Mechanisms Of The Protective Effect Of Glutamine On Lipopolysaccharide-induced Rat Myocardial Dysfunction

Objective:Cardiovascular dysfunction was found at the early stage of sepsis.50% of patients with severe sepsis and septic shock had left ventricular systolic/diastolic dysfunction. Mechanisms of cardiac dysfunction secon-dary to sepsis were complex and diverse. The primary mechanism was the activation of vascular endothehial cells, neutrophils, monocyte-macrophage system, as well as blood coagulation, complement and kinin systems by bacterial endotoxin(major component was lipopolysaccharide, LPS), proceeded with the release of various inflammatory mediators and cytokines (TNF-a, IL-β,lysosome, NO etc). these myocardial depressant factors (MDS) directly damaged myocardial cells and the metabolic functions of myocardial mitchondria and endoplasmic reticulum. In addition, calcium circulatory disorder of myocardial cells in sepsis and TNF-a-induced myocardial apoptosis promotes the occurrence of cardiac dysfunction. Factors involved in the mechanisms were so complex and various to find one single part to block. Toll-like receptors (TLRs) Could recognize the pathogen initiate innate immune response at the early stage of pathogen invasion, promote maturation and classification of immune cells, regulate immune response, and trigger inflammatory response. LPS could activate the TLR4 signaling pathways resulting in the activation of NF-κB, and then large amounts of inflammatory factors would be expressed, Leading to the occurrence of systemic inflammatory response and multiple organ failure. A20,a NF-KB-dependant cytosolic protein, Could block NF-κB activation via a negative feedback loop, and participate in the regulation of inflammatory response in vivo. Therefore, screening and downregulation of TLR4 to inhibit the source of infection, together with upregulation of the A20 expression and inhibition of LPS signaling transduction to protect cardiac function has become the hot spot in recent years.Gin, as the body's most abundant free amino acids, was a multi-target drug for myocardial protection, which could enhance the capacity of antioxidant activity and inhibition of nitric oxide production through the synthesis of GSH and induction of heat shock protein 70 expression.Therefore, Gin had been used in clinical as its protection for myocardial ischemia and reperfusion injury, as well as protection for intestinal mucosa immunological barrier. It could improve the prognosis of sepsis and had been proved safe. Studies had confirmed that Gin could downregulate the expression of TLR4 signaling pathway in sepsis to reduce the mucosal injury. Gin could also reduce the decomposition of I-KB and inhibit the activation of NF-κB to reduce the level of pro-inflammatory cytokines. In short, gln could inhibit TLR4 signaling by influnce the expression of TLR4 in upstream and activation of NF-κB in downstream, and thereby reduced inflammatory injury, meanwhile Gin had been used in isolated rat heart against aortic perfusion, and was proved protective effects on LPS induced myocardial injury as follows, improving the LPS-induced myocardial injury in monophosic activating amplitude, decreasing the extention of MAPD and MAPD 20,and reducing myocardial tension and heart rate. In our study, we constructed a LPS induced septic model, to find the protective effects of Gin on myocardial injury through the inhibition of TLR4-NF-κB signalling and LPS signalling. We hope to find an effective adjuvant treatment to prevent cardiac dysfunction secondary to sepsis.METHOEDS:1 Animal model and experimental groups:healthy and clean SD rat 90,male, body weight (350±25)g were randomly divided into 3groups: control group 40:saline 5ml/only;LPS group 40:LPS 10mg/Kg (in normal saline diluted to 5ml) LPS+Gln group 40:LPS 10mg/Kg+Glnlg/Kg in normal saline diluted to 5ml, intraperitoneally we sacrificed 6,24,48,72h after the animals injected.2 Intubate a catheter through the right carotid artery to the left ventricle at 6,24,48 and 72h after intraperitoneal injection.RM6240 BD multifunctional physiological recorders,was used to record the average blood pressure (MAP),left ventricular end systolic pressure,pressure rise and fall of left the maximum rate of change (±dp/dtmax) and other cardcial function.3 Apical tissues were fixed, dehydrated, embeded, HE stained, and we oberseved the changes of cardiac pathology.4 RT-PCR was used to detect myocardial TLR4,A20 and TNF-a mRNA expression,and Western-blotting was used to detect the expression of TLR4, A20,NF-κB P65 protein.5 RT-PCR was used to detect cardiac Bax,Bcl-2 mRNA expression, immunohistochemistry was used to measure myocardial Bax Bcl-2 protein expression TUNEL was used to examine the level of cardiomyocyte apoptosis,and colorimetric technique was used to determine caspase-3 activity.6 ELISA was used to measure the content of myocardial TNF-a serum CTnl,NT-proBNP.Results:1 Toxemia performance in rats:6h after injection of LPS, rats appeared fatigue sleepiness, reduction in the consumption of water,shortness of breath,increasing of eyes and nose's bloody dischange,diarrhea and weight loss. Heart enlargement and lung congestion were observed anatomically.2 Cardiac functional measurements:compared with controlled group, LVESP,+dp/dtmax,-dp/dtmax decreased 6h after LPS injection, and progressively decline and then rebounded till 72h. Gln increased +dp/dtmax,+dp/dtmax at different time points (p<0.05).3 Pathology:LPS included myocardical cell edema, degeneration and necrosis. Gin treatment group reduced myocardial cell injury.4 LPS group:myocardial TLR4, A20 and TNF-a mRNA and TLR4,A20, NF-κB 65protein expression increased; compared with LPS group, LPS+Glngroup decreased.The difference was significant (p<0.05).5 LPS led to decline in myocardial Bcl-2/Bax,caspase-3 activity and apoptosis rate Gln could significantly improve Bcl-2/Bax,and reduce the caspase-3 activity and apoptosis rat (p<0.05).6 In LPS group, myocardial TNF-a,serum cTnI,BNP concentration increased significantly.Gln could decrease TNF-a, CTnl,NT-ProBNP concentration (p<0.05).Conclusion:1 LPS signal pathway could induce expression of inflammatory mediators and myocardial apoptosis through TLR4, leading to myocardial injury.2 Gln could inhibit the TLR4/NF-κB signal pathway, and had a protective effect on the LPS induced myocardial injury.3 Gln could partially inhibit LPS-induced myocardial apoptosis, and partially improve cardiac function.4 Expression of the A20 was self regulation of NF-kappaB. Gln couldn't upregulate the expression of A20.