The Regulation Role Of TLR4/JNK Signaling Pathway In The Sepsis-induced Myocardial Injury

Part 1:Establishment and evaluation of mouse models of lipololysaccharide-induced septic myocardial injuryObjective To establish mouse models of sepsis-induced myocardial injury by intraperitoneal injection of lipopolysaccharide?LPS?in order to provide a reliable method for the research of pathogenesis of sepsis-induced myocardial injury.Methods According to the method of random number table,a total of 150 mice were randomly divided into five groups:NC group,Sham group,and LPS 10,12,15mg/kg groups,30 mice in each group.Septic myocardial injury was induced by LPS in mice;Mice of Sham group were injected intraperitoneally?i.p.?with equal 0.9%saline;while there was no treatment on mice of NC group.15 of the 30 mice in each group were used to observe the general status of mice before and after LPS or saline injection.24 hours after LPS or saline injection,the left ventricular function was assessed by echocardiography,serum levels of cardiac troponin?cTnI?were determined by enzyme linked immunosorbent assays?ELISA?,and the cardiac histomorphology and ultrastructure were observed;the other 15 mice were used to monitor the 7-day mortality after LPS or saline injection.Results The mice subjected to LPS presented symptoms of sepsis,such as depression,ruffled fur,diarrhea.Compared with NC group,left ventricular ejection fraction?LVEF?,left ventricular fraction shortening?LVFS?were significantly decreased at 24 hours after LPS administration in LPS 10,12,15 mg/kg groups[LVEF:0.459±0.044,0.432±0.034,0.348±0.064 vs.0.588±0.019,LVFS:?22.36±2.60?%,?20.78±1.91?%,?16.27±3.31?%vs.?30.55±1.30?%,all P<0.01].cTnI were significantly increased?ng/L:270.40±43.50,281.14±41.79,298.39±42.05vs.192.59±16.90,all P<0.01?.Myocardium injury were observed in three LPS groups,myocardial fibrosis,interstitial edema,erythrocyte leakage and infiltrating inflammatory cells were observed under light-microscope;ultrastructural changes in disorderly arranged cardiac muscle fibers,mitochondrial swelling and even partly missing mitochondria cristae were found under transmission electron microscope?TEM?,and the higher of the dose,the heavier of the damage.There was no significant difference between sham group and NC group.The 7-day mortality in LPS 10,12,15 mg/kg groups were 33%,53%and 87%,respectively,while no deaths in the NC group and sham group.Conclusions For establishing the mouse model of sepsis-induced myocardial injury by intraperitoneal injection with LPS,12 mg/kg is the preferable choice in our research.Part 2:The regulation role of TLR4/JNK signaling pathway in the sepsis-induced myocardial injuryObjective Tumor necrosis factor-alpha?TNF-??plays an important role in sepsis-induced myocardial injury.The aim of our study was to investigate the regulation role of TLR4/c-Jun NH₂-terminal kinases?JNK?signaling in TNF-?expression and myocardial injury during LPS stimulation.Methods According to the method of random number table,mice were randomly divided into four groups:Normal Control?NC?group?n=6?,Normal Saline?NS?group?n=6?,LPS group?n=18?and TAK-242 group?n=18?.For time course experiments,mice of LPS group and TAK-242 group were sacrificed at 3h,12h and24h?n=6 for each time point?.Mice of LPS group were injected intraperitoneally?i.p.?with LPS?12mg/kg?.Mice of TAK-242 group were treated intraperitoneally with a TLR4 inhibitor TAK-242,1 hour later,followed by LPS?12mg/kg?.Mice of NS group were injected with equal 0.9%saline;while there was no treatment on mice of NC group.The general status of mice were observed before and after LPS or saline injection.Left ventricular?LV?function was assessed 3h,12h and 24h after LPS or saline injection during anesthesia.The morphological changes of myocardium were examined by pathological methods.Serum level of cardiac troponin?cTnI?and TNF-?were determined by ELISA.TLR4?JNK and c-jun mRNA levels were analysed by real-time RT-PCR.TLR4?JNK?p-JNK?c-jun and p-c-jun protein were analysed by western blot.Results1?changes of myocardial function:compared with NC group,LVEF?LVFS were significantly decreased in LPS groups?all P<0.01?;LVESD?LVESV were significantly increased at 3h and 12h after LPS injection?P<0.05?;LVEDD?LVEDV were significantly reduced at 12h and 24h after LPS injection?P<0.05?.Compared with LPS groups,pretreatment of TAK-242 induced a significant increase of LVEF and LVFS?P<0.01?,and significantly decreased LVESD and LVESV?P<0.05?at3h after LPS injection,and reduced a significant increase of LVEDD?LVEDV at 12h and 24h?P<0.05?but a significant decrease at 3h?P<0.01?after LPS injection.2?serum levels of cTnI and TNF-?:serum cTnI and TNF-?protein levels were significantly increased by LPS stimulation compared to NC group?P<0.05?;while compared with LPS groups,pretreatment of TAK-242 induced a significant decrease of serum cTnI and TNF-?protein levels?P<0.05?.3?Myocardium injury were observed in LPS groups:myocardial fibrosis,interstitial edema,erythrocyte leakage and infiltrating inflammatory cells were observed under light-microscope;ultrastructural changes in disorderly arranged cardiac muscle fibers,mitochondrial swelling and even partly missing mitochondria cristae were found under transmission electron microscope?TEM?.Compared with LPS groups,pretreatment of TAK-242 ameliorated LPS-induced myocardium injury.4?TLR4 expression in the myocardium:TLR4 mRNA and protein expression in response to LPS were significantly increased?P<0.05?,and reached the maximal levels around 3h;while pretreatment of TAK-242 significantly decreased TLR4mRNA and protein expression at 3h after LPS injection?P<0.05?.5?JNK and p-JNK expression in the myocardium:JNK mRNA and protein expression in response to LPS were significantly increased?P<0.05?,while pretreatment of TAK-242 significantly decreased JNK mRNA and protein expression at 12h after LPS injection?P<0.05?.A significant increase of phosphorylation of JNK in respond to LPS started within 3h and reached the peak around 12h.6?c-jun and p-c-jun expression in the myocardium:at 3h after LPS injection,c-jun mRNA expression was significantly increased in LPS group?P<0.05?,but significantly decreased in TAK-242 group?P<0.05?;while c-jun protein expression was significantly increased in respond to LPS,but decreased by TAK-242 at 12h after LPS injection.Phosphorylation of c-jun was significantly increased in LPS groups compared with NC group?P<0.05?;however pretreatment of TAK-242 significantly increased phosphorylation of c-jun induced by LPS rather than decreased the level of p-c-jun.Conclusions Our data suggest that JNK activation induced by LPS/TLR4 may represent an important mechanism resulting in TNF-?production and myocardial injury during sepsis in mice.Therefore,TLR4/JNK signaling pathway plays an important role in the sepsis-induced myocardial injury.