Effect Of TLR4/JNK Signaling Pathway On Apoptosis In Septic Myocardial Injury

Objective:A mouse model of sepsis myocardial injury was established by intraperitoneal injection of lipopolysaccharide.To investigate the role of TLR4/JNK signaling pathway in the process of apoptosis in septic myocardial injury,the expressions of pathway proteins,inflammatory factors,and apoptotic proteins were detected.Methods:1.54 SPF male C57BL/6 mice were randomly divided into three groups: Normal Saline group(NS group),sepsis group(LPS group)and TLR4 inhibitor group(TAK-242 group).To establish a mouse model of septic myocardial injury by intraperitoneal injection of lipopolysaccharide(LPS)12 mg/kg,TAK-242 groups were injected TAK-242 2 mg/kg 1 hour later followed by LPS.NS groups were injected with the same volume of normal saline.2.At 3 h,12 h and 24 h after injection,the pathological changes of myocardial tissue were observed by hematoxylin-eosin staining(HE),and the changes of myocardial ultrastructure were observed by electron microscope.3.Apoptosis were determined by terminal deoxynucleotidyl transferase mediated d UTP Nick end labeling(TUNEL)at 3 h,12 h and 24 h after injection.And calculated the apoptotic index(AI).4.The expression of toll like receptor 4(TLR4),c-jun amino-terminal kinase(JNK),p-JNK,nuclear transcription factor-κB(NF-κB),activating aspartate specific cysteine proteinase3(cleaved-caspase-3),B-lymphoma-2 gene(Bcl-2),Bcl-2-related X protein(bax),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)protein were determined by Immunohistochemical staining(IHC).5.ANOVA was used for multiple groups,t-test was used for two groups,and Pearson correlation coefficient method was used for correlation analysis among various indicators.P < 0.05 indicated that the difference was statistically significant.Results:1.Under the light microscope,the structure of myocardial cells in LPS group was loose and disordered,the fibers were broken and dissolved,the size of nucleus wasdifferent,the inflammatory cells infiltrated in the myocardial interstitium,the pathological changes increased with the prolongation of the molding time,and the pathological changes were the most obvious in 24 hours;the degree of myocardial injury in TAK-242 group was lighter than that in LPS group at the same time point.Electron microscopy showed that in LPS group,mitochondria were not arranged orderly,membrane rupture,cristae disappearing,matrix outflow,vacuole deformation,myocardial fiber rupture,nuclear pyknosis and fragmentation of some myocardium were observed.The organelle structure changed earlier,and pathological changes increased with the prolongation of modeling time;the above myocardial ultrastructural changes in TAK-242 group were less than LPS group at the same time point.2.The results of TUNEL semi quantitative analysis showed that the AI increased with the prolongation of modeling time,and reaching the peak value at 24 hours;the AI of NS group,TAK-242 group and NS group were [(74.3±8.6)% vs(59.1±5.4)%vs(15.1±3.6)%] at 24 hour.Among them,AI in LPS group was significantly higher than that in NS group,and AI in TAK-242 group was significantly lower than that in LPS group,and the difference was statistically significant(P< 0.05).3.The results of IHC staining showed that TLR4,JNK,p-JNK,NF-κB,cleaved-caspase-3,bax,TNF-α and IL-6 levels in LPS group were significantly higher than those in NS group at the same time point,TLR4 and IL-6 levels were the highest at 3 hours(TLR4: 0.171 ± 0.014;IL-6: 0.083 ± 0.022),JNK,p-JNK,cleaved-caspase-3 and TNF-α levels were the highest at 12 hours(JNK: 0.133 ±0.015;p-JNK: 1.148 ± 0.222;cleaved-caspase-3: 0.118 ± 0.023;TNF-α: 0.086 ±0.012),NF-κB and bax levels were the highest at 24 hours(NF-κB: 0.160 ± 0.012;bax: 0.132 ± 0.016).The TLR4,JNK,p-JNK,NF-κB,cleaved-caspase-3,bax,TNF-αand IL-6 levels in TAK-242 group were significantly lower than those in LPS group at the same time point,among which the JNK,NF-κB,bax and TNF-α levels were the lowest at 3 hours(JNK: 0.070 ± 0.020;NF-κB: 0.089 ± 0.018;bax: 0.075 ± 0.010;TNF-α: 0.055 ±0.007),and the TLR4,p-JNK,cleaved-caspase-3,and IL-6 levels were the lowest at 24 hours(TLR4: 0.055 ± 0.016;p-JNK: 0.701 ± 0.144;cleaved-caspase-3: 0.059 ± 0.015;IL-6: 0.057 ± 0.013).The bcl-2 and bcl-2/baxlevels in LPS group and TAK-242 group were significantly lower than those in NS group at the same time point,and reached the lowest at 24 hours(bcl-2: 0.061 ± 0.015,0.086 ± 0.011 vs.0.110 ± 0.012,bcl-2/bax: 0.469 ± 0.124,0.786 ± 0.217 vs.1.802 ±0.282,all P < 0.05).4.Correlation analysis results showed that the expression of TNF-α(r = 0.710,P< 0.01;r = 0.868,P < 0.01)and IL-6(r = 0.581,P < 0.05;r = 0.731,P < 0.01)were positively correlated with the p-JNK protein;p-JNK was positively correlated with AI(r = 0.583,P < 0.05;r = 0.686,P <0.01);TNF-α was positively correlated with AI(r= 0.834,P < 0.01;r = 0.715,P <0.01)and cleaved-caspase-3(r = 0.815,P < 0.01;r= 0.849,P < 0.01),TNF-α with bcl-2(r =﹣0.747,P <0.01;r =﹣0.722,P < 0.01)was negatively correlated;IL-6 was positively correlated with AI(r = 0.596,P < 0.01;r = 0.598,P < 0.01)and cleaved-caspase-3(r = 0.596,P < 0.01;r = 0.819,P < 0.01),with bcl-2(r =﹣0.523,P <0.05;r =﹣0.684,P <0.01)was negatively correlated.Conclusion:LPS can active TLR4/JNK signaling pathway and lead to inflammatory reaction and apoptosis.Inhibiting TLR4 and JNK signaling pathway,the apoptosis can be reduced and the degree of myocardial damage in sepsis can be alleviated through decreasing the expression of inflammatory factors.