本文選題：KB + LPS��； 參考：《第三軍醫(yī)大學(xué)》2013年博士論文

【摘要】：1．目的 膿毒癥(Sepsis)是由感染因素引起的全身性炎癥反應(yīng)綜合征(systemic inflammatoryresponse syndrome, SIRS)，是臨床病人發(fā)生感染后的常見并發(fā)癥，并已成為導(dǎo)致臨床危重癥患者死亡的首要因素，目前尚無有效的藥物用于臨床治療。 為研發(fā)有效的治療膿毒癥藥物，本實驗室以介導(dǎo)細菌膿毒癥的主要病原體相關(guān)分子——細菌脂多糖/內(nèi)毒素(lipopolysaccharide, LPS)和細菌CpG DNA為靶標，應(yīng)用基于生物傳感器的篩選和定向分離技術(shù)，從地骨皮藥材中定向分離得到一個能同時拮抗LPS和CpG DNA的生物堿類化合物苦柯胺B。 由于天然提取物產(chǎn)率低，無法滿足臨床用藥需求，為此我們對該化合物進行了全化學(xué)合成，制備獲得了該化合物的藥用鹽——甲磺酸苦柯胺B(Kukoamine B，KB)。前期研究證實，該化合物能夠顯著提高膿毒癥模型小鼠的存活率，降低其血中LPS和炎癥介質(zhì)TNF-α的水平。 由于從小鼠模型中獲取的標本(尤其是血液樣品)不足以支持治療膿毒癥藥物深入的藥效學(xué)和作用機制的研究，因此，為滿足KB作為原創(chuàng)新藥申報國家1.1類化藥的藥效學(xué)研究的需要，本研究通過探索、改進建模方法，建立了成熟穩(wěn)定的盲腸結(jié)扎穿孔(cecal ligation and puncture, CLP)大鼠膿毒癥模型，并應(yīng)用該模型研究了KB治療膿毒癥的藥理作用及機制，為其臨床前研究奠定基礎(chǔ)。 2．方法 2.1標準化CLP膿毒癥大鼠模型的建立及首次使用抗菌藥物時間的確定 2.1.1穩(wěn)定的CLP膿毒癥模型術(shù)式的篩選與建立：比較自制三棱針與傳統(tǒng)穿刺針、傳統(tǒng)穿刺針+引流條、傳統(tǒng)穿刺針+引流管等不同穿刺方法建模后大鼠死亡率的穩(wěn)定性差別。 2.1.2不同死亡率CLP膿毒癥模型大鼠的建立：采用3、4、5號三棱針(邊徑分別為3、4和5mm)穿刺建模，觀察大鼠死亡率；確定死亡率為70%－90%CLP大鼠膿毒癥模型用于藥物保護實驗；確定死亡率為30%－50%的CLP大鼠膿毒癥模型用于藥物治療實驗。 2.1.3CLP膿毒癥大鼠模型首次抗菌藥物使用時間點的篩選：在死亡率為70%－90%的CLP大鼠膿毒癥模型中，分別于術(shù)后0、1、2、3、4、5、6、7、8h首次靜脈注射抗菌藥物(100mg/kg舒氨西林+甲硝唑5mg/kg)，觀察大鼠死亡率，確定利于藥物療效觀察的抗菌藥物首次給藥時間。 2.2KB對CLP膿毒癥大鼠模型的保護作用：建立死亡率為70%－90%的CLP大鼠模型，在抗菌、補液治療的基礎(chǔ)上，給予低、中、高劑量(0.3、1和3μg/kg)的KB，選用烏司他丁(10000U/kg)作為對照藥物，觀察KB對CLP膿毒癥大鼠模型的保護作用。 2.3KB對CLP膿毒癥模型大鼠的治療作用：建立死亡率為30%－50%的CLP膿毒癥大鼠模型，在抗菌、補液治療的基礎(chǔ)上，分別給予中、高劑量(1和3μg/kg)的KB以及烏司他丁(10000U/kg)。在CLP術(shù)后4、8、12、24h采集血液標本，檢測各時相點血中的細菌量、LPS和炎癥介質(zhì)(TNF-α和IL-6)、血常規(guī)、凝血和血生化指標的變化情況。在術(shù)后24h采集肺組織和小腸組織，光鏡下觀察組織病理改變，以評價KB對CLP膿毒癥模型大鼠的治療作用。 2.4KB改善CLP膿毒癥大鼠模型急性肺損傷的作用及其機制研究 2.4.1體外實驗：分離大鼠外周血嗜中性粒細胞，檢測分析KB與烏司他丁干預(yù)LPS刺激嗜中性粒細胞后，對炎癥介質(zhì)信號分子和轉(zhuǎn)錄因子活化以及炎癥介質(zhì)TNF-α、IL-6和彈性蛋白酶的表達的影響，探討KB改善CLP膿毒癥模型大鼠急性肺損傷的機制。 2.4.2體內(nèi)實驗：建立死亡率為30%－50%的CLP膿毒癥大鼠模型，在抗菌、補液治療的基礎(chǔ)上，分別給予高劑量(3μg/kg)的KB以及烏司他丁(10000U/kg)。術(shù)后12、24h取肺組織，檢測肺干濕重，檢測肺組織中信號分子(IκB-α和p38)和轉(zhuǎn)錄因子(NF-κB)活化情況、炎癥介質(zhì)TNF-α、IL-6和彈性蛋白酶的表達變化。 3.結(jié)果 3.1自制三棱針較傳統(tǒng)穿刺針可更好地建立穩(wěn)定的CLP膿毒癥大鼠模型 3.1.1自制三棱針較傳統(tǒng)穿刺針建立的CLP膿毒癥大鼠模型動物組間死亡率差異�。鹤灾迫忉槾┐探⒌腃LP膿毒癥大鼠模型動物組間死亡率差異不超過10%，而采用傳統(tǒng)穿刺針、穿刺針+引流條、穿刺針+引流管和錐形三棱針的方法建立的CLP膿毒癥大鼠模型動物的組間死亡率差異可高達50%、60%、50%和40%。說明采用自制三棱針較傳統(tǒng)穿刺針可更好地建立穩(wěn)定的CLP膿毒癥大鼠模型。 3.1.2自制三棱針的邊徑與模型動物的死亡率成正相關(guān)：3#針組為20%－30%，4#針組為30%－50%，5#針組為70%－90%。 建立死亡率為70%-90%的大鼠CLP膿毒癥模型主要條件為：盲腸結(jié)扎1.6cm，用5號三棱針穿刺1孔；建立死亡率為30%－50%的大鼠CLP膿毒癥模型主要條件為：盲腸結(jié)扎1.6cm，用4號三棱針穿刺1孔。 3.1.3在CLP膿毒癥模型大鼠中確定首次使用抗菌藥物的時間為術(shù)后7h：在死亡率為70%－90%的CLP膿毒癥模型大鼠中，抗菌藥物首次使用時間為術(shù)后5h以內(nèi)，可顯著提高大鼠存活率(P0.05或0.01)，當抗菌藥物首次使用時間為術(shù)后6h－8h，單獨抗菌已不能提高大鼠的存活率(P0.05)，可模擬臨床抗菌藥物無效的情況，有利于目標藥物藥理作用的觀察，因此本研究中將抗菌藥物首次使用時間確定為術(shù)后7h。 3.2KB可顯著提高CLP膿毒癥模型大鼠的生存率 生理鹽水(NS)組大鼠7天生存率為13.33%，抗菌藥物組為36.67%，二者無統(tǒng)計學(xué)差異(P＞0.05)。 對照藥物烏司他丁組大鼠生存率為63.33%，與抗菌藥物組比較具有顯著的統(tǒng)計學(xué)差異(P0.05)。 低、中、高劑量的KB(0.3、1和3μg/kg)組可顯著提高大鼠生存率，由36.67%提高至63.33%、70.00%和86.67%，具有顯著和非常顯著的統(tǒng)計學(xué)差異(P 0.05或0.01vs抗菌藥物組)。KB低劑量組的大鼠生存率與烏司他丁組無統(tǒng)計學(xué)差異(P＞0.05)，，而中高劑量組保護作用優(yōu)于烏司他丁(P 0.05)。 3.3KB對CLP膿毒癥大鼠模型具有顯著的治療作用 相對于假手術(shù)組，NS組大鼠血中可檢出細菌，血LPS和炎癥介質(zhì)TNF-α和IL-6水平顯著升高，并出現(xiàn)了動脈血pH下降、凝血功能異常(APTT↑、PT↑、PLT↓)、肝、腎功能異常(ALT、AST、TBIL、Cr和BUN↑)和心肌損傷(CK-MB↑)，病理學(xué)觀察發(fā)現(xiàn)肺組織出現(xiàn)出血、水腫等改變，小腸組織出現(xiàn)上皮細胞壞死、脫落、嗜中性粒細胞的浸潤為主要特征的急性炎癥性損傷。 抗菌藥物可有效降低大鼠血細菌計數(shù)外，但不能改善其他觀察指標。但在抗菌、補液治療的基礎(chǔ)上給予KB，可顯著降低CLP模型大鼠血中LPS水平、抑制炎癥介質(zhì)TNF-α和IL-6的釋放，還可有效減輕大鼠的凝血功能障礙、抑制動脈血pH值降低，并減輕腎臟和心肌功能異常，減輕肺和小腸組織炎癥性損傷等癥狀，效果優(yōu)于對照藥物烏司他丁。 3.4KB可減輕CLP膿毒癥大鼠模型的急性肺損傷，其機制與抑制炎癥介質(zhì)密切相關(guān)。 體外實驗結(jié)果顯示，KB可抑制LPS誘導(dǎo)的嗜中性粒細胞胞TNF-α、IL-6、彈性蛋白酶和TLR4等炎癥介質(zhì)和受體的表達，抑制LPS誘導(dǎo)的胞內(nèi)重要信號轉(zhuǎn)導(dǎo)分子p38、IκB-α活化和轉(zhuǎn)錄因子NF-κB活化。體內(nèi)實驗結(jié)果顯示，KB能顯著降低CLP模型大鼠肺干濕重比值，抑制肺組織信號分子p38和IκB-α的活化以及TNF-α、IL-6和彈性蛋白酶的升高。對照藥物烏司他丁對大鼠肺損傷也具有一定的減輕作用，但KB的作用強于烏司他丁。 4．結(jié)論： 4.1成功建立了穩(wěn)定的CLP膿毒癥大鼠模型自制三棱針較傳統(tǒng)穿刺針可更好地建立穩(wěn)定的CLP膿毒癥大鼠模型，解決了傳統(tǒng)CLP膿毒癥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膿毒癥模型重復(fù)性差的難題。本方法操作簡單、可控，模型大鼠的死亡率穩(wěn)定、重現(xiàn)性好，可用于后續(xù)的藥物研究。 4.2KB可顯著提高CLP膿毒癥大鼠模型的生存率，且效果優(yōu)于烏司他丁。 4.3KB可顯著降低CLP膿毒癥模型大鼠血中LPS濃度，減少炎癥介質(zhì)TNF-α和IL-6的產(chǎn)生，緩解酸中毒，改善凝血狀況，對心和腎臟等臟器功能具有顯著的保護作用，且效果優(yōu)于烏司他丁。 4.4KB可減輕CLP膿毒癥大鼠模型的急性肺損傷，其機制與抑制炎癥介質(zhì)密切相關(guān)。
[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 - 偽 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 - 偽 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 - 偽 , 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 - 偽 and p38 ) and transcription factor ( NF - 魏B ) in lung tissues was detected , and the expression of TNF - 偽 , 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 - 偽 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 - 偽 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 - 偽 , IL - 6 , elastase , and the activation of NF - 魏B , and inhibit the activation of p38 , I魏B - 偽 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 - 偽 in lung tissue signal molecules , and increase the TNF - 偽 , 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 - 偽 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 .
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R-332;R459.7

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