FTO Attenuates Myocardial Ischemia-Reperfusion Injury By Inhibiting NLRP3-Mediated Pyroptosis Via CBL/β-Catenin Pathway

Ischemia-reperfusion is the most effective treatment for acute myocardial infarction,but it can cause myocardial ischemia-reperfusion injury(IRI).Limited by the imperfect mechanism of myocardial IRI,there is currently no effective prevention and treatment for myocardial IRI.Therefore,in-depth study of its pathological mechanism is the basis for the development of myocardial IRI treatment.The previous literature analysis of this study found that NLRP3-mediated pyroptosis can promote myocardial IRI,while β-catenin can regulate NLRP3-mediated pyroptosis and is abnormally expressed in myocardial IRI.CBL can regulate the stability of β-catenin,and silencing CBL can alleviate myocardial IRI;the fat mass and obesity-associated(FTO)gene may regulate the expression of CBL,and overexpression of FTO can alleviate IRI.It is suggested that FTO,CBL and β-catenin plays important roles in the pathological mechanism of myocardial IRI.Therefore,this study proposed a hypothesis that FTO may regulate pyroptosis through CBL/β-catenin and participate in the course of myocardial IRI.To verify this hypothesis,this study carried out the following four parts:(1)To clarify the effect of FTO on NLRP3-mediated pyroptosis in myocardial IRI.The specific experiments include: constructing in vitro myocardial IRI model and detecting the expression level of FTO in myocardial IRI model,then constructing myocardial IRI cell model with FTO overexpression or knockdown to evaluate the effects of FTO on the viability,apoptosis and pyroptosis of ischemia-reperfusion cardiomyocytes.(2)To verify whether FTO affects pyroptosis in myocardial IRI via CBL and to explore its molecular mechanism.The specific experiments include: detecting the expression level of CBL in myocardial IRI model;the effects of CBL on the viability,apoptosis and pyroptosis of ischemia-reperfusion cardiomyocytes were evaluated by constructing CBL overexpression or knockdown myocardial IRI cell models.The expression of FTO and CBL,the interaction between IGF2BP3 and CBL were detected.The myocardial IRI cell model with simultaneous overexpression of FTO and CBL was constructed to verify whether FTO regulates pyroptosis through CBL.(3)To determine whether CBL regulates the expression of β-catenin and whether β-catenin affects NLRP3-mediated pyroptosis in myocardial IRI.Specific experiments include:detecting the expression level of β-catenin in myocardial IRI model;the myocardial IRI cell model of β-catenin overexpression or knockdown was constructed to evaluate the effect of β-catenin on the viability,apoptosis and pyroptosis of myocardial cells after ischemia-reperfusion;established FTO overexpression and silencing myocardial IRI model in vitro,the m6 A level of CBL mRNA was detected by Me RIP,and the CBL mRNA level was detected by RT-PCR to evaluate the effect of FTO on m6 A modification and mRNA transcription of CBL mRNA;the cell viability was detected by CCK-8,the apoptosis was detected by TUNEL fluorescence,and the levels of IL-18 and IL-1β were detected by ELISA to verify that FTO regulates pyroptosis through CBL to affect myocardial IRI.(4)To verify whether FTO inhibits pyroptosis and alleviates myocardial IRI by CBL/β-catenin in vitro and in vivo.The specific experiments include: constructing a myocardial IRI cell model with simultaneous overexpression of FTO and knockdown of β-catenin to evaluate the effects of FTO/β-catenin on the viability,apoptosis and pyroptosis of ischemia-reperfusion cardiomyocytes;the rat model of myocardial IRI with FTO overexpression or knockdown was constructed to verify the effect of FTO on myocardial IRI in vivo.The m6 A level of CBL,the expression levels of FTO,CBL,β-catenin and NLRP3-mediated pyroptosis-related proteins in myocardial tissue were detected.The results of this study showed that:(1)The expression of FTO was down-regulated in myocardial IRI model,and overexpression of FTO reduces myocardial IRI by inhibiting NLRP3-mediated pyroptosis.(2)CBL was up-regulated in myocardial IRI model,silencing CBL could attenuate myocardial IRI by inhibiting NLRP3-mediated pyroptosis,and FTO could inhibit IGF2BP3-mediated up-regulation of CBL expression to inhibit pyroptosis.(3)The expression of β-catenin was down-regulated in myocardial IRI model,overexpression of β-catenin reduced myocardial IRI by inhibiting NLRP3-mediated pyroptosis,and CBL inhibited the expression of β-catenin by promoting the ubiquitination degradation of CBL.(4)In vitro and in vivo,it was verified that FTO could alleviate myocardial IRI by down-regulating the expression of CBL,up-regulating the expression of β-catenin,and inhibiting pyroptosis.The results of this study showed that FTO could inhibit pyroptosis and alleviate myocardial IRI through CBL/β-catenin pathway.