标准化大鼠CLP脓毒症模型的建立及甲磺酸苦柯胺B对其的保护作用与机制研究

发布时间：2018-06-24 15:39

本文选题：KB + LPS　；参考：《第三军医大学》2013年博士论文

【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(P0.05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(P0.05)，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(P0.05)。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。
[Abstract]:1 . Purpose

sepsis is a systemic inflammatory response syndrome caused by infection factors , which is a common complication after infection in clinical patients and has become the primary factor leading to death in critically ill patients . There is no effective drug for clinical treatment .

In order to develop an effective drug for the treatment of sepsis , this laboratory is targeted to the main pathogen - related molecules _ bacterial lipopolysaccharide / endotoxin ( LPS ) and bacterial CpG DNA which mediate bacterial sepsis , and the application of the biological sensor - based screening and orientation separation technique is used to directionally separate the alkaloids from the medicinal materials of the cortex and bark to obtain an alkaloid - like alkaloid compound B which can simultaneously antagonise LPS and CpG DNA .

Due to the low yield of natural extraction and the inability to meet the needs of clinical medication , we carried out an all - chemical synthesis of the compound to prepare a pharmaceutically acceptable salt of the compound _ ( Kukoamine B , KB ) . Previous studies have shown that the compound can significantly improve the survival rate of mice with sepsis and reduce the levels of LPS and inflammatory mediators TNF - 伪 in its blood .

In order to satisfy KB ' s need for pharmacodynamic research on drug application , this study has established mature and stable sepsis model of cecal ligation and puncture ( CLP ) rats by exploring and improving the modeling method . The model is used to study the pharmacological action and mechanism of KB in treating sepsis , which lays a foundation for its pre - clinical research .

2 . Method

2.1 Establishment of rat model of standardized CLP sepsis and determination of the time of first use of antibacterial drug

2.1 . 1 Selection and establishment of stable CLP sepsis model : compare the stability difference between self - made three - edge needle and traditional puncture needle , traditional puncture needle , drainage strip , traditional puncture needle and drainage tube after modeling .

2.1 . 2 Establishment of rats with different mortality CLP sepsis model : 3 , 4 , 5 ( 3 , 4 and 5 mm lateral diameters ) were used to model the rats , and the mortality of rats was observed .
determining the mortality rate of 70 % -90 % CLP rat sepsis model for the drug protection experiment ;
A CLP rat sepsis model with a mortality rate of 30 % to 50 % was established for the drug treatment experiment .

2.1 . 3Screening of the time point for the first antibacterial drug use in the rat model of sepsis in CLP : In the CLP rat sepsis model with the mortality rate of 70 % -90 % , the antibacterial drugs ( 100 mg / kg Shu - ampicillin + metronidazole 5 mg / kg ) were injected intravenously at 0 , 1 , 2 , 3 , 4 , 5 , 6 , 7 and 8 h after operation respectively , and the mortality of rats was observed , and the time of the first administration of antibacterial drugs for the observation of drug efficacy was determined .

The protective effect of 2 . 2KB on the rat model of CLP sepsis was established : a CLP rat model with a mortality rate of 70 % -90 % was established . On the basis of antibiotic and tonic treatment , KB of low , medium and high doses ( 0.3 , 1 and 3 渭g / kg ) were given . The protective effect of KB on the rat model of CLP sepsis was observed .

2 . The therapeutic effect of 3KB on CLP sepsis model rats was established . The rats model of CLP sepsis with mortality rate of 30 % -50 % was established . Blood samples were collected at 4 , 8 , 12 and 24 hours after CLP . The changes of bacterial quantity , LPS and inflammatory mediators ( TNF - 伪 and IL - 6 ) , blood routine , blood coagulation and blood biochemical indexes were measured at 4 , 8 , 12 and 24 hours after CLP .

Effect of 2.4KB on acute lung injury in rats with CLP sepsis and its mechanism

2.4 . 1 In vitro experiments : The mechanism of KB improving the acute lung injury in rats with CLP sepsis model was investigated by analyzing the effects of KB and Ulinastatin on the activation of inflammatory mediators and the expression of TNF - 伪 , IL - 6 and elastase in rat peripheral blood neutrophils .

2.4 . 2 In vivo experiments : A rat model of CLP sepsis with a mortality rate of 30 % -50 % was established . On the basis of anti - bacterial and tonic therapy , high - dose ( 3 渭g / kg ) KB and Ulinastatin ( 10000U / kg ) were given respectively . After 12 and 24 hours after operation , lung tissue was taken to detect the lung dry and wet weight , and the expression of signal molecules ( I魏B - 伪 and p38 ) and transcription factor ( NF - 魏B ) in lung tissues was detected , and the expression of TNF - 伪 , IL - 6 and elastase was detected in the inflammatory mediators .

3 . Results

3.1 A stable model of CLP sepsis can be established with a self - made three - edge needle compared with the traditional puncture needle .

3.1 . 1 Compared with the traditional puncture needle , the mortality of CLP sepsis rat model was less than 10 % , and the mortality of CLP sepsis rat model was up to 50 % , 60 % , 50 % and 40 % with the traditional puncture needle , puncture needle , drainage strip , puncture needle + drainage tube and tapered triangular needle .

3.1 . 2 The side diameter of the self - made triangular needle is positively related to the mortality of the model animal : 20 % -30 % of 3 # needle group , 30 % -50 % of 4 # needle group and 70 % -90 % of 5 # needle group .

The establishment of the rat CLP sepsis model with the mortality rate of 70 % -90 % was as follows : the cecal ligation was 1.6 cm , and the 1 - hole was punctured with the third - edge needle No . 5 ;
Establishment of the rat CLP sepsis model with the mortality rate of 30 % -50 % was the main condition : the cecal ligation was 1.6 cm , and the 1 - hole was punctured with the three - edged needle .

3.1 . 3 In CLP sepsis model rats , it was determined that the first time to use the antibacterial drugs was 7 hours after the operation . The first time of use of the antibacterial drugs was within 5 hours after the operation , and the survival rate of the rats ( P 0.05 or 0.01 ) was significantly improved . When the first time of use of the antibacterial drugs was 6 hours to 8 hours after the operation , the survival rate of the rats was significantly improved ( P0.05 ) .

3.2 KB can significantly improve the survival rate of CLP sepsis model rats

The survival rate of 7 - day survival rate was 13.33 % in NS group , 36.67 % in group B , and no statistical difference between them ( P > 0.05 ) .

The survival rate of the control drug was 63.33 % , which was significantly different from that of the antibacterial drug group ( P0.05 ) .

Low , medium and high doses of KB ( 0.3 , 1 , and 3 渭g / kg ) significantly increased the survival rate of rats , increased from 36.67 % to 63.33 % , 70.00 % and 86.67 % , with significant and very significant statistical difference ( P 0.05 or 0.01 vs antibacterial drug group ) . The survival rate of rats in low - dose group was significantly higher than that in Ulinastatin group ( P > 0.05 ) , while the protective effect of medium - high dose group was better than that of Ulinastatin ( P 0.05 ) .

3 . 3KB plays an important role in the treatment of CLP sepsis rat model .

Compared with sham operation group , the levels of TNF - 伪 and IL - 6 in blood of NS group were significantly higher than those in sham operation group and NS group , and the changes of arterial blood pH , abnormal blood coagulation function ( PT & lt ; PT & gt ; , PLT ) , liver and renal function ( ALT , AST , TBIL , Cr and BUN & lt ; 3 & gt ; ) and myocardial injury ( CK - MB & lt ; 3 & gt ; ) were observed .

The antibacterial medicine can effectively reduce the blood bacterial count of rats , but can not improve other observation indexes , but can remarkably reduce LPS levels in the blood of the CLP model rats , inhibit the release of the TNF - 伪 and IL - 6 in the inflammatory mediators , reduce the blood coagulation dysfunction of the rats , reduce the pH value of the arterial blood , and reduce the function abnormality of the kidney and the myocardium , reduce the inflammatory injury of the lung and the small intestine tissue , and the like , and has the advantages of better effect than the control drug Ulinastatin .

3.4KB can alleviate acute lung injury of CLP sepsis rat model , and its mechanism is closely related to the inhibition of inflammatory mediators .

The results showed that KB could inhibit the expression of inflammatory mediators and receptors , such as TNF - 伪 , IL - 6 , elastase , and the activation of NF - 魏B , and inhibit the activation of p38 , I魏B - 伪 activation and NF - 魏B activation in LPS - induced cells . The results showed that KB could significantly decrease the lung dry - wet weight ratio in CLP model rats , inhibit the activation of p38 and I魏B - 伪 in lung tissue signal molecules , and increase the TNF - 伪 , IL - 6 and elastase .

4 . Conclusion :

4.1 A stable CLP sepsis rat model was established successfully in rats with stable CLP sepsis model , and the problem of poor reproducibility of CLP sepsis model caused by improper control of the amount of bacteria in the abdominal cavity caused by puncture was solved . The method was simple and controllable , the mortality of the model rats was stable , the reproducibility was good , and the model rats could be used for the subsequent drug research .

4.2KB can significantly improve the survival rate of the rat model of CLP sepsis , and the effect is better than that of Ulinastatin .

4.3KB can significantly reduce the LPS concentration in the blood of CLP sepsis model rats , reduce the production of TNF - 伪 and IL - 6 in inflammatory mediators , alleviate acidosis , improve the blood coagulation condition , have a significant protective effect on organ function such as heart and kidney , and the effect is better than that of Ulinastatin .

4 . 4KB can alleviate acute lung injury of CLP sepsis rat model , and its mechanism is closely related to the inhibition of inflammatory mediators .
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R-332;R459.7

【参考文献】