摘要
1．目的
脓毒症(Sepsis)是由感染因素引起的全身性炎症反应综合征(systemic inflammatoryresponse syndrome, SIRS)，是临床病人发生感染后的常见并发症，并已成为导致临床危重症患者死亡的首要因素，目前尚无有效的药物用于临床治疗。
为研发有效的治疗脓毒症药物，本实验室以介导细菌脓毒症的主要病原体相关分子——细菌脂多糖/内毒素(lipopolysaccharide, LPS)和细菌CpG DNA为靶标，应用基于生物传感器的筛选和定向分离技术，从地骨皮药材中定向分离得到一个能同时拮抗LPS和CpG DNA的生物碱类化合物苦柯胺B。
由于天然提取物产率低，无法满足临床用药需求，为此我们对该化合物进行了全化学合成，制备获得了该化合物的药用盐——甲磺酸苦柯胺B(Kukoamine B，KB)。前期研究证实，该化合物能够显著提高脓毒症模型小鼠的存活率，降低其血中LPS和炎症介质TNF-α的水平。
由于从小鼠模型中获取的标本(尤其是血液样品)不足以支持治疗脓毒症药物深入的药效学和作用机制的研究，因此，为满足KB作为原创新药申报国家1.1类化药的药效学研究的需要，本研究通过探索、改进建模方法，建立了成熟稳定的盲肠结扎穿孔(cecal ligation and puncture, CLP)大鼠脓毒症模型，并应用该模型研究了KB治疗脓毒症的药理作用及机制，为其临床前研究奠定基础。
2．方法
2.1标准化CLP脓毒症大鼠模型的建立及首次使用抗菌药物时间的确定
2.1.1稳定的CLP脓毒症模型术式的筛选与建立：比较自制三棱针与传统穿刺针、传统穿刺针+引流条、传统穿刺针+引流管等不同穿刺方法建模后大鼠死亡率的稳定性差别。
2.1.2不同死亡率CLP脓毒症模型大鼠的建立：采用3、4、5号三棱针(边径分别为3、4和5mm)穿刺建模，观察大鼠死亡率；确定死亡率为70%－90%CLP大鼠脓毒症模型用于药物保护实验；确定死亡率为30%－50%的CLP大鼠脓毒症模型用于药物治疗实验。
2.1.3CLP脓毒症大鼠模型首次抗菌药物使用时间点的筛选：在死亡率为70%－90%的CLP大鼠脓毒症模型中，分别于术后0、1、2、3、4、5、6、7、8h首次静脉注射抗菌药物(100mg/kg舒氨西林+甲硝唑5mg/kg)，观察大鼠死亡率，确定利于药物疗效观察的抗菌药物首次给药时间。
2.2KB对CLP脓毒症大鼠模型的保护作用：建立死亡率为70%－90%的CLP大鼠模型，在抗菌、补液治疗的基础上，给予低、中、高剂量(0.3、1和3μg/kg)的KB，选用乌司他丁(10000U/kg)作为对照药物，观察KB对CLP脓毒症大鼠模型的保护作用。
2.3KB对CLP脓毒症模型大鼠的治疗作用：建立死亡率为30%－50%的CLP脓毒症大鼠模型，在抗菌、补液治疗的基础上，分别给予中、高剂量(1和3μg/kg)的KB以及乌司他丁(10000U/kg)。在CLP术后4、8、12、24h采集血液标本，检测各时相点血中的细菌量、LPS和炎症介质(TNF-α和IL-6)、血常规、凝血和血生化指标的变化情况。在术后24h采集肺组织和小肠组织，光镜下观察组织病理改变，以评价KB对CLP脓毒症模型大鼠的治疗作用。
2.4KB改善CLP脓毒症大鼠模型急性肺损伤的作用及其机制研究
2.4.1体外实验：分离大鼠外周血嗜中性粒细胞，检测分析KB与乌司他丁干预LPS刺激嗜中性粒细胞后，对炎症介质信号分子和转录因子活化以及炎症介质TNF-α、IL-6和弹性蛋白酶的表达的影响，探讨KB改善CLP脓毒症模型大鼠急性肺损伤的机制。
2.4.2体内实验：建立死亡率为30%－50%的CLP脓毒症大鼠模型，在抗菌、补液治疗的基础上，分别给予高剂量(3μg/kg)的KB以及乌司他丁(10000U/kg)。术后12、24h取肺组织，检测肺干湿重，检测肺组织中信号分子(IκB-α和p38)和转录因子(NF-κB)活化情况、炎症介质TNF-α、IL-6和弹性蛋白酶的表达变化。
3.结果
3.1自制三棱针较传统穿刺针可更好地建立稳定的CLP脓毒症大鼠模型
3.1.1自制三棱针较传统穿刺针建立的CLP脓毒症大鼠模型动物组间死亡率差异小：自制三棱针穿刺建立的CLP脓毒症大鼠模型动物组间死亡率差异不超过10%，而采用传统穿刺针、穿刺针+引流条、穿刺针+引流管和锥形三棱针的方法建立的CLP脓毒症大鼠模型动物的组间死亡率差异可高达50%、60%、50%和40%。说明采用自制三棱针较传统穿刺针可更好地建立稳定的CLP脓毒症大鼠模型。
3.1.2自制三棱针的边径与模型动物的死亡率成正相关：3#针组为20%－30%，4#针组为30%－50%，5#针组为70%－90%。
建立死亡率为70%-90%的大鼠CLP脓毒症模型主要条件为：盲肠结扎1.6cm，用5号三棱针穿刺1孔；建立死亡率为30%－50%的大鼠CLP脓毒症模型主要条件为：盲肠结扎1.6cm，用4号三棱针穿刺1孔。
3.1.3在CLP脓毒症模型大鼠中确定首次使用抗菌药物的时间为术后7h：在死亡率为70%－90%的CLP脓毒症模型大鼠中，抗菌药物首次使用时间为术后5h以内，可显著提高大鼠存活率(P<0.05或0.01)，当抗菌药物首次使用时间为术后6h－8h，单独抗菌已不能提高大鼠的存活率(P>0.05)，可模拟临床抗菌药物无效的情况，有利于目标药物药理作用的观察，因此本研究中将抗菌药物首次使用时间确定为术后7h。
3.2KB可显著提高CLP脓毒症模型大鼠的生存率
生理盐水(NS)组大鼠7天生存率为13.33%，抗菌药物组为36.67%，二者无统计学差异(P＞0.05)。
对照药物乌司他丁组大鼠生存率为63.33%，与抗菌药物组比较具有显著的统计学差异(P<0.05)。
低、中、高剂量的KB(0.3、1和3μg/kg)组可显著提高大鼠生存率，由36.67%提高至63.33%、70.00%和86.67%，具有显著和非常显著的统计学差异(P <0.05或0.01vs抗菌药物组)。KB低剂量组的大鼠生存率与乌司他丁组无统计学差异(P＞0.05)，而中高剂量组保护作用优于乌司他丁(P <0.05)。
3.3KB对CLP脓毒症大鼠模型具有显著的治疗作用
相对于假手术组，NS组大鼠血中可检出细菌，血LPS和炎症介质TNF-α和IL-6水平显著升高，并出现了动脉血pH下降、凝血功能异常(APTT↑、PT↑、PLT↓)、肝、肾功能异常(ALT、AST、TBIL、Cr和BUN↑)和心肌损伤(CK-MB↑)，病理学观察发现肺组织出现出血、水肿等改变，小肠组织出现上皮细胞坏死、脱落、嗜中性粒细胞的浸润为主要特征的急性炎症性损伤。
抗菌药物可有效降低大鼠血细菌计数外，但不能改善其他观察指标。但在抗菌、补液治疗的基础上给予KB，可显著降低CLP模型大鼠血中LPS水平、抑制炎症介质TNF-α和IL-6的释放，还可有效减轻大鼠的凝血功能障碍、抑制动脉血pH值降低，并减轻肾脏和心肌功能异常，减轻肺和小肠组织炎症性损伤等症状，效果优于对照药物乌司他丁。
3.4KB可减轻CLP脓毒症大鼠模型的急性肺损伤，其机制与抑制炎症介质密切相关。
体外实验结果显示，KB可抑制LPS诱导的嗜中性粒细胞胞TNF-α、IL-6、弹性蛋白酶和TLR4等炎症介质和受体的表达，抑制LPS诱导的胞内重要信号转导分子p38、IκB-α活化和转录因子NF-κB活化。体内实验结果显示，KB能显著降低CLP模型大鼠肺干湿重比值，抑制肺组织信号分子p38和IκB-α的活化以及TNF-α、IL-6和弹性蛋白酶的升高。对照药物乌司他丁对大鼠肺损伤也具有一定的减轻作用，但KB的作用强于乌司他丁。
4．结论：
4.1成功建立了稳定的CLP脓毒症大鼠模型自制三棱针较传统穿刺针可更好地建立稳定的CLP脓毒症大鼠模型，解决了传统CLP脓毒症模型大鼠中由于穿刺引流入腹腔中的菌量不易控制而造成的CLP脓毒症模型重复性差的难题。本方法操作简单、可控，模型大鼠的死亡率稳定、重现性好，可用于后续的药物研究。
4.2KB可显著提高CLP脓毒症大鼠模型的生存率，且效果优于乌司他丁。
4.3KB可显著降低CLP脓毒症模型大鼠血中LPS浓度，减少炎症介质TNF-α和IL-6的产生，缓解酸中毒，改善凝血状况，对心和肾脏等脏器功能具有显著的保护作用，且效果优于乌司他丁。
4.4KB可减轻CLP脓毒症大鼠模型的急性肺损伤，其机制与抑制炎症介质密切相关。
1. Objectives
Sepsis 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. Methods
2.1Establishment of the standardized CLP-induced rat model of sepsis anddetermination of the time of the first dose of antibacterials
2.1.1Evaluation 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 mechanisms
2.4.1In 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. Results
3.1Three-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. Conclusions
4.1Stable 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.
引文
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