【作者】 贺常亮；

【导师】 韦旭斌；

【作者基本信息】 吉林大学 ， 临床兽医学， 2011， 博士

【摘要】 鸡大肠杆菌病(Chicken colibacillosis)是由某些血清型的致病性大肠埃希氏菌(Escherichia coli,简称大肠杆菌E.coli)所引起的传染病。各日龄的鸡均可发病,以雏鸡和中鸡居多。临床表现为多种症型,较常见的有败血症、肉芽肿、心包炎、肝周炎、气囊炎、肠炎、脐炎、全眼球炎、卵黄性腹膜炎、肿头综合征等,发病率为11%-69%,致死率为40.2%-90.3%,与其它病原混合感染时,死淘率会更高,其危害居细菌性疾病之首,常给养鸡业带来巨大经济的损失。目前,鸡大肠杆菌病的防治还存在许多亟待解决的重大问题：一是致病性大肠杆菌血清型众多,我国现已报道的鸡致病性大肠杆菌血清型有60多种,现有的疫苗不能有效预防；二是养鸡场综合防控措施不够完善,为鸡致病性大肠杆菌的滋生和传播提供了外部条件；三是由于抗生素和化学抗菌药物的大量使用,导致病原产生了广泛的耐药性,使治疗效果不断下降,临床又不得不加大剂量并延长给药时间,大幅度提高了治疗难度与成本;四是抗生素和其他化学抗菌药在鸡肉、内脏和鸡蛋等产品中形成有害残留危害食品安全。近年来,采用中药防治鸡大肠杆菌病的报道较多,且多数认为有较好效果。但尚存在下列问题：一是多数研究未从现代传染病学角度证明其有效性；二是未从现代药理学角度阐明其作用机理。为此,研究与开发更加安全、有效的中药制剂并阐明其作用机理将具有重要的理论研究与临床应用价值。我们在中兽医药学理论指导下,根据相关文献报道,确定了清热类、祛湿类、理血类、理气类等114味中草药,以鸡大肠杆菌078作为目标菌株,进行了体外的抑菌试验,筛选出6种具有一定抗菌作用的中药,包括有芦荟、穿心莲、大蒜、锦灯笼、乌梅、石榴皮,其抑菌圈分别是15 mm、12 mm、12 mm、11 mm、11 mm、7 mm。运用硅胶柱层析、凝胶柱层析、ODS柱层析对芦荟的抗菌成分进行了分离、纯化,经过质谱、红外光谱和核磁共振分析了得到的抗菌单体物质,确定为延胡索酸。进一步的抗菌活性研究结果显示,延胡索酸对金黄色葡萄球、链球菌、大肠杆菌和沙门氏菌的MIC分别是50、50、780和780 gg/ml,MBC分别是100、100、1560和780μg/ml。利用建立的人工感染鸡大肠杆菌病理模型,对39个备选方剂进行了筛选,发现方4、方5、方24与方26的效果较好,死亡率分别是30%、40%、26.7%、26.7%。为了进一步确证这些方剂的有效性,对方4、5、24、26进行了2次重复实验,结果显示4个方剂效果稳定,对实验性鸡大肠杆菌病均有治疗效果,死亡率在46.7%以下。相比之下,26号方效果最好,2次死亡率均为26.7%。通过正交试验对26号方组成药物的用量进行了筛选,确定其最佳配比为香附40g、穿心莲30 g、黄芪30 g,并将该方命名为“香芪汤”。利用人工感染鸡大肠杆菌的病理模型,进一步考察了中药香芪汤的疗效。结果表明,中药香芪汤能改善感染大肠杆菌鸡只的饮水量和采食量。感染大肠杆菌鸡只的死亡率为50%,香芪汤中、高剂量能显著降低死亡率,分别是20%(P<0.05)和13.3%(P<0.01),且二组对感染鸡只的体增重也有较好的改善作用。血常规指标分析结果显示,攻毒对照组白细胞数极显著高于健康对照组(P<0.01),中药香芪汤能有效抑制血液白细胞过度的增加,低、中、高剂量组的白细胞数分别显著(P<0.05)和极显著(P<0.01)低于攻毒对照组：攻毒对照组的凝血细胞数显著低于健康对照组(P<0.05),高剂量的香芪汤能保护凝血细胞免受破坏,其数量显著高于攻毒对照组(P<0.05),香芪汤对红细胞也具有一定的保护作用；香芪汤能减轻接种大肠杆菌鸡只的病理变化,降低心包炎、肝周炎的发生率,并改善心、肝、脾、肾的脏体指数；通过ELISA法检测各组鸡只血清炎症介质TNF-α、IL-1和sEPCR的水平,结果显示,在相应的时间段内香芪汤能降低这些炎症介质的水平。以上结果表明,香芪汤对人工感染大肠杆菌鸡只具有良好的疗效.并且有可能是通过抗炎和抗凝血两条途径来发挥治疗作用的。我们推测在鸡大肠杆菌致病过程中,微血管内皮细胞(Microvascular endothelial cells,MVECs起到了关键性的作用,因此,在本实验中,通过建立LPS诱导MVECs的炎症模型,考察了香芪汤及其有效成分对MVECs凝血途径相关因子的影响。MVECs长成融合状态的单层时,加入香芪汤、α-香附酮、黄芪甲苷和穿心莲内酯的低、中、高剂量作用细胞3h后,加入LPS刺激,18h收集培养细胞上清液,采用ELISA法测定PAI-1和TF的含量。结果,香芪汤和α-香附酮的低、中、高剂量组,黄芪甲苷中、高剂量组,穿心莲内酯低剂量组的PAI-1含量显著低于LPS对照组(P<0.05或P<0.01)；香芪汤低、中、高剂量组,α-香附酮中、高剂量组,黄芪甲苷高剂量组,穿心莲内酯高剂量组的TF含量显著低于LPS对照组(P<0.05或P<0.01),表明,香芪汤及其有效成分能抑制LPS诱导MVECs的PAI-1和TF的分泌,减轻凝血反应。半定量RT-PCR结果显示,LPS作用MVECs 6 h后,能过多的提高PAI-1和TF基因mRNA表达的水平、降低上游调控基因KLF2 mRNA表达的水平。香芪汤组能降低PAI-1基因mRNA的表达水平,与LPS对照组比较差异显著(P<0.01),而穿心莲内酯组,黄芪甲苷和α-香附酮组的PAI-1基因mRNA表达水平与LPS对照组没有显著的差异(P>0.05)；香芪汤组和α-香附酮组的TF基因mRNA表达水平与LPS对照组比较差异极显著(P<0.01),黄芪甲苷、穿心莲内酯组的TF基因mRNA表达水平与LPS对照组比较差异不显著(P>0.05)。此外,香芪汤组、α-香附酮组、黄芪甲苷组和穿心莲内酯组的KLF2基因mRNA表达水平与LPS对照组比较差异显著(P<0.01)。以上结果表明,香芪汤及其有效成分能够一定程度上抑制LPS诱导的MVECs PAI-1和TF基因mRNA水平的影响,并且对这两个基因的上游调控基因KLF2也有一定的调节作用。利用Western blot方法检测了香芪汤及其有效成分对MVECs分泌TNF-α和表达ICAM-1蛋白以及上游调控蛋白IκB-α、NF-κB p65的影响。结果显示,香芪汤和α-香附酮的低、中、高剂量组,黄芪甲苷的高剂量组,穿心莲内酯中、高剂量组均能极显著降低LPS诱导的MVECs TNF-α的分泌量(P<0.01)；香芪汤、α-香附酮、黄芪甲苷、穿心莲内酯能降低ICAM-1蛋白的表达量,与LPS对照组相比差异极显著(P<0.01),以上结果表明香芪汤及其有效成分具有较好的抗炎作用。另外,香芪汤及其有效成分不能抑制LPS诱导的MVECs IκB-α蛋白表达量的下降,能够抑制NF-κB p65蛋白表达量的增加,表明,香芪汤及其有效成分的抗炎作用是通过抑制NF-κB p65来介导的。综上所述,本课题利用体外抑菌试验对114味中药进行了筛选,利用人工感染鸡大肠杆菌的病理模型对39个中药复方进行了筛选,得到一个效果最好的中药复方,命名为香芪汤。通过进一步的疗效观察,证明香芪汤对感染鸡只的饮水量、采食量、死亡率、体增重、血液学指标、典型病理变化、脏体指数以及血清炎症介质水平等指标具有良好的改善作用。香芪汤的疗效可能是通过调节KLF2介导的凝血反应和NF-κB介导的炎症反应来完成的。更多还原

【Abstract】 Chicken colibacillosis. which is caused by avian pathogenic Escherichia coli (APEC), is one of the most significant and widespread infectious diseases occurring in different varieties and age of chicken. It is responsible for large financial losses for the poultry industry each year due to high morbility and mortality. It is also a deuteropathy of New castle disease. avian influenza and other chronic respiratory disease. Antibacterial agents. and inactivated and subunit vaccines are applied to curing or preventing this disease at present. However. the vaccines and antibacterial agents are often invalid due to the variform serotypes of APEC and complicated pathogenic mechanisms. In addition. because of the unreasonable usage of antibacterial agents for long time. the human food security and public health problem are caused. The traditional Chinese medicine has lots of advantages in curative effect. security. and controlling drug resistance and residue. so it is more important for prevention and treatment of livestock infectious disease. Therefore. in this study, we had screened an effective Chinese medical formula through a serial of experiments in vitro and in vivo, then further observed its curative effect and studied its action mechanism from cellular and molecular perspectives. According to these, we hoped to provide a theoretical basis for further research on this Chinese medical formula and the application in prevention or therapy for chicken colibacillosis.According to pertinent literature, we chose 114 kinds of Chinese materia medica including heat-clearing drugs, dampness-eliminating drugs. blood-regulating drugs and others to examine their antibacterial activity against APEC through bacteriostasis test in vitro.6 kinds of Chinese material medica had good antibacterial activity. such as aloe. andrographitis. garlic. calyx seu fructus physalis. fructus mume and granati cortex. Their inhibition zone were respectively 15.12.12.11.11 and 7 mm. Then, we isolated and purified the antibacterial components of aloe by silica gel column chromatography, gel column chromatography and ODS column chromatography. At last, the antibacterial component was identified as fumaric acid through MS. IR and NMR analysis. Then, we further studied the activity of fumaric acid against Escherichia coli. Staphylococcus aureus, Streptococcus. Salmonella. The MIC was respectively 50,50,780 and 780μg/ml and MBC was respectively 100.100,1560 and 780μg/ml.We had screened 39 Chinese medical formula through chicken model infected by APEC. The results showed that No.4. No.5. No.24 and NO.26 had better effect on decreasing mortality. and their mortality were respectively 30%.40%.26.7%. 26.7%. To confirm the effects of these Chinese medical formula. we carried out 2 repeated tests and found that they had stable effects on chicken infected APEC. the mortality were below 46.7%. And No.26 is the best one which the mortality was 26.7% in 2 repeated tests. Then we defined the usage of the combined drugs of No.26. that is Rhizoma Cyperi 40 g. Andrographis paniculata 30 g. Astragalus membranaceus 30 g. and designated it as "Xiang-Qi-Tang"We had further detected the effects of XQT using chicken model infected by APEC. The results showed that XQT could improve food intake and water consumption of infected chicken. The mortality of APEC group was 50%. the middle dose group and high dose group of XQT were respectively 20%(P<0.05) and 13.3% (P<0.01). and the two XQT groups also could improve weight gain. The blood routine results showed that the WBC of APEC group were significantly higher than that of control group (P<0.01). the WBC of low. middle and high dose group of XQT were significantly lower than that of APEC group (P<0.05 or P<0.01):the number of thrombocyte of APEC were significantly lower than that of control group (P<0.05). and the number of high dose group of XQT were higher than that of APEC group (P<0.05):XQT also could protect red blood cell. Then. XQT could decrease the typical pathological change of infected chicken, including decreasing incidence of pericarditis and perihepatitis and improving organ indexes of heart, liver, spleen and kidney. Through ELISA method, we detected the levels of TNF-a. IL-1 and sEPCR in serum of infected chicken, the results showed that XQT could decrease these inflammation mediator. These results indicated that XQT had better effect on chicken infected by APEC. which might be through anti-inflammation and anti-coagulation to play its role.Microvascular endothelial cells play an important role in pathogenic process of APEC. Therefore, in this study, we detected the effects of XQT and its active components on coagulation. through the inflammation model of MVECs induced by LPS. MVECs was treated with XQT and its active components for 3h. then challenged with 1μg/mL LPS. the cells were incubated at 37℃in a cell incubator for 18h. The supernatants were collected and analyzed the contents of PAI-1 and TF by ELISA kits. The results showed that as compared with LPS control group, the PAI-1 contents were significantly decreased in the low. middle and high dose group of XQT andα-Cyperone. in the middle and high dose group of AstragalosideⅣ, and in the low dose Andrographolide;the TF contents were significantly decreased in the low. middle and high dose group of XQT. in the middle and high dose group ofα-Cyperone. and in the high dose group of AstragalosideⅣand Andrographolide. It suggested that XQT and its active components could inhibite the secretion of PAI-1 and TF, which could decrease coagulation.We analyzed effect of XQT and its active components on mRNA levels of PAI-1, TF and KLF2 using semi-quantitive RT-PCR. The results showed that, after MVECs challenged with LPS for 6h. as compared with LPS control group. the levels of PAI-1 mRNA significantly decreased in XQT group. while the levels did not decrease inα-Cyperone. AstragalosideⅣand Andrographolide group; the levels of TF mRNA significantly decreased in XQT andα-Cyperone group. while the levels did not decrease in AstragalosideⅣand Andrographolide group:the levels of KLF2 mRNA significantly decreased in XQT.α-Cyperone. AstragalosideⅣ. Andrographolide group. These results suggested that XQT and its active components could inhibit the levels of mRNA of PAI-1 and TF induced bv LPS. and regulated the levels of mRNA of KLF2 which was the regulator of PAI-1 and TF. We analyzed effect of XQT and its active components on expression of ICAM-1, IκB-αand NF-κB p65 using ELISA and western blot. The results showed that as compared with LPS control group. the levels of mRNA TNF-a significantly decreased in low, middle and high dose group of XQT and a-Cyperone. in middle and high dose group of Astragaloside IV and Andrographolide. The expression of ICAM-1 protein significantly decreased in XQT. a-Cyperone. Astragaloside IV and Andrographolide. These results suggested that XQT and its active components had better anti-inflammation. In addition. XQT and its active components did not increase the expression of IκB-α. while they could decrease the expression of NF-κB p65. It suggested that the anti-inflammation action of XQT and its active components was mediated by decreasing the expression of NF-κB p65.Based on all the information above, we obtained an effective Chinese medical formula. XQT. curing chicken colibacillosis through in vitro and in vivo screening. XQT could improve water consumption. food intake, mortality, weight gain. blood routine. typical pathological change. organ indexes and inflammation mediator. And the effect of XQT might be performed through regulating the coagulation mediated by KLF2 and inflammation mediated by NF-κB.更多还原