| 1. ObjectivesSepsis is defined as systemic inflammatory response syndrome (SIRS) caused byinfection. It is a common complication in patients with infection, and has become theleading cause of death in critically ill patients. But there are no effective drugs for clinicaluse at present.To develop medications for the treatment of sepsis, we screen and isolate drugstargeting lipopolysaccharide (LPS) and CpG DNA which are the main pathogen-associatedmolecular patterns (PAMPs) using biosensor, and an alkaloid compound Kukoamine B (KB)that can simultaneously neutralize LPS and CpG DNA was isolated from Lycii Cortex. KBwas subsequently synthesized because of the low product yield from natural resources, andthe salt Kukoamine B Mesilate (KB) was obtained. Previous study has shown that KB couldsignificantly improve the survival rate in mice with sepsis and reduce the serum LPS andTNF-α levels.Because it is insufficient for the further study of pharmacology and mechanism usingmouse model of sepsis, and to meet the requirement of new drug application (chemicaldrugs in class1.1), a mature and stable CLP-induced rat model of sepsis was established byimproving the procedure of cecal ligation and puncture (CLP) operation. We provided thescientific basis for the pre-clinical study of KB via using this model in pharmacology andmechanism study of KB.2. Methods2.1Establishment of the standardized CLP-induced rat model of sepsis anddetermination of the time of the first dose of antibacterials2.1.1Evaluation and establishment of stable CLP surgery performed in rats: tocompare the stability of mortality rate of rats punctured by three-edged needle, traditionalpuncture needle, traditional puncture needle with drainage strip and traditional puncture needle with drainage tube, respectively,2.1.2Establishment of CLP-induced rat model of sepsis with different mortality:3,4and5#(the width of edge is3,4and5mm) three-edged needle were used for the puncture.The model with mortality rate ranging from70%to90%was used for the protectionexperiments and30%to50%was used for the therapeutic experiments.2.1.3Comparison of the time of the first dose of antibacterials: the antibacterials (100mg/kg ampicillin sodium-sulbactam sodium and5mg/kg metronidazole) were firstintravenously injected at0,1,2,3,4,5,6,7and8h after CLP operation in the rats withmortality rate ranging from70%to90%. The mortality rate was observed to estimate thetime of the first dose of antibacterials.2.2Protective effect of KB in CLP-induced rat model of sepsis: to demonstrate theprotective effect of KB in CLP-induced rat model of sepsis, on the basis of antibacterialsand resuicitation treatment, KB (0.3,1and3μg/kg) was injected in the rats with mortalityrate ranging from70%to90%. Ulinastatin (10,000U/kg) was used as positive control.2.3Therapeutical effect of KB in CLP-induced rat model of sepsis: to demonstrate thetherapeutical effect of KB in CLP-induced rat model of sepsis, on the basis of antibacterialsand resuicitation treatment, KB (1and3μg/kg) and ulinastatin (10000U/kg) were injectedin the rats with mortality rate ranging from30%to50%, respectively. Blood samples werecollected at4,8,12and24h after CLP operation for the detection of bacterial count, LPS,pro-inflammatory mediators (TNF-α and IL-6), blood tests, blood coagulation andbiochemical indicators. Lung and intestine tissues were collected at24h after CLPoperation for the pathological examination.2.4Amelioration of acute lung injury by KB in CLP-induced rat model of sepsis andthe mechanisms2.4.1In vitro experiment: neutrophilic granulocytes were isolated from peripheralblood of rats, and the inhibition on LPS-induced expression of inflammatory signalingmolecules, activation of transcription factor and expression of elastase by KB andulinastatin was analysed to explore the mechanism of ameliorating acute lung injury by KBin CLP-induced rat model of sepsis.2.4.2In vivo experiment: on the basis of antibacterials and resuicitation treatment, KB(3μg/kg) and ulinastatin (10,000U/kg) were injected in the rats with mortality rate ranging from30%to50%, respectively. Lung tissue was collected at12and24h after CLPoperation for the detection of lung wet/dry weight ration, activation of signaling molecules(IκB-α and p38) and transcription factors (NF-κB) and expression of inflammatorymediators (TNF-α and IL-6) and elastase.3. Results3.1Three-edged needle was qualified for the establishment of a more stableCLP-induced rat model of sepsis.3.1.1There was no significant difference between the groups using three-edged needle,the variation of which was less than10%, while the variation of mortality between groupswas50%(using traditional puncture needle),60%(using traditional puncture needle withdrainage strip),50%(using traditional puncture needle with drainage tube) and40%(usingconical shape three edged needle). Thus, three-edged needle is better than other needles forthe establishment of stable CLP-induced rat model of sepsis.3.1.2There was a direct correlation between the mortality rate of rats and the width ofthe edge of three-edged needle: the mortality rate of groups using3#,4#and5#three-edgedneedle was20%－30%,30%－50%and70%－90%, respectively.The condition of CLP rat model with mortality rate ranging from70%－90%was asfollows: the cecum was ligated at1.6cm away from the tip and punctured by5#three-edged needle for once between the ligation and the tip. The condition of CLP ratmodel with mortality rate ranging from30%-50%was as follows: the cecum was ligated at1.6cm away from the tip and punctured by4#three-edged needle for once between theligation and the tip.3.1.3The time of first dose of antibacterials was7h after CLP operation: The survivalrate of rats could be significantly improved if the antibacterials were first administeredwithin5h after CLP operation (P<0.05or0.01). But the administration of antibacterials at6－8h after CLP operation was ineffective (P>0.05), thus demonstrating some mimics inclinical practice for the evaluation of pharmacological activity. Therefor, the time of firstdose of antibacterials was7h after CLP operation in the present study.3.2KB significantly improved the survival rate of CLP rats.There was no significant difference between the survival rate of rats receivedphysiological saline (13.33%) and antibacterials (36.67%, P>0.05). There was significant difference between the survival rate of rats received Ulinastatin(63.33%) and only antibacterials (P<0.05).On the basis of antibacterials and resuicitation treatment, KB (0.3,1and3μg/kg)significantly increase the survival rate from36.67%to63.33%,70.00%and86.673%,respectively (P<0.05or0.01vs antibacterials). There was significant difference betweenKB (1and3μg/kg) and Ulinastatin (P<0.05), but no significant difference between KB (0.3μg/kg) and ulinastatin (P>0.05).3.3KB showed significant therapeutical effect in CLP-induced rat model of sepsis.Compare to the sham operated group, the abnormal of bacterial count, serum LPS,inflammatory mediators (TNF-α and IL-6), blood pH, coagulation (APTT, PT and PLT),liver function, renal function (ALT, AST, TBIL, Cr and BUN) and myocardial damage(CK-MB) were observed in the rats received physiological saline. Pathological examinationshowed hemorrhage and edema in lung tissue, epithelial cell shedding and necrosis as wellas neutrophil infiltration in intestine tissue.Antibacterials were effective in decreasing the bacterial load, but ineffective for others.On the basis of antibacterials and resuicitation treatment, KB can significantly reduce theserum levels of LPS, TNF-α and IL-6, alleviate coagulation disorders and decrease in bloodpH, ameliorate the renal and liver dysfunction and improve the lung and intestine tissueinjury, and it showed better efficacy than ulinastatin.3.4KB ameliorated acute lung injury in CLP-induced rat model of sepsis, and it isassociated with the inhibition of inflammatory mediators.In vitro, KB inhibited the expression and production of TNF-α, IL-6, elastase andTLR4from neutrophils induced by LPS, and it also suppressed the activation ofintracellular signal transduction molecules p38and IκB-α and transcription factor NF-κB.In vivo, KB significantly reduced the lung wet/dry weight ratio, inhibited the activation ofsignaling molecules such as p38and IκB-α and the expression of TNF-α, IL-6and elastasein lung tissue.KB exhibited better efficacy than ulinastatin, although ulinastatin was also found toameliorate the lung injury in rats. 4. Conclusions4.1Stable CLP-induced rat model of sepsis was successfully established. Three-edgedneedle is better for the establishment of stable CLP-induced rat model of sepsis. It solvedthe problem that it was hard to control the bacterial load in abdominal cavity usingtraditional puncture needle. The procedure was easy to control; it kept the mortality ratestable and had a good reproducibility.4.2KB significantly improved the survival rate of CLP rat and showed better efficacythan ulinastatin.4.3KB significantly reduced the serum levels of LPS, TNF-α and IL-6, alleviatedacidosis, ameliorated coagulation disorders and protected heart and renal tissue againstsepsis, and exhibited better efficacy than ulinastatin.4.4KB ameliorated acute lung injury in CLP-induced rat model of sepsis, and themechanism was tightly associated with the inhibition of inflammatory mediators. |
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