Alamandine Attenuates Sepsis-associated Cardiac Dysfunction Via Inhibiting MAPKs Signaling Pathways

Background: Sepsis is a systemic inflammatory response syndrome caused by infection.It is one of the most common fatal diseases in Intensive Care Unit.Epidemiological investigation shows that 40% of sepsis patients are complicated with myocardial injury and cardiac dysfunction.Despite effective treatment including anti-infection,circulatory support and enhanced myocardial contractility,the mortality rate of patients with sepsis myocardial injury is still as high as 70% to90%.Therefore,it is of great significance to clarify the pathogenesis of sepsis myocardial injury and to discover new specific therapeutic targets for improving clinical prognosis.RAS system plays an increasingly important role in the pathogenesis of sepsis in recent years.It is shown in some studies that RAS system is involved in LPS-induced sepsis inflammation and myocardial cell autophagy and apoptosis.As a new member of the RAS system,Alamandine display the function in opposite to angiotensin II,such as diastolic blood vessels,lower blood pressure,inhibit cardiomyocyte hypertrophy and fibrosis,inhibiting inflammatory reaction and cell autophagy apoptosis.MAPK/NOS signaling pathway is an important signal transduction pathway taking part in the pathological process of sepsis,and many of the mechanisms of Alamandine are also mediated by the MAPK/NOS signaling pathway.Therefore,this study proposed the hypothesis that Alamandine could improve LPS-induced myocardial injury by MAPKs signaling pathway.Objective: 1.To observe the effect of Alamandine on cardiac function in LPS-induced sepsis mice.2.To observe the effect of Alamandine on myocardial inflammatory response induced by LPS.3.To observe the effect of Alamandine on autophagy apoptosis of cardiac myocytes induced by LPS.4.To observe whether Alamandine can improve LPS-induced myocardial injury by MAPKs signaling pathway.Methods: 1.To select the optimal injection dosage of Alamandine,Alamandine(0.01,0.1,1.0,10um/kg)was injected into the tail vein and LPS(10mg/kg)was intraperitoneally injected to construct the mouse sepsis model.After modeling for 12 hours,the expression of serum inflammatory factor TNF-α and IL-1β in each group was detected to evaluate the pharmacodynamic effect of Alamandine.2.Animal experiment groups: saline group,LPS group,Ala group,Ala+LPS group;Drug dosage: Alamandine(1.0um /kg),LPS(10mg/kg).After 12 hours modeling,echocardiography was used to evaluate the cardiac function of mice.Blood pressure were monitored by transcaudal artery.Mouse was killed,cardiac and serum samples were retained.3.Cell experiment groups: PBS group,LPS group,Ala group,Ala+LPS group;Drug dosage: Alamandine(1ug/ml),LPS(100ng /ml).12 hours After drug treatment and cell culture,protein and m RNA were extracted.4.Cell experiment groups for MAPKs:PBS group,LPS group,db-camp group,Ala+LPS group,db-camp+Ala+LPS group;Dosage:db-camp(10u M/ml),Alamandine(1ug/ml),LPS(100ng/ml).12 hours After drug treatment and cell culture,protein and m RNA were extracted.5.Electron microscope was used to observe abnormal cardiac tissue structure;Cardiac apoptosis was observed by CC3 immunofluorescence and TUNEL staining;Western Blot was used to detect the expression of i NOS/e NOS,MHC-α/β,LC3-I/II,Bax,Bcl2 and other indicators in protein levels.RT-PCR technology was used to detect the m RNA level expression of MHC-α/β,S100A8/S100A9,Atg3/5and other indicators.Results: 1.In addition to the Alamandine(0.01 u M/kg)group,all Alamandine concentrations inhibited the verexpression of TNF-α and IL-1β in the serum of mouse with LPS-induced sepsis.Considering the dose-pharmacodynamic relationship of Alamandine and the possible side effects,1.0 u M/kg was selected as the experimental dose of Alamandine.2.Echocardiography showed that Alamandine(1.0um /kg)pretreatment could effectively improve cardiac dysfunction caused by LPS,and improve EF and FS values.3.Alamandine inhibited abnormal expression of apoptosis indicators TUNEL,CC3,Bax,Bax/Bcl2,and autophagy indicators lc3-I/II,atg3/5 induced by LPS,In both animal and cell experiments.4.Alamandine inhibited abnormal expression of cardiac injury indicators MHC-α/β,S100A8,S100A9 induced by LPS,In both animal and cell experiments.5.LPS leads to the overexpression of i NOS/e NOS,and the reduction of blood pressure in mice,while Alamandine,which has the function of relaxing blood vessels,does not aggravate this process.6.In the MAPKs pathway,Alamandine can improve the abnormal expression of ERK,JNK and p38 caused by LPS,while db-c AMP can antagonize the effects of Alamandine.Conclusions: 1.Alamandine inhibited the myocardial inflammatory response and autophagy apoptosis induced by LPS;2.Alamandine can improve cardiac dysfunction and myocardial injury induced by LPS in mice;3.Alamandine does not aggravate LPS induced hypotension and vascular endothelial dysfunction;4.The effect of Alamandine on LPS may be related to the inhibition of MAPKS signaling pathway.