Modulation Of Aminooxyacetic Acid On Macrophage Polarization And Cardiac Function After Myocardial Infarction In Mice

BackgroundIn the past 20 years,owing to the implementation of primary percutaneous coronary intervention and the prescription of evidence-based drug treatments,the mortality of acute myocardial infarction has decreased significantly.However,acute myocardial infarction leads to irreversible loss of myocardial cell and adverse ventricular remodeling.Some patients will suffer from heart failure later,which seriously impairs the long-term prognosis of patients with myocardial infarction.Excessive activation of pro-inflammatory M1 macrophages following myocardial infarction aggravates adverse cardiac remodeling and heart dysfunction.There are two breakpoints in the tricarboxylic acid cycle of M1 macrophages,and aspartate-arginosuccinate shunt compensates them and continues the interrupted cycle.Aminooxyacetic acid(AOAA)is a strong inhibitor of aspartate aminotransferase in this shunt.Previous studies showed that manipulating macrophage metabolic reprogramming may control macrophage polarization and inflammatory response.Therefore,we speculate that AOAA may regulate macrophage polarization by modulating macrophage energy metabolism,and then improve cardiac function after myocardial infarction.Objective(1)In the present study,we set out to determine the effects of AOAA on macrophage energy metabolism and macrophage polarization by using primary mice bone marrow derived macrophages(BMDMs)and RAW264.7 cells at the cellular level.(2)Then,we will further clarify the effect of AOAA on cardiac function after myocardial infarction in a mouse model and preliminarily explore the potential mechanism.Methods and Results(1)BMDMs were induced and differentiated from mice bone marrow cells by M-CSF stimulation for 7 days in vitro.BMDMs were identified by morphological observation and flow cytometry analysis.BMDMs were then treated with 10ng/mL LPS and 10ng/mL IFN-y or 20ng/mL IL-4 for 24 hours,respectively.Morphological observation,flow cytometry analysis,immunofluorescence staining,and qPCR analysis were used to determine whether M1 and M2 macrophages were successfully polarized.The results showed that the bone marrow cells exhibited spindle or irregular shape and highly expressed CD11b and F4/80 after M-CSF induction for 7 days,suggesting that these cells were successfully differentiated into BMDMs.After stimulation with LPS+IFN-y or IL-4 for 24 hours,M1 macrophages turned into round or oval,while M2 macrophages were slightly elongated and still spindle or irregular shape.M1 macrophages were highly expressed CD86,iNOS,TNF-?,IL-6,and IL-1?,while M2 macrophages were highly expressed CD206,Argl,Ym1,and IL-10,indicating that M1 and M2 macrophages were induced successfully.At the same time,we also detected the changes of RAW264.7 cells after polarization stimulation.Though there had no significant change in morphology,the expression of markers and related cytokines were the same as BMDMs,which confirmed the successful induction of M1 and M2 macrophages in RAW264.7 cells.(2)After successful extraction,induction,and polarization of macrophages,we clarified the characteristics of macrophages energy metabolism by observing culture supernatant color,measuring pH value,lactate,ATP,and glycolysis levels,and detecting expression of glucose metabolism rate-limiting enzyme by qPCR.Then the effects of AOAA on macrophages energy metabolism were observed.The results showed that compared with M0,the supernatant turned into lemon yellow,pH value decreased,lactate level increased,ATP production decreased,glycolysis level increased,and expression of glucose metabolism rate-limiting enzyme increased in M1 macrophages;on the other hand,lactate level decreased significantly,ATP production increased,and glycolysis and expression of glucose metabolism rate-limiting enzyme were all inhibited in M2 macrophages,although supernatant color and pH value did not change significantly.At the same time,we detected the level of lactate and ATP after macrophage polarization in RAW264.7 cells,and the changing trend was consistent with BMDMs,which further confirmed that M1 and M2 macrophages had different metabolic characteristics.Interestingly,AOAA treatment increased the pH value,reduced the levels of lactate and glycolysis,increased ATP production in a dose-dependent manner,and inhibited the increase of glucose metabolism rate-limiting enzyme in M1 macrophages,suggesting that AOAA switch the metabolism of M1 macrophages towards M2-like metabolism.(3)Manipulating macrophage metabolism may control macrophage polarization.Therefore,we evaluated the effect of AOAA on macrophage polarization by immunofluorescence staining,Western blot and qPCR analysis in both BMDMs and RAW264.7 cells.We found that AOAA regulates macrophage polarization by restraining M1 macrophage phenotype and boosting M2 phenotype.These data suggest that AOAA may regulate macrophage polarization by regulating macrophage metabolizing reprogramming.(4)Then,we constructed a mouse model of myocardial infarction,and evaluated the effects of AOAA on cardiac function,myocardial infarction area,inflammatory response,and macrophage polarization in hearts by echocardiography,Masson's trichrome staining,H&E staining,immunofluorescence staining,Western blot,and qPCR analysis.We found that AOAA reduced the area of myocardial infarction and improved cardiac function.Further study showed that AOAA reduced the proportion of M1 macrophages and boosted the proportion of M2 macrophages in the ischemic border zone.Accordingly,we found that AOAA regulated the expression of macrophage associated pro-inflammatory and anti-inflammatory cytokines in the hearts.(5)NLRP3 inflammasome play an important role in the pathophysiology of myocardial infarction,and they are activated predominantly in macrophages.Therefore,we investigated the effects of AOAA on NLRP3-caspase 1/IL-1? signaling pathway in both cell and animal levels by using Western blot and qPCR analysis.The results showed that AOAA restrained the excessive activation of NLRP3,caspasel,and IL-1? in both M1 macrophages and the hearts of myocardial infarction mice.Conclusion(1)Short-term AOAA treatment during the peak inflammatory phase of the immune response remarkably improves cardiac function in mice with myocardial infarction.(2)The cardioprotection of AOAA may be achieved by balancing macrophage polarization through modulating macrophage energy metabolism,and at least in part by inhibiting the excessive activation ofNLRP3-Caspase1/IL-1? pathway.The results confirm the feasibility of modulating macrophage polarization by regulating macrophage metabolic reprogramming,emphasize the important role of macrophages in the pathophysiology of myocardial infarction,and provide a new idea for exploring strategies of regulating macrophage polarization and improving cardiac function of patients with myocardial infarction.