Study On The Effect Of Triptolide On Myocardial Injury In Septic Rats And Its Molecular Mechanism

Objective:To replicate a rat model of sepsis by cecal ligation and puncture（CLP）,and to explore the effect of triptolide（TP）on myocardial injury caused by sepsis and its possible mechanism.Method:1.In vivo experimentMale SPF rats replicated the sepsis rat model by cecal ligation and puncture（CLP）.The TP intervention group and the polymyxin group were pretreated by intraperitoneal injection of drugs 4h and 28h after the modeling of the rats.The rats were randomly divided into 6 groups（n=10）:sham operation group（SHAME group）,Sepsis model group（CLP group）,TP low concentration intervention group（CLP+TP-L group,50μg/kg）,TP medium concentration intervention group（CLP+TP-M group,100μg/kg）,TP high concentration intervention group（CLP+TP-H group,200μg/kg）and polymyxin B group（CLP+PMX-B group,1 mg/kg）.The rats in each group were intubated with the common carotid artery 48 hours after the operation to monitor the mean arterial blood pressure（MABP）and hemodynamic parameters:left ventricular systolic peak pressure（LVPSP）,Left ventricular end diastolic pressure（left ventricular end diastolic pressure,LVEDP）,heart rate（HR）,left ventricular pressure maximum rise/decrease rate(maximal rate of the increase/decrease of left ventricular pressure,±dp/dt_max).The HE staining method was used to observe the morphological changes of rat myocardium and coronary vessels.ELISA method was used to detect the contents of CK-MB and c Tn-I in rat plasma,and the contents of TNF-αand IL-6 in serum.RT-q PCR detects the amount of TLR4,TAK1,NF-κB p65,TNF-α,IL-6 m RNA in the myocardium.Western Blot method was used to detect the content of TLR4,TAK1,NF-κB p65 protein in rat myocardium.2.In vitro experimentsMale SPF rats,the experimental groupings and their drug interventions are the same in vivo experiments,randomly divided into 6 groups（n=10）:sham operation group（SHAME group）,sepsis model group（CLP group）,low TP Concentration intervention group（CLP+TP-L group,50μg/kg）,TP medium concentration intervention group（CLP+TP-M group,100μg/kg）,TP high concentration intervention group（CLP+TP-H group,200μg/kg）and polymyxin B group（CLP+PMX-B group,1 mg/kg）.Forty-eight hours after the operation,rats in each group were subjected to isolated cardiac perfusion experiments using the Langendorff device.After 30 minutes of stability,the I channel of the Power Lab system was used to determine the isolated cardiac function indicators of each group of rats:heart rate（HR）,left ventricular development pressure（Left ventricular developed pressure,LVDP）,the maximum rate of the increase/decrease of left ventricular pressure(±dp/dt_max)and coronary flow（CF）.The coronary vessels on the myocardium were taken under a stereomicroscope,and the amounts of TLR4,TAK1,NF-κB,TNF-αand IL-6 m RNA in the coronary vessels were detected by RT-q PCR,and TLR4 in the rat coronary vessels was detected by Western Blot.,TAK1,NF-κB p65 protein expression.Result:1.CLP method to establish sepsis model identification and 72h survival rate analysisThe rats in the SHAME group were in good spirits,the abdominal fluid was light yellow,and there was no death within 72 hours;The rats in the CLP group were mentally wilted,decreased in activity,decreased appetite,intestinal adhesions,edema and necrosis,and had bloody fluid in the abdominal cavity,and the survival rate was significantly decreased（P<0.05）.Compared with the CLP group,rats in the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group improved their mental status,reduced intestinal edema and necrosis,and increased the survival rate of rats（P<0.05）.2.Changes of MABP and cardiac function indexes of rats in each groupCompared with the SHAME group,the hemodynamic indexes MABP,LVDP×HR,±dp/dt_maxof rats in the CLP group were significantly decreased,and myocardial function was weakened（P<0.05）.Compared with the CLP group,the CLP+TP-L group had no significant difference in LVDP×HR,-dp/dt_max（P>0.05）;compared with the CLP+TP-L group,the CLP+TP-M group,CLP+TP-H The MABP,LVDP×HR and±dp/dt_maxof rats in the CLP+PMX-B group and the CLP+PMX-B group were significantly improved（P<0.05）.3.Changes of isolated heart function indexes and coronary flow（CF）Compared with the SHAME group,the isolated heart function indexes LVDP×HR and±dp/dt_maxof the rats in the CLP group decreased significantly,and the coronary flow decreased significantly（P<0.05）.Compared with the CLP group,LVDP×HR and+dp/dt_maxin the CLP+TP-L group were significantly improved（P<0.05）,there was no significant difference in-dp/dt_max,and there was no significant difference in coronary flow(（P>0.05）;Compared with the CLP+TP-L group,the LVDP×HR and±dp/dt_maxof the CLP+TP-H group and the CLP+PMX-B group were significantly improved,and the coronary flow was also significantly restored（P<0.05）.4.Pathological changes of myocardial tissue and blood vessels in each groupThe results of HE staining showed that the vascular morphology of the SHAME group was normal,the cardiomyocytes were intact,the muscle fibers were clearly arranged,and there was no inflammatory cell infiltration;the CLP group showed vasodilation and congestion,focal degeneration and necrosis of cardiomyocytes,and inflammatory cell infiltration.Compared with the CLP group,there was no obvious inflammatory cell infiltration in the CLP+TP-L group,but cardiomyocytes swelled,striae disappeared,and cell nuclei contracted.The pathological changes of blood vessels and cardiomyocytes in the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group improved and tended to be normal.5.Changes in biochemical indicators of rats in each group（1）The content of CK-MB and c Tn-I in serum of rats in each groupThe levels of CK-MB and c Tn-I in the serum of rats in the CLP group were higher than those in the SHAME group（P<0.05）.The levels of CK-MB and c Tn-I in the serum of rats in the TP intervention groups and the CLP+PMX-B group were lower than those in the SHAME group.CLP group（P<0.05）.Compared with CLP+TP-L,the serum levels of CK-MB and c Tn-I in the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group were significantly reduced（P<0.05）.（2）The content of TNF-αand IL-6 in plasma of rats in each groupCompared with the SHAME group,the plasma levels of TNF-a and IL-6 in the CLP group were significantly higher（P<0.05）;compared with the CLP group,the plasma TNF-a and IL-6 in the TP intervention groups and the CLP+PMX-B group-a and IL-6 levels were significantly reduced（P<0.05）;compared with CLP+TP-L,TNF-a in the plasma of rats in the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group And IL-6 content decreased significantly（P<0.05）.6.Expression of TNF-αand IL-6 m RNA in myocardium and coronary vessels of rats in each groupThe expression levels of TNF-a and IL--6 m RNA in myocardium and coronary vessels in CLP group were higher than those in SHAME group（P<0.05）,and TNF-a and IL in myocardium and coronary vessels in TP intervention group and CLP+PMX-B group-6m RNA expression was lower than that of CLP group（P<0.05）;compared with CLP+TP-L,TNF-a and IL-6 m RNA expression in myocardium and coronary vessels in CLP+TP-H and CLP+PMX-B groups The content was significantly reduced（P<0.05）.7.The expression of TLR4,TAK1 and NF-κB p65 m RNA in the myocardium and coronary vessels of rats in each group（1）Expression of TLR4,TAK1 and NF-κB p65 m RNA in rat myocardiumThe expression levels of TLR4,TAK1,and NF-κB p65 m RNA in the myocardium of the CLP group were significantly higher than those of the SHAME group（P<0.05）;The expression levels of TAK1 and NF-κB p65 m RNA were lower than those of the CLP group（P<0.05）.（2）The expression of TLR4,TAK1 and NF-κB p65 m RNA in rat coronary vesselsThe expression levels of TLR4,TAK1 and NF-κB p65 m RNA in the coronary vessels of the CLP group were significantly higher than those of the SHAME group（P<0.05）;the coronary arteries of the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group The expression levels of TAK1 and NF-κB p65 m RNA in blood vessels were lower than those in the CLP group（P<0.05）.There was no significant difference in the expression of TLR4m RNA in coronary vessels between the CLP+TP-M group and the CLP group（P>0.05）.8.The expression of TLR4,TAK1 and NF-κB p65 protein in myocardium and coronary vessels of rats in each group（1）Expression of TLR4,TAK1 and NF-κB p65 protein in rat myocardiumThe expression of TLR4,TAK1 and NF-κB p65 protein in the myocardium of CLP group was significantly higher than that of SHAME group（P<0.05）;TLR4,TLR4,TAK1and NF-κB p65 protein expression in myocardium of CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group The expression of TAK1 and NF-κB p65 protein was significantly lower than that of the CLP group（P<0.05）.（2）The expression of TLR4,TAK1 and NF-κB p65 protein in the coronary vessels of ratsThe expression of TLR4,TAK1 and NF-κB p65 protein in the coronary vessels of the CLP group was significantly higher than that of the SHAME group（P<0.05）;the coronary arteries of the CLP+TP-M group,CLP+TP-H group and CLP+PMX-B group The expression of TAK1 and NF-κB p65 protein in blood vessels was significantly lower than that in the CLP group（P<0.05）.There was no significant difference in the expression of TLR4 protein in coronary vessels between the CLP+TP-M group and the CLP group（P>0.05）.Conclusion:1.CLP method successfully replicated the rat model of sepsis;2.TP has obvious protective effects on in vivo and isolated cardiac function and myocardial morphology and structure of septic rats;3.The protective effect of TP on myocardial injury in septic rats may be through down-regulating the expression of TLR4/TAK1/NF-κB pathway related proteins,thereby reducing the inflammatory factors in myocardium and coronary vessels.