S1PR3 Agonist RY-15 Promotes Bacterial Clearance Effect Of Macrophage Through The Phosphorylation Of ERK Pathway

Background:Sepsis is a clinically common life-threatening organ dysfunction caused by the body's abnormal response to infection.It is a common complication after severe burn trauma and major surgery.According to the latest data,more than 19 million people worldwide suffer from sepsis each year,including more than 5.3 million deaths,which is the leading cause of death in ICU patients.In the early stage,our team has completed a prospective survey on the epidemiological status of sepsis in China.The results show that the incidence of severe sepsis in China is 8.68%,which is equivalent to that in Europe and the United States.50%.Therefore,exploring the prevention and treatment of sepsis has extremely important clinical significance.The pathogenesis of sepsis is very complicated,and pathogenic microbial infection and inflammatory response are the basis of its pathophysiology.Pathogenic microbial infection and sustained inflammatory reaction in the body lead to tissue and organ damage in patients with sepsis,followed by acute lung injury or acute respiratory distress syndrome,septic shock,multiple organ failure,etc.,resulting in decreased ability of the body to remove pathogenic microorganisms.If the condition is not controlled,excessive inflammatory reactions will consume a large amount of cytokines and inflammatory mediators,leading to immunosuppression of the body,causing the pathogenic microorganisms to multiply,and the disease worsens or even dies.Therefore,early control and effective removal of pathogenic microorganisms have become the key to reducing the incidence of severe sepsis(multiple organ failure,DIC,etc.)and increasing the survival rate of sepsis.When the pathogenic microorganism invades the body,the innate immune system first makes a rapid immune response.Macrophages,as key effector cells in the body's innate immune response,are highly regarded in the early immunotherapy of sepsis.The way in which macrophages exert anti-infective effects is mainly three steps:the formation of phagosomes to take up pathogenic microorganisms,the phagosomes to obtain antibacterial ability,the secretion of cytokines to recruit inflammatory cells to the infected site to further eliminate pathogenic microorganisms,and the ERK signaling pathway as MAPK One of the subtypes is involved.S1P receptors(sphingosine 1-phosphate receptors,S1PRs)are G protein-coupled receptors,widely distributed in cardiomyocytes,endothelial cells,nerve cells,macrophages,T cells and other immune cells.It is a hotspot in the study of non-TLRs in sepsis at present.It binds to sphingosine 1-phosphate(S1P),a phospholipid molecule,to initiate cell transmembrane signal transduction and fine regulation.Cell proliferation,apoptosis,angiogenesis and inflammatory response.S1PR3 is an important member of the S1PRs family.It is coupled with three G proteins(Gi,Gq,G12/G13)to activate downstream PI3K/Akt,MAPK signaling pathways.On the one hand,it mediates macrophage recruitment to inflammatory lesions and release of inflammatory factors,on the other hand,it mediates macrophage clearance of bacteria.Previous studies have confirmed that the survival rate of mice infected with E.coli and CLP-induced sepsis in abdominal cavity of S1PR3 gene knockout group is significantly lower than that of wild mice,and the bacterial load of blood,lung,spleen and liver is increased.It is also confirmed that "S1PR3-G protein-Vps34-NOX2/EEA1 signaling pathway" is involved in the regulation of macrophage phage function by S1PR3.Previous clinical studies also showed that the expression of S1PR3 in ICU sepsis patients was significantly lower than that in sepsis survivors.The serum concentration of SIP(ligand substance of S1PR3 receptor)in severe sepsis patients was significantly lower than that in other sepsis patients and healthy volunteers.The low level of S1P in vivo was related to the mortality of sepsis patients.Therefore,we speculate that the application of specific targeted stimulation of S1PR3 in the early stage of sepsis will provide a new strategy for the prevention and treatment of sepsis.Objective:Previous studies have shown that KRX-725 is a short peptide consisting of a 9-amino acid sequence(143M-RPYDANK-R151)homologous to the second loop of S1PR3,which can specifically activate S1PR3 and enhance macrophages.Bacterial clearance can improve the prognosis of sepsis,but its transmembrane cell entry time is longer,and the stability of the amino acid sequence of the drug itself is insufficient.In this study,KRX-725 was further optimized to synthesize a new generation of S1PR3 specific agonist,RY-15,and to study its role in promoting macrophage clearance of bacteria and its mechanism,providing laboratory basis for clinical transformation research.Methods:KRX-725 is a short peptide with 9 amino acid sequences derived from the second intracellular loop of S1PR3.Our previous study showed that this short peptide can specifically activate S1PR3,enhance the bacteria clearance of macrophage and improve the prognosis of sepsis.On this basis,we further optimized the structure of the peptide,and studied its ability to enter cells,clear bacteria and phosphorylation of ERK pathway in THP-1 cells.Results:1.Molecular formulas gy-5 and RY-15 of S1PR3 specific agonist were synthesized.2.The laser confocal microscopy showed that the cells of THP-1 were inserted 10 min after the effect of RY-15.After 30 min,the cell entry effect was significant.3.The protective experiment of gentamicin showed that the number of surviving escherichia coli in macrophages after phagocytosis could be significantly reduced by RY 15(p<0.05),while GY-5 had no effect on the scavenging effect of macrophages.4.Western Blot results showed that GY-5 and RY-15 could phosphorylate the ERK pathway,and the activation effect of RY-15 was more significant.Conclusion:S1PR3 specific activator RY-15 can enter cells and promotes bacterial clearance of macrophage,which may be achieved through the phosphorylation of ERK pathway.