盐酸小檗碱对猪胸膜肺炎放线杆菌作用机制的研究

发布时间：2018-09-05 17:44

【摘要】：胸膜肺炎放线杆菌是无芽孢多形性球状短杆菌,能引起猪呼吸系统疾病,具有高死亡和高传染等特点,临床上主要表现为肺部炎症和呼吸系统功能障碍,常呈暴发性流行,并常与多种细菌混合感染。盐酸小檗碱是从黄连等植物中提取的季胺型异喹啉类生物碱,对多种革兰氏阳性、阴性菌均有较好的抑制和杀灭效果。本文拟研究盐酸小檗碱对该菌的抑制和杀灭效果并初步阐明作用机制。主要研究结果如下：1.盐酸小檗碱的体外抑菌效果的研究：采用二倍稀释法,测定盐酸小檗碱对猪胸膜肺炎放线杆菌的最低抑菌浓度(Minimal inhibitory concentration,MIC)和最小杀菌浓度(Minimal bactericidal concentration,MBC),并绘制其抑菌曲线。结果表明：盐酸小檗碱对猪胸膜肺炎放线杆菌具有较好的体外杀灭效果,其MIC和MBC均为0.3125 mg/mL；当盐酸小檗碱浓度为MIC时,细菌在8 h内全部死亡,浓度为1/2 MIC及以下时,细菌生长受到明显抑制,但生长曲线与正常组相似,表明盐酸小檗碱对猪胸膜肺炎放线杆菌存在剂量依赖而非时间依性。以上结果表明盐酸小檗碱对猪的胸膜肺炎放线杆菌具有较好的体外杀菌效果。2.盐酸小檗碱对猪胸膜肺炎放线杆菌细胞膜的影响：通过测定大分子物质和Ca,Mg,K离子的泄漏判断细胞膜的完整性,并对膜上蛋白进行荧光分析；测定还原糖的利用情况和采用氮苯基-1-萘胺(N-Phenyl-1-Napthylamine,NPN)法判断细胞膜的渗透性；通过测量乳酸脱氢酶(Alkaline phosphatase,LDH)和碱性磷酸酶(Alkaline phosphatase,AKP)的体外活性判断药物对细菌细胞壁完整性的影响。结果表明：盐酸小檗碱作用于细菌后,在前10 min内,大分子物质DNA胞外浓度变化不显著,1-12h内,胞外DNA浓度和蛋白质浓度先上升后下降,Ca,Mg,K离子胞外浓度升高,离子泄露,表明盐酸小檗碱能够破坏细胞膜的完整性和渗透性。胞外LDH和AKP的活性与对照组相比变化不明显。膜蛋白荧光结果显示,盐酸小檗碱能与细胞膜上的色氨酸残基相结合,引起细胞膜结构发生改变,其淬灭方式为静态淬灭。以上结果表明盐酸小檗碱能够影响胸膜肺炎放线杆菌细胞膜的完整性和渗透性,与膜上的某些蛋白质结合从而影响蛋白质的结构与功能。3.盐酸小檗碱对猪胸膜肺炎放线杆菌蛋白质表达的影响：通过测定细菌总蛋白含量的变化及SDS-聚丙烯酰胺凝胶电泳(Sodium dodecyl sμLphate polyacrylamide gel-electrophoresis,SDS-PAGE)测定细菌蛋白表达的差异,研究盐酸小檗碱对细菌蛋白质合成的影响。结果表明：盐酸小檗碱引起细菌总蛋白的表达量先升高后降低正常组蛋白质条带多而清晰,药物作用后蛋白表达出现差异,一些蛋白条带变暗甚至消失,推测44.3kDa以下的蛋白质可能为盐酸小檗碱的作用靶点。以上结果表明盐酸小檗碱使得细菌蛋白质的合成和表达下降。4.盐酸小檗碱对猪胸膜肺炎放线杆菌DNA合成的影响：通过4',6-二脒基-2-苯基吲哚(4'、6-diamidino-2-phenylindole,DAPI)染色,紫外吸收光谱,琼脂糖凝胶电泳,EB-DNA(溴化乙锭-DNA)体系,热溶解曲线等方法检测细菌DNA在药物作用前后表达量的变化及DNA与盐酸小檗碱的体外结合状态,研究盐酸小檗碱对细菌DNA的影响。荧光染色结果表明,正常组荧光强度较强,数目较多，药物作用组荧光强度降低,表明盐酸小檗碱能够影响细菌DNA的表达。紫外吸收光谱的改变和琼脂糖凝胶电泳结果表明,盐酸小檗碱能够与DNA结合。EB-DNA和热溶解曲线结果表明盐酸小檗碱能够与DNA在体外结合,其结合方式与EB相同但作用强度更高。以上结果表明盐酸小檗碱能够影响细菌DNA的表达,在体外与DNA结合,其结合方式为嵌插作用。综合以上结果,盐酸小檗碱对猪的胸膜肺炎放线杆菌具有良好的杀灭效果,作用机理为引起细菌细胞膜完整性的改变,影响细菌蛋白质和DNA的合成。
[Abstract]:Actinobacillus pleuropneumoniae is a non spore forming * short form Bacillus brevis, which can cause respiratory diseases in pigs. It is characterized by high mortality and high infection. Clinically, it is mainly manifested as pulmonary inflammation and respiratory dysfunction. It is often shown as an outbreak epidemic and is often mixed with a variety of bacteria. Berberine hydrochloride is extracted from plants such as Coptis chinensis. Quaternary isoquinoline alkaloids have good inhibitory and killing effects on a variety of gram-positive and gram-negative bacteria. In this paper, the inhibitory and killing effects of berberine hydrochloride on the bacteria were studied and its mechanism was preliminarily elucidated. The main results are as follows: 1. In vitro inhibitory effects of berberine hydrochloride on bacteria were studied by double dilution method. Berberine hydrochloride * Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (Minimal bactericidal concentration, MBC) of Actinobacillus pleuropneumoniae, and their bacteriostatic curves were drawn. The results showed that berberine hydrochloride had a good in vitro killing effect on * swine Actinobacillus pleuropneumoniae, MIC and MBC. They were all 0.3125 mg/mL; when the berberine hydrochloride concentration was MIC, the bacteria died within 8 h, and the growth of bacteria was significantly inhibited when the concentration was 1/2 MIC and below. However, the growth curve was similar to that of the normal group * indicating that berberine hydrochloride had a dose dependence rather than time dependence on the swine Actinobacillus pleuropneumoniae. * the effect of.2. berberine on the cell membrane of Actinobacillus pleuropneumoniae * cell membrane was determined by measuring the leakage of porcine Actinobacillus pleuropneumoniae. The integrity of the membrane was determined by measuring the leakage of macromolecules and Ca, Mg and K ions, and fluorescence analysis of the protein on the membrane was performed. The utilization of reducing sugar and nitrogen benzol were determined. The permeability of cell membrane was determined by N-Phenyl-1-Napthylamine (NPN) method, and the effect of the drug on the integrity of cell wall was evaluated by measuring the activities of lactate dehydrogenase (LDH) and alkaline phosphatase (AKP). Within 1-12 hours, the concentration of extracellular DNA and protein increased first and then decreased. The concentration of Ca, Mg, K ions increased and the ion leakage showed that berberine hydrochloride could destroy the integrity and permeability of cell membrane. The activity of extracellular LDH and AKP had no significant change compared with the control group. The results showed that berberine hydrochloride could bind to the tryptophan residues on the cell membrane, resulting in the change of cell membrane structure, and the quenching mode was static quenching. Effects of berberine hydrochloride and function.3. * on the protein expression of Actinobacillus pleuropneumoniae protein: the changes of bacterial total protein content and SDS- dodecyl Sodium Lphate polyacrylamide gel-electrophoresis (SDS-PAGE) were used to detect the difference of bacterial protein expression, and the effects of berberine hydrochloride on bacterial protein expression were studied. The results showed that the expression of total bacterial protein increased first and then decreased in normal group. The protein expression was different after the drug action. Some protein bands became dark or even disappeared. It was speculated that the protein below 44.3 kDa might be the target of berberine hydrochloride. The results showed that berberine hydrochloride reduced the protein synthesis and expression of.4. * the effect of berberine hydrochloride on the DNA synthesis of Actinobacillus pleuropneumoniae DNA: 4', 6- two amipryl -2- phenyl indole (4', 6-diamidino-2-phenylindole, DAPI) staining, UV absorption spectrum, agarose gel electrophoresis, EB-DNA (ethidium bromide -DNA) system, thermal dissolution. The effect of berberine hydrochloride on bacterial DNA was studied. The results of fluorescence staining showed that the fluorescence intensity of normal group was stronger, the number of bacterial DNA was larger, the fluorescence intensity of drug group was lower, indicating that berberine hydrochloride could affect bacterial DN. The results of ultraviolet absorption spectroscopy and agarose gel electrophoresis showed that berberine hydrochloride could bind to DNA. The results of EB-DNA and thermolysis curves showed that berberine hydrochloride could bind to DNA in vitro, and the binding mode was the same as that of EB, but the binding intensity was higher. In combination with DNA in vitro, the binding mode is inserted. In conclusion, berberine hydrochloride has good killing effect on * Actinobacillus pleuropneumoniae, and the mechanism of action is to cause changes in the integrity of bacterial cell membrane and affect the synthesis of bacterial protein and DNA.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.28

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