Effect Of Sphingosine Kinase 1 In Myocardial Injury

?Introduction?Myocardial infarction(MI)is the critical performance type of coronary heart disease(CHD).Recently,studies have shown that inflammatory response performed an important role in cardiac tissue injury after myocardial infarction.When Myocardial infarction occurs,a large number of cardiomyocytes encountered irreversible damage and necrosis due to ischemia or hypoxia,released cell membrane components and endogenous alarming signals,and activating the immune system,triggering an intense inflammatory reaction.During acute myocardial infarction(MI)progress inflammatory response could serve a dual effect.On the one hand,the inflammatory cells is necessity to remove necrotic cells in the infarct regions and contributes importantly to activate the endogenous reparative pathways.On the other hand,increased pro-inflammatory cytokines expression and persistent inflammation will lead to the degradation of matrix and the apoptosis of cardiomyocytes,thus cause infarction area expansion and d cardiac function deterioration.It is necessary to understand the inhibition or removal mechanism of the inflammatory response after myocardial infarction,and then to explore possible inflammation negative regulation mechanisms.Sphingosine kinase(SPHK),is an intracellular signaling enzyme that catalyzes thephosphorylation of sphingosine to sphingosine-1-phosphate(S1P).S1 P has been shown to be involved in many normal physiological processes,as well as in disease development and progression.Recently,it was proposed that SPHK1 play essential roles in many pathological processes that possess important inflammatory components.It is has been implicated in the regulation of immune cells,including neutrophils,monocytes and macrophages.Those critical roles highlights the therapeutic potential of targeting the SPHK1/S1 P pathway.And also facilitates us to decipher more physiological roles of this enzyme in different disease models.?Objectives?1? To investigate the effect of SPHK1 in cardiac tissue injury,pro-inflammatory cytokines level,macrophage infiltration and fibrosis after myocardial infarction.2? To analyse roles and preliminary mechanisms of SPHK1 in regulating macrophage.?Methods?1? The mice model of acute myocardial infarction was established by ligaturing the left anterior descending coronary artery.With or without the intervention of an isoenzyme-specific SPHK1 inhibitor,SK1-I,by subcutaneously implanted capsule osmotic pump.2? Western blot to detect proteins expression;To evaluate myocardial injury,HE staining was used to observe the myocardial tissue pathological changes,and ELISA method to detect serum cTnT;To estimate the inflammation of myocardial tissue and macrophages infiltration,immunohistochemical staining was chosen to observe the expression of CD68,and RT-qPCR to detect inflammation cytokines and macrophage surface molecular F4/80 mRNA level;Masson staining and collagen type?and ? mRNA level detection to assess myocardial fibrosis.3? Mice macrophage cells Ana-1 was induced by LPS as the cell model,using SK1-I and CAY10444 to inhibit the macrophages SPHK1 and S1P3,respectively.Westernblot to detect proteins expression;Transwell assay to examine the impact on the migration ability;RT-qPCR to detect macrophages TNF alpha,IL-1beta,IL-6 mRNA level;Enzyme-linked immunosorbent assay to test inflammatory cytokines concentration in culture medium.?Results?1.Mice with the adminastration of SPHK1 inhibitior--SK1-I had markedly diminished pathological myocardial injury estimated by HE staining performance and lower serum concentration of cTnT.Furthermore,cardiac tissue expression of the cytokines TNF-alpha,IL-1beta,IL-6 was decreased and macrophages infiltration was lower evidenced by decreased CD68 expression and F4/80 mRNA level.In addition,SPHK1 inhibition diminished myofibroblast accumulation and collagen production.2.LPS could promote macrophages migration,and induce the expression of SPHK1S1P3 and p-AKT/AKT protein.SK1-I reduced the migration ability of macrophage,lessened the expression of cytokines,the same effect was observed after the useage of S1P3 antagonist and also p-AKT/AKT protein expression was partially attenuated when S1P3 was blocked.?Conclusions?1? The intervention of SPHK1 inhibitor has certain protective effect in myocardial injury,that may be related to alleviated myocardial tissue local inflammatory response after myocardial infarction.2? SPHK1 mediated macrophages migration and inflammatory cytokines secretion,S1P3-p-AKT/AKT may be involved in this regulation pathway.