HFstl1-Fc Attenuates Myocardial Ischemia/reperfusion Injury By Regulating The Activation Of Macrophages

Background:Sudden death caused by the quick pace of modern life and the high pressure of routine work becomes prevalent in recent times.Acute myocardial infarction(AMI)is a disease of cardiomyocytes death resulted from transient or chronic myocardial ischemia-hypoxia in the coronary artery,a pathophysiological process involving many factors.Currently,not only the incidence rate of AMI is increasing year by year,but also the patients of it are getting younger and younger.Ill-informed of first-aid measures,some patients are not rescued timely and properly,making AMI a life-tlreatening disease.Common treatment strategies for AMI are thrombolytic therapy and percutaneous coronary intervention,both procedures aim to improve blood flow to the heart.While the reflow brings oxygen cardiomyocytes survive on,it also induces oxidative stress,activates inflammation pathway and inhibits ion pump activity,aggravating infarction.This cascade of injury is called ischemia/reperfusion(I/R)injury.During this process,damaged tissues and cells release dangerous molecular patterns(DAMPs),a kind of dangenous molecules which recruit and activate perpheral inflammatory cells to infiltrate targets such as cardiomyocytes,fibroblasts and vascular cells,resulting in excessive inflammation.Macrophages have target point called pattern recognition receptors(PRRs)that bind to DAMPs,thus the cells participate in various acute and chronic inflammations and are closely related to the repair of cardiac injury.Owing to different stimuli,macrophages will differentiate into two phenotypes:M1 macrophages(classical,pro-inflammatory)and M2 macrophages(alternative,anti-inflammatory).Recently,there is evidence that macrophage-mediated inflammatory response plays an important role in I/R.Therefore,targeting macrophage activation may be a significant therapeutic strategy to reduce I/R injury and salvage damaged cardiomyocytesFollistatin-like 1(Fstll)is a secreted glycoprotein induced by TGF-?1.It was reported that Fstll functioned as an inhibitor in inflammation,and that recombinant Fstll protein and Fstll-targeted gene therapy might be effective in rheumatoid arthritis and allogeneic heart transplantation.However,the regulation of Fstll in macrophage activation and its role in myocardial I/R injury remains unclear.Here,we used a kind of fusion protein hFstll-Fc,which was prepared from the whole length of human Fstll and the Fc fragment of IgG1,to exaimine the effect of this protein on I/R injury in C57BL/6 mice,and to explore its regulating role in macrophage activation in vitroPurpose:A fusion protein hFstll-Fc was prepared to exaimine the effect of this protein on I/R injury and to explore its role in macrophage activationMethods:1.hFstll-Fc plasmid was constructed from the full length of human Fstll and Fc fragment of IgGl.The fusion protein hFstll-Fc was expressed and purified by Mammalian expression system.2 In vivo,a model of myocardial Ischemia 40 minutes/Reperfusion 24 hours(I40min/R24h)was established in C57BL/6 mice.The protein was injected into tail vein at a dose of 100 pg/kg five minutes before reperfusion.Evans blue-TTC double staining was utilized to measure the area of myocardial infarction,and statistical analysis was performed.Fstl1+/-mice were used to establish the model of I40min/R24h,Evans blue-TTC double staining was utilized to measure the area of myocardial infarction,and statistical analysis was performed.3.In vitro,the main research objects were mouse macrophage cell line RAW264.7,mouse bone marrow derived macrophages(BMDMs)and primary peritoneal macrophages(PMs).The effects of hFstll-Fc on the chemotaxis of macrophages under Hypoxia/Reoxygenation(H/R)were investigated by transwell.The effects of hFstll-Fc on the reative oxygen species(ROS)release and the polarization of macrophages stimulated by lipopolysaccharide(LPS)were investigated by flow cytometry.The effects of hFstll-Fc on the release of inflammatory cytokines from macrophages stimulated by H/R and LPS were investigated by Real-Time PCR(RT-PCR).The role of hFstl1-Fc in the activation of NF-?B signaling pathway(p65 and its phosphorylation level)in macrophages stimulated by LPS was examined by Western blot.The effects of hFstl1-Fc on the entry of p65 into the nucleus of LPS-stimulated macrophages were examined by Western blot and Immuno fluorescence.Results:The fusion protein hFstl1-Fc was successfully prepared.In vivo,the model of I40min/R24h was established in C57BL/6 mice,and 100 ?g/kg hFstl1-Fc was given 5 minutes before reperfusion.The results of Evans blue-TTC double staining showed that hFstl1-Fc reduced the area of myocardial infarction by 21%compared with phosphate buffer saline(PBS)group.The model of myocardial I40min/R24h was established in Fstl1+/-mice and wild type(WT)mice.Results showed that the area increased by 43%in Fstl1+/-mice compared with WT mice.In vitro,the transwell results showed that hFstl1-Fc inhibited macrophage chemotaxis under H/R conditions.Flow cytometry results showed that hFstl1-Fc inhibited ROS release from macrophages,and reduced M1 type macrophages.RT-PCR results showed that hFstl1-Fc inhibited the release of inflammatory cytokines from macrophages under H/R.Western blot showed that hFstl1-Fc reduced the phosphorylation level of p65 in macrophages.Western blot and unmunofluorescence results showed that hFstl1-Fc inhibited the entry of p65 into the nucleus of macrophages stimulated by LPS.Conclusions:hFstl1-Fc may alleviate myocardial I/R injury by inhibiting macrophage-mediated inflammatory response via the deactivation of NF-?B signaling pathway.