group,and TGF-β1 and p-Smad2/3 protein expressions in PTX3 group were decreased after Ang II induction compared with no-load group.Conclusion: PTX3 attenuated Ang II-induced cardiac remodeling in mice and significantly reduced Ang II-induced cardiac hypertrophy,fibrosis,and inflammatory responses,and recovery of systolic dysfunction via TGF-β1/Smad2/3 and Ikk-alpha /NF-κB.Part Four PTX3 alleviates myocardial dysfunction in hypertensive heart failure by regulating CXCL1Objective: To explore the molecular mechanism of the effect of PTX3 on Ang II induced cardiac remodeling and dysfunction through in vivo and in vitro experiments.Methods: The overexpressed plasmid of PTX3 or CXCL1 was constructed and transient transfected into cardiomyocytes,and divided into no-load group and overexpressed group.Myocardial cells were treated with Ang II or normal Saline and divided into saline group and Ang II group.The m RNA and protein expressions of myocardial fibrosis and myocardial hypertrophy were detected by q RT-PCR and Western blot,as well as the expression differences of related signaling pathways.Results:1.Compared with control group and Saline group,CXCL1 m RNA and protein expressions in Ang II group were significantly increased(P<0.05),and after Ang II induction,CXCL1 m RNA and protein expressions in PTX3 group were significantly decreased compared with Vehicle group or no-load group(P<0.05).2.The inhibitory effect of PTX3 overexpression on Ang II-induced cardiomyocyte hypertrophy was reversed by CXCL1 overexpression;The inhibitory effect of PTX3 overexpression on Ang II-stimulated CFs differentiation was reversed by CXCL1 overexpression.Conclusion: PTX3 can alleviate myocardial dysfunction in patients with hypertensive heart failure by regulating CXCL1. |
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