| Objective: The most effective treatment for acute myocardial ischemia is timely and effective reperfusion therapy to reduce mortality,while reperfusion therapy improves myocardial blood supply accompanied by a series of pathological and physiological reactions,including peroxidation,inflammation,intracellular calcium overload,and finally irreversible cell apoptosis and necrosis.This reperfusion injury is called myocardial ischemia reperfusion injury(MI/RI).FOXO1 is an important transcription factor involved in myocardial ischemia reperfusion injury.The mechanism of the FOXO1 lactylation involved in myocardial ischemia reperfusion injury has not been clearly studied.Therefore,the involvement of lactate in myocardial ischemia reperfusion injury was studied in this paper,and the mechanism of FOXO1 lactylation in myocardial ischemia reperfusion injury was discussed,providing a new research target for improving myocardial ischemia reperfusion injury.Methods: In this study,the myocardial ischemia reperfusion injury model of SD rats was constructed,and the success of the modeling and the change of the global protein level of the new acylation were detected by WB experiment,and the optimal acylation modification was selected as the research target;The anoxia and reoxygenation model of rat cardiomyocytes was established in vitro to test whether the model was successfully constructed in vitro and whether the changes of the optimal acylation modified global protein level were consistent with those in vivo.WB test and RT-q PCR were mainly used to detect myocardial apoptosis indicators and inflammatory indicators after myocardial ischemia reperfusion injury to judge the condition of heart injury;The FOXO1 lactylation level of in myocardial ischemia reperfusion injury models in vivo and in vitro was detected by CO-IP assay;To verify the involvement of lactate in myocardial ischemia reperfusion injury by providing lactate group donor sodium lactate and inhibiting the generation of lactate,WB experiment and cck-8 experiment were mainly used;The downstream target of FOXO1 transcriptional regulation was determined by WB assay;The FOXO1 lactylation level of was studied by overexpression of HDAC1.Results:(1)Myocardial ischemia reperfusion injury in rats can cause myocardial injury and changes in the level of novel PTM protein.The ischemia reperfusion model(I/R)was established in vivo.The samples were collected after 45 min of hypoxia and 2h of reoxygenation,and protein was extracted for Western Blot test.The expression of apoptosis index BAX and Cleved-Caspase-3 protein increased,and myocardial infarction area increased significantly.The overall protein levels of lactate(Klac),succinylation(Ksu)and butyacylation(Kbu)were increased in myocardial ischemia-reperfusion injury model,and the number of acylation bands was enriched.Propionylation(Kpr)decreased,the number of bands was more,and the bands were enriched.Crotonylation(Kcr)and glutarylation(Kglu)had little change,and the number of bands was small.(2)Hypoxia and reoxygenation can cause myocardial cell injury and lactate protein level change in rats.Myocardial anoxia and reoxygenation model(H/R)was established in vitro.After anoxia for 9h/ reoxygenation for 3h,protein samples were extracted for Western Blot assay.Apoptotic indicators BAX,Cleaved Caspse-3 and NLRP3 protein expression were increased significantly.The m RNA expressions of BAX and NLRP3 were also increased.The changes of lactated protein levels were directly verified in the in vitro model,and the results were consistent with those of the animal I/R model in vivo,with overall lactated protein levels increased.(3)The myocardial ischemia reperfusion injury model of rats was established in vivo and in vitro,and lactate content expression in myocardial cells,heart tissue and serum of rats was detected at different time periods.The results showed that lactate content increased after hypoxia,and lactate level remained high after reoxygenation.Lactate content in heart tissue and serum increased after ischemia,but decreased after reperfusion.(4)Rat cardiomyocytes were treated with lactate modified donor sodium lactate at different concentration gradients,and the results showed that sodium lactate induced a concentration dependent increase in the expression of BAX and NLRP3 proteins,as well as an increase in the overall lactate level.(5)Glycolytic process related inhibitors can reverse H/R induced lactation,inflammation,and apoptosis.Under H/R conditions,the use of inhibitors such as 2-Deoxy-D-glucose(2-DG)and Sodium oxamate(SO)can significantly reduce the increase of the overall level of lactate induced by H/R,and reduce the protein expressions of BAX and NLRP3.It is proved that lactate is involved in myocardial ischemia reperfusion injury.(6)FOXO1 lactylation was induced after myocardial ischemia reperfusion.In vivo and in vitro models of myocardial ischemia reperfusion injury in rats were constructed,and the FOXO1 lactylation was detected by CO-IP assay.It was found that the FOXO1 lactylation increased significantly after myocardial ischemia reperfusion injury.(7)Inhibition of FOXO1 transcriptional activity can inhibit NLRP3 production and alleviate H/R induced apoptosis.NLRP3 is a downstream target of FOXO1 and is regulated by FOXO1 transcription.In the presence of H/R,inhibition of FOXO1 transcriptional activity by AS1842856 significantly inhibited the production of NLRP3,decreased the expression of Cleved-Caspase-3 protein and increased the cell activity.(8)HDAC1 as a lactated eraser can alleviate FOXO1 lactylation.Overexpression of HDAC1 was involved in sodium lactate and H/R regulation of FOXO1 lactylation.The results showed that overexpression of HDAC1 could reduce the FOXO1 lactylation by Nala.Overexpression of HDAC1 also decreased H/R induced FOXO1 lactylation.Conclusion: Myocardial ischemia reperfusion injury can induce lactate modification of FOXO1,and increase the expression levels of apoptosis factor BAX and Cleved-Caspase-3 protein.Inhibition of FOXO1 transcriptional activity can inhibit NLRP3 production.Meanwhile,the H/R-induced increased expression of Cleved-Caspase-3 protein and decreased cell activity were reversed,suggesting that FOXO1 regulate NLRP3 production through post-translational modification to mediate myocardial injury.HDAC1 can regulate FOXO1 lactylation process and provide theoretical data to improve the treatment of myocardial ischemia reperfusion by inhibiting FOXO1 lactate modification. |
