The Role And Mechanism Of CREG1 Truncated Peptide NC51 In Acute Myocardial Infarction

Part I: Activity analysis of CREG1 truncated polypeptide and its effect on macrophage inflammationObjective: Inflammation is an important pathophysiological reaction during the occurrence and evolution of myocardial tissue injury.Moderate inflammatory response can help the injured site to resist the invasion of pathogens,remove the necrotic tissue and promote the repair of the damaged tissue.On the contrary,excessive inflammatory response will lead to delayed repair of the injured site,aggravating cardiac pathological injury and ultimately worsening cardiac function.As a kind of important innate immune cells,mononuclear macrophages not only initiate inflammatory response,but also promote tissue regeneration and inflammatory repair.Previous studies have found that the cellular repressor of E1 A stimulated genes 1(CREG1)protein has an anti-inflammatory effect,but the effective structural region of its anti-inflammatory effect is still unclear.This part aims to identify the active region of CREG1 in macrophages where it plays an antiinflammatory role,so as to obtain smaller molecular weight fragments that can effectively inhibit inflammatory response.Methods: In this part,CREG1 is truncated into peptides of different lengths according to its domain.The human CREG1 recombinant protein or peptides were added to macrophage cell line RAW264.7,and lipopolysaccharide(LPS)stimulation was used to induce inflammation model.RT-PCR and ELISA were used to detectethe expression of IL-1β and IL-6.The effect of NC51 on macrophage migration were detected by scratch test.The expression of i NOS and CD206 were detected through RT-PCR and Western Blot.The effect of NC51 on cell viability was detected through CCK-8 assay.GFP-NC51 fusion plasmid was constructed,and NC51 was overexpressed in RAW264.7 cells by cell transfection technique.After LPS stimulation,the expression of IL-6 was detected by Western Blot.Results: The N-terminal 30-80 amino acid(NC51)polypeptide fragment of CREG1 significantly decreased the expression of IL-1β and IL-6 after LPS stimulation,but the other fragments had no significant effect on LPS induced macrophage inflammation.In addition,the inhibition effect of NC51 peptide on IL-1β and IL-6 expression in macrophages increased in a dose-dependent manner.NC51 peptide significantly reduced the migration area of macrophages.NC51 peptide down-regulated the expression of i NOS while up-regulated the expression of CD206 in macrophages.CCK-8 assay showed that NC51 peptide had no effect on macrophage viability within the concentration range where NC51 peptide exerts anti-inflammatory effects.After endogenous overexpression of GFPNC51 peptide fragments in RAW264.7 cells,IL-6 expression was significantly inhibited.Conclusion: The 30-80 amino acid polypeptide(NC51)in the N terminal of CREG1 protein has definite anti-inflammatory effect in macrophages.NC51 peptides inhibit the migration of macrophages and their transformation into M1 macrophages.NC51 polypeptide had no obvious toxic effect on macrophages.Part II: The role of NC51 peptide in acute myocardial infarctionObjective: The inflammatory reaction of macrophages ran through the whole process of(acute myocardial infarction,AMI),affecting the prognosis of patients with AMI.Our previous studies have demonstrated that CREG1 protein can alleviate myocardial cell damage,myocardial fibrosis and slow the occurrence of heart failure after AMI.In the first part of this study,NC51 was found to play an anti-inflammatory role in CREG1 in macrophages.This part of the study attempted to investigate whether NC51 peptide can alleviate myocardial injury in AMI model mice,and whether its cardiac protective effect is related to its anti-macrophage inflammation.Methods: AMI model was constructed in C57 mice.After 1 d of AMI operation,different CREG1 peptide fragments or human CREG1 recombinant protein were administered by subcutaneous implantable osmotic pressure sustained release pump.After 28 day of AMI operation,cardiac function related indexes were detected in each group.Echocardiographic assessment was used to assess cardiac function in mice.The heart weight,body weight,and tibial length of mice were measured to calculate the HW/BW and HW/TL.Then ELISA kit was used to detect the contents of CKMB,AST and ALT in serum,and IL-1β and IL-6 in myocardial tissue at the edge of infarction.HE,MASSON and CD68 immunofluorescence staining were used to detect the infarct size,cardiac fibrosis and macrophage infiltration in each group.RT-PCR and Western Blot were used to detect the expression of IL-1β,IL-6,i NOS,and CD206.Results: Compared with sham group,the EF% and FS% of heart in MI group were decreased,LVEDd,LVESd,HW/BW and HW/TL were significantly increased,the myocardial infarction area,fibrosis area and CD68-positive macrophage were also increased.The levels of IL-1β and IL-6 in myocardial tissue at the edge of infarction were significantly increased.Compared with MI group,the EF% and FS% of heart in NC51 and CREG1 groups were significantly increased,HW/BW and HW/TL were significantly decreased,and the area of myocardial infarction and fibrosis were also significantly decreased.However,other peptide fragments had no significant effect on AMI model.NC51 group and CREG1 group also reduced the content of CKMB in serum of mice,but had no significant effect on AST and ALT.In addition,the NC51 and CREG1 groups reduced the infiltration of CD68-positive macrophages and the expression of IL-1β,IL-6 and i NOS in myocardial tissue at the edge of infarction,but increased the expression of CD206.Compared with CREG1 group,NC51 group and CREG1 group showed no significant difference in the above indexes.Conclusions:(1)Exogenous administration of NC51 peptide reduced myocardial infarction and fibrosis area and improved cardiac function in AMI mice.(2)Exogenous administration of NC51 peptides inhibited macrophage inflammatory response after myocardial infarction;(3)There was no significant difference between NC51 peptide and CREG1 in the treatment of AMI.Part III: Mechanism of NC51 peptide in macrophage inflammatory responseObjective: After clarifying the role of NC51 peptide on macrophage inflammatory response after AMI,we aim to further elucidate the molecular mechanism of NC51 peptide on macrophage inflammatory response.Methods:(1)The ability of FITC-NC51 peptides to enter macrophages was detected by flow cytometry,full-wavelength enzymoscope and laser confocal scanning microscope.(2)Cell membrane proteins were extracted from bone-marrow macrophages of CREG1 completely knocked out mice and supplemented with NC51 polypeptide.The target proteins affecting peptide entry were screened by tandem mass spectrometry quantitative proteomics,and the mass spectrometry results were verified by Co IP assay.(3)In order to clarify the necessity of the target protein for NC51 polypeptide to enter cells,the target protein was silenced by cell RNA interference technology,and then the peptide entry into cells was observed by laser confocal microscopy,and the expressions of IL-1β and IL-6 were detected by Western Blot.(4)The effect of NC51 peptide on Sirt1 expression in macrophages and MI marginal region was detected by Western Blot.(5)Sirt1 was silenced by cellular RNA interference technique,and the expressions of IL-1β and IL-6 were detected by Western Blot.Results:(1)After adding NC51 polypeptide to the bone marrow derived macrophages of CREG1 knockout mice,the cell membrane proteins were extracted,and 24 different proteins were obtained by mass spectrometry.These proteins are mainly concentrated in pathways related to vesicle transport and protein endocytosis.(2)Flow cytometry showed that about 80% of RAW264.7 cells were fluorescence stimulated after FITC-NC51 was added into the cells.The fluorescence of cells in the FITC-NC51 group was also found to be significantly increased.(3)After observing the cells by laser confocal microscope,it was found that a large number of green round fluorescent substances appeared in the perinuclear nuclei of FITC-NC51 group.(4)Co-immunoprecipitation confirmed the association of NC51 polypeptide with ANXA1 in macrophages.(5)After the silencing of ANXA1 in macrophages,the entry of FITC-NC51 polypeptide into macrophages was significantly reduced.At the same time,the inhibition effect of NC51 peptide on IL-1β and IL-6 induced by LPS disappeared.(6)The expression of Sirt1 was down-regulated in LPS stimulated macrophages and cardiac marginal tissues,and NC51 peptide significantly reduced the expression of Sirt1.After silencing Sirt1,the inhibition of p65 acetylation by NC51 peptide and the inhibition of IL-1β and IL-6 disappeared.