| BackgroundHeart failure following myocardial infarction(MI)remains a major public health concern worldwide,exerting a substantial economic burden,has become a major threat to human health and is the leading cause of death in the world.The pathological process of myocardial infarction includes inflammatory reaction,granulation tissue formation and myocardial fibrosis.Cardiac remodeling is viewed as a key determinant of the clinical outcomes in heart diseases.Myocardial fibrosis is a common process of cardiac remodeling after myocardial infarction,characterized by excessive proliferation of cardiac interstitial fibroblasts,excessive deposition and abnormal distribution of collagen.At present,the specific pathogenesis of myocardial fibrosis is not clear,mainly believed to be closely related to the renin-angiotensin-aldosterone system,apoptosis,regulatory cytokines,oxidative stress,inflammatory factors and so on.These factors affect the occurrence and development of myocardial fibrosis through the same or different pathways.Recently,it has become an important task to determine therapeutic targets for inhibiting or reversing myocardial fibrosis.Therefore,finding drugs to inhibit ventricular remodeling has become one of the important research objectives in the prevention and treatment of cardiac fibrosis in heart failure.Treatment options to block or reverse fibrosis are scarce.Lcz696(valsartan/sacubitril),the first of the new ARNI(dual-acting angiotensin-receptor-neprilysin inhibitor)drug class,contains equimolar amounts of valsartan which is an angiotensin-receptor blocker,and sacubitril,which is aprodrug for the neprilysin inhibitor,and ARNI has been extensively reported to approve for the treatment of heart failure patients with reduced ejection fraction.ARNI,apart from blocking angiotensin Ⅱ(AngⅡ)-signaling,also augments natriuretic peptides by inhibiting their breakdown by neprilysin,an endopeptidase that degrades various vasoactive peptides,including ANP(atrial natriuretic peptide)and BNP(brain natriuretic peptide).Natriuretic peptides,activated in cardiac dysfunction and HF,counteract the RAAS and promote vasodilation,natriuresis,and inhibit fibrosis and hypertrophy.The addition of the neprilysin component in Lcz696 augments plasma levels of natriuretic peptides.Multiple signaling pathways have been demonstrated to be involved in cardiac remodeling by myocardial fibrosis.Wnt/β-catenin signaling is relatively silent in adult cardiac tissue,but reactivated after various cardiac injuries such as myocardial infarction,ranging from acute ischemic injury to chronic stress overload.Abnormal activation of Wnt/β-catenin signaling pathway may be related to the occurrence and development of myocardial fibrosis.Studies have demonstrated that inhibition of Wnt/β-catenin signaling can improve infarct healing area and prevent heart failure after myocardial infarction,suggesting that blocking Wnt/β-catenin signaling pathway may be a potential treatment for preventing adverse cardiac remodeling after myocardial infarction,effectively slowing down myocardial fibrosis and remodeling,thereby improving cardiac function.Soluble frizzled related protein1(sFRP-1)is a soluble frizzled related proteinl secreted as an antagonist of the Wnt/β-catenin signaling pathway.The level of β-catenin decreased significantly after sFRP-1 blocked the binding of Wnt ligand to frizzled receptors on the plasma membrane,suggesting that sFRP-1 mainly inhibited the Wnt/β-catenin signaling pathway.sFRP-1 overexpression inhibited the activation of Wnt/β-catenin signaling pathway,significantly inhibited myocardial fibrosis,slowed cardiac remodeling and improved cardiac function.Based on the above research background,our group intends to explore the role and molecular mechanism of ARNI in improving myocardial remodeling and cardiac dysfunction after myocardial infarction by constructing a mouse model of myocardial infarction,and propose a hypothesis that ARNI inhibits Wnt/β-catenin signaling pathway by regulating the expression of sFRP-1.Thus,it can reduce collagen secretion and deposition,inhibit the expression of fibrosis molecules,and improve myocardial fibrosis after myocardial infarction,providing new theoretical support and experimental basis for ARNI to improve myocardial fibrosis after myocardial infarction.The role and mechanism of ARNI in myocardial infarction were evaluated by in vivo and in vitro,and the potential role of ARNI on Wnt/β-catenin signaling pathway was explored through sFRP-1 inhibition.Objective1.By constructing mouse myocardial infarction model,to explore the effects of ARNI and valsartan on cardiac dysfunction and myocardial fibrosis in experimental mice with myocardial infarction.2.To investigate whether ARNI and valsartan can improve myocardial fibrosis and play a protective role in the heart through Wnt/β-catenin signaling pathway.3.In vitro,sFRP-1 inhibitor Way316606 was used to further explore whether ARNI regulates Wnt/β-catenin signaling pathway through sFRP-1.Methods1.Animal model construction and grouping120 eight-week-old male C57BL/6 mice were selected.The mice were induced by ligation of left anterior descending branch and divided into four groups:Sham group,MI group,ARNI group,VAL group.2.EchocardiographyThe cardiac function was then evaluated using the Vevo770 imaging system.The left LVEF,FS,early(E)peak,late(A)peak,LVED d,and LV mass were measured.The ratios of early-to-late mitral flow velocity(E/A)and diastolic velocity ratio(E’/A’)were also calculated.3.Animal processingMice were anesthetized with 0.8%pentobarbital sodium intraperitoneally,and their body weight was measured and recorded after ideal anesthesia.Blood was collected through the apex of the heart and perfused throughout the body.The heart was then cut out.The heart tissue,which was planned to undergo molecular biological testing,was stored in tissue cryopreservation tubes and placed in liquid nitrogen.Cardiac tissue planned for histopathological examination was fixed in a PBS solution containing 4%formaldehyde and stored at room temperature4.Serum measurementsEnzyme-linked immunosorbent assay(ELISA)-Mouse NT-proBNP ELISA kit was used to determine the concentration of mouse NT-pro BNP.5.TTC stainingThe heart was rinsed with normal saline and frozen at-20℃ for 30 min before being taken out.Then,itswere crosscut into slices of about 2 mm and stained with TTC to evaluate the size of myocardial infarction in mice.6.Histological examinationThe mouse tissues were collected,the weight of mice and the weight of heart were measured,and the image of the whole heart was obtained by camera.3-5mm coronal surface of ventricular papillary muscle was taken from mouse heart tissue for fixation with 4%formaldehyde tissue fixative.After fixation for 48-72 hours,paraffin embedded and sectioned,dewaxing and other treatments were followed by HE,Masson,Sirius Red,TUNEL,Immunohistochemistry and Immunofluorescence histological staining.7.Extraction and culture of primary myocardial fibroblasts from Suckling mice Fibroblasts(CFs)were extracted from the hearts of newborn mice at 1-3 days.The cells were cultured in DMEM medium containing 10%fetal bovine serum and double antibiotics,and placed in a cell incubator containing 5%C02 at a constant temperature of 37℃.8.Real-time quantitative RT-PCR The mRNA expressions of β-catenin、sFRP-1、GSK-3β、Dvl-1、α-SMA、TGF-β、ANP and BNP were detected by real-time quantitative RT-PCR.9.Western blotThe protein expression of β-catenin、active-β-catenin、sFRP-1、GSK-3β、α-SMA、TGF-βand Coll Ⅲ proteins were detected by western blot.10.Statistical AnalysisAll data are expressed as mean ± SEM derived from at least three independent experiments.Statistical analysis was performed using GraphPad Prism 8.The Shapiro-Wilk test was applied for normality assumption.Thereafter,normally distributed data were analyzed using the unpaired two-tailed Student’s t-tests(two-group comparisons),or the one-way ANOVA followed by Dunnett’s or Tukey’s post hoc tests(comparisons between multiple groups).p<0.05 was considered statistically significant.Results1.ARNI improves myocardial remodeling and cardiac dysfunction after myocardial infarctionHistological staining showed that left ventricular volume and cross-sectional area decreased significantly in mice with myocardial infarction after drug treatment,especially in the ARNI group.The results of echocardiography and NT-proBNP serology indicated that ARNI improved the cardiac dysfunction of myocardial infarction significantly than valsartan.2.ARNI improves myocardial fibrosis after myocardial infarctionMasson and Sirius Red staining of myocardial tissue showed obvious fibrosis in the infarct area and surrounding area in the MI group,and decreased fibrosis in the VAL group,but more obvious in the ARNI group than in the VAL group.At the same time,the expression ofα-SMA,TGF-β,collagen Ⅰ and Ⅲ in myocardial tissue of mice in MI group increased,and the expression of these molecules showed a downward trend after drug intervention,which was more obvious in ARNI group than in VAL group.3.ARNI improves apoptosis induced by myocardial infarctionTUNEL staining of myocardial tissue sections of mice in each group showed that compared with the control group,DNA damage of myocardial cells was significantly increased and the proportion of TUNEL positive cells was significantly increased after myocardial infarction,while DNA damage of myocardial cells in ARNI and VAL groups was decreased and the proportion of TNNEL positive cells was decreased.And the ARNI group was more significant.4.ARNI may inhibit Wnt/β-catenin signaling pathway after myocardial infarction by sFRP-1After 4 weeks of myocardial infarction,the expression levels of β-catenin and activatedβ-catenin in myocardium of mice were higher than those of sham operation group.After ARNI intervention,the expression levels of β-catenin and activated β-catenin were decreased and significantly higher than that of valsartan.The expression trend of Dvl-1 was the same asβ-catenin.The expression trend of GSK-3β was opposite to that of β-catenin.In order to further verify the effect of ARNI on Wnt/β-catenin signaling pathway in vitro experiments,after Ang II stimulation,the expression levels of β-catenin and Dvl-1 in fibroblasts were increased,and the expression levels of β-catenin and Dvl-1 in fibroblasts were inhibited by ARNI and significantly higher than that of valsartan.These results suggest that Wnt/β-catenin signaling pathway is activated in the process of myocardial fibrosis,and ARNI can inhibit Wnt/β-catenin signaling pathway significantly more than valsartan.At the same time,we found that the expression of sFRP-1 molecule in myocardial tissue of mice after myocardial infarction was increased after drug treatment,and ARNI was more significant than valsartan.We believe that the inhibition of ARNI on Wnt/β-catenin signaling pathway after myocardial infarction may be related to sFRP-1.5.ARNI can improve collagen deposition and fibrosis by regulating sFRP-1The expression levels of α-SMA and TGF-β in fibroblasts stimulated by Ang Ⅱ were significantly higher than those in the blank control group,and the expression levels of α-SMA and TGF-β in fibroblasts treated with ARNI(Lcz696)were significantly lower than those in the Ang Ⅱ stimulated group.α-SMA expression level in ARNI group was significantly different from that in VAL group,but TGF-β expression level in ARNI group and VAL group was not statistically significant.The expression of collagen Ⅲ,α-SMA and TGF-β in fibroblasts treated with sFRP-1 inhibitor Way316606 was lower than that in Ang Ⅱstimulation group,while the expression of collagen Ⅲ,α-SMA and TGF-β in sFRP-1 inhibitor Way316606 was higher than that in ARNI group.These results suggest that ARNI inhibits collagen deposition in cardiac fibroblasts and improves fibrosis by regulating the expression of sFRP-1.6.ARNI inhibits the Wnt/β-catenin signaling pathway of fibroblasts by sFRP-1In vivo experiments,we found that the expression of sFRP-1 increased after ARNI intervention in mice with myocardial infarction.In order to explore the effect of ARNI on the expression of sFRP-1 in vitro experiments,we found that after ARNI or valsartan intervention,the expression of sFRP-1 increased significantly in the ARNI intervention group compared with the valsartan group.In order to further verify that ARNI regulates Wnt/β-catenin signaling pathway through sFRP-1,sFRP-1 inhibitor Way316606 was used to inhibit sFRP-1 expression in vitro.It was found that the expression of β-catenin protein in THE ARNI intervention group was significantly lower than that in the AngⅡ group.However,after sFRP-1 inhibitor Way316606 was added,the expression of β-catenin protein was higher than that in the ARNI group.The results showed that ARNI inhibited the activation of Wnt/β-catenin signaling pathway in fibroblasts through sFRP-1.Conclusion1.ARNI can improve left ventricular remodeling and function in experimental mouse myocardial infarction model,and has advantages over valsartan.2.ARNI may reverse myocardial fibrosis after myocardial infarction through Wnt/β-catenin signaling pathway.3.ARNI inhibited the Wnt/β-catenin signaling pathway of AngⅡ-stimulated cardiac fibroblasts by sFRP-1,and inhibited collagen deposition and fibrosis of cardiac fibroblasts... |
