| Objective:This study aims to explore the molecular mechanism of soluble frizzled related protein1(Sfrp1)during aging acute myocardial ischemia injury through inhibiting Wnt/?-catenin signaling pathway activity.At the animal level,to investigate Sfrp1 whether improve heart function and reduce the degree of myocardial fibrosis,meanwhile,whether inhibit myocardial apoptosis and reduce acute ischemic injury of aging myocardium by inhibiting Wnt/?-catenin signaling pathway as a gene therapy drug.At the cellular level,to investigate relative molecular mechanisms about Sfrp1 inhibit the activity of Wnt/?-catenin signaling pathway to reduce the apoptosis of myocardial cells and the proliferation of cardiac fibroblasts;To explore the potential role of SNPs of Wnt/?-catenin signaling pathway core genes in myocardial infarction(MI)susceptibility and to provide further evidence for MI diagnosis and prevention.Methods:Part I:15 months old C57BL/6J mice were selected for the current study and dsAAV9-Sfrp1 was injected into mice by tail vein.We have established acute myocardial infarction model in mice at the peak time of Sfrp1 expression.Further,we detected the effect of Sfrp1 expression on the histological changes of heart function parameters,extracellular matrix,myocardial cell apoptosis and Wnt/?-catenin signaling pathway activity on aging mice after acute myocardial infarction.Part II:dsAAV9-Sfrp1 was transfected into H9c2 myocardial cells and cardiac fibroblasts,respectively,at the peak time of Sfrp1 expression,hypoxia/reoxygenation(H/R)induced apoptosis of H9c2 cells and TGF-?1 stimulated proliferation of cardiac fibroblasts.To test the role of Sfrp1 overexpression wuich leads to inhibiting the Wnt/?-catenin signaling pathway activity on H9c2 cardiac cells apoptosis and mitochondrial apoptosis pathway,as well as on fibroblast proliferation,collagen synthesis,and differentiation into myofibroblasts.Part III:SNPs of Wnt/?-catenin signaling pathway core genes SFRP1,CTNNB1,and WISP1 were genotyped for 465 MI cases and 485 controls by PCR-RFLP assays and gene sequencing assays.Differences in the frequencies of alleles and genotypes between cases and controls were evaluated by?~2test.Univariate and multivariate logistic regression models were applied to calculate crude and adjusted odds ratios(ORs)and 95%confidence intervals(CIs),respectively.Results:Part I:(1)Echocardiography showed that Sfrp1 overexpression was associated with a reduction in LVEDd compared to the corresponding days after myocardial infarction(all P<0.05),while Pwdth and FS values were increased(all P<0.05).(2)HE staining showed that overexpression of Sfrp1,inflammatory cell infiltration was decreased at the third day after myocardial infarction,myocardial necrosis and fibrous tissue hyperplasia symptoms were significantly decreased at the seventh ay after myocardial infarction.The results of Masson staining showed that overexpression of Sfrp1,the expression of collagen fibers in infarct area was significantly lower than when compared to the third day and the seventh day after myocardial infarction(all P<0.05).(3)Immunohistochemical staining showed that overexpression of Sfrp1,the expression of extracellular matrix Col-1 and Col-3 were significantly decreased at the third day and the seventh day after myocardial infarction(all P<0.05).(4)Tunel staining was used to detect the apoptosis rate of myocardial cells,the results showed that myocardial cell apoptosis rate were significantly lower than the corresponding to the third day and the seventh day after myocardial infarction due to Sfrp1 over expression(32.15±3.43 vs.21.76±2.22%?47.33±5.12%vs.23.34±2.86%,all P<0.05).Western blot showed that over expression of Sfrp1 inhibit the expression of Bax and increase the expression of Bcl-2 in both the third day and the seventh day after myocardial infarction(all P<0.05).(5)Western bolt and Real time PCR showed that Dvl-1,?-catenin and Wisp1 protein level and mRNA level were increased.In addition,While Sfrp1 was over expressing,both the third day and the seventh day after myocardial infarction was significantly decreased(all P<0.05).Part II:(1)AnnexinV-FITC/PI double staining assay showed that overexpression of Sfrp1 decreased the percentage of H/R by inducing apoptosis in H9c2 cells,56.67±3.49 vs.31.48±5.18(P<0.05).The results of Caspase-3/7 activity showed that Sfrp1 overexpression significantly decreased Caspase-3/7 activity when compared to the H/R group(P<0.05).Western blot showed that Sfrp1 overexpression,significantly increased Bcl-2/Bax ratio and the expression of Cleaved.caspase-3 compared with the H/R group(all P<0.05).However,when Wnt/?-catenin signaling pathway specific activator Licl simultaneous action,the effect of Sfrp1 on H9c2 cells apoptosis was inhibited.(2)The results of MTT showed that Sfrp1overexpression decreased the proliferation of cardiac fibroblasts induced by TGF-?1,and the MTT-OD value was 0.532±0.062 vs.0.293±0.025(P<0.05).Flow cytometry was used to detect the distribution of proliferative cycle,the results showed that Sfrp1overexpression significantly increased the percentage of cells in G0/G1 phase and G2/M phase compared to the TGF-?1 group,53.65±1.78 vs.63.61±1.96%and 9.24±0.82vs.11.80±0.25%respectively,decreased the percentage of the S period,37.42±0.78vs.24.57±0.66%(all P<0.05).The results of ELISA detection showed that Sfrp1overexpression was decreased the production of I and III collagen when compared to the TGF-?1 group in cell supernatant,2123.65±543.73 vs.1127.92±194.73pg/ml?10.535±1.206 vs.5.524±1.236ng/ml(all P<0.05).Western blot showed that Sfrp1overexpression was significantly decreased the level of?-SMA protein expression when compared to the TGF-?1 group(P<0.05).However,while the Licl was acting at the same time,the role of above anti-fibrosis of Sfrp1 were inhibited.Part III:Genotype distributions were significantly different for SFRP1 rs7832767(P=0.022),CTNNB1rs2293303(P=0.009)and WISP1 rs16893344(P=0.007)between the cases and controls.Then we found that the SFRP1 rs7832767 variant allele(T)was associated with a significantly increased risk of MI[TT vs.CC:OR=3.13,95%CI=1.78-5.51;CT/TT vs.CC:OR=1.53,95%CI=1.12-2.08].The significant association with MI risk was also found for the CTNNB1 rs2293303(CT vs.CC:OR=3.48,95%CI=2.28-5.33;TT vs.CC:OR=7.37,95%CI=2.08-26.16;CT/TT vs.CC:OR=3.72,95%CI=2.46-5.62),and WISP1 rs16893344polymorphisms(CT vs.CC:OR=2.43,95%CI=1.70-3.47;TT vs.CC:OR=5.17,95%CI=1.85-14.41;CT/TT vs.CC:OR=2.58,95%CI=1.83-3.66).The associations remain significant in stratified analysis by demographic and clinical characteristics of participants,with few exceptions.Conclusion:(1)Wnt/?-catenin signaling pathway is a key target on aging acute myocardial ischemia,early after acute myocardial infarction,Wnt/?-catenin signaling pathway was continuously activated.Sfrp1 might be used as a small molecule gene therapy drug,improved the heart function and reduced the degree of myocardial fibrosis,meanwhile,inhibited myocardial cell apoptosis and reduced the acute ischemic injury of aging myocardium by inhibiting Wnt/?-catenin signaling pathway,therefore effectively protect the aging of acute myocardial ischemia.(2)At the cellular level,it was proved that Sfrp1 inhibited the Wnt/?-catenin signaling pathway and regulated the mitochondrial pathway in order to resist the apoptosis of cardiomyocytes.It was also demonstrated that Sfrp1 inhibited the Wnt/?-catenin signaling pathway,and decreased the proliferation of cardiac fibroblasts,collagen synthesis,and the phenotype transformation of muscle fibroblasts,thus alleviating the development of myocardial fibrosis.(3)The SFRP1 rs7832767 C>T,CTNNB1 rs2293303C>T and WISP1 rs16893344C>T variant genotypes may contribute to the risk of myocardial infarction.Larger,well-designed,prospective studies with different ethnic populations are warranted to validate our findings. |
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