家畜疯草中毒解毒药物的研制及疯草中活性成分苦马豆素的抗病毒作用机制研究

发布时间：2018-06-25 01:28

本文选题：疯草中毒 + 解毒药物　；参考：《甘肃农业大学》2017年博士论文

【摘要】：“疯草”(Locoweed)是棘豆属、黄芪属和苦马豆素属中有毒植物的统称,动物长期误食能引起中毒。现已查明,吲哚里西啶生物碱苦马豆素是其主要致毒物质。另有研究表明苦马豆素具有抗病毒感染、增强免疫、抗肿瘤、抑制肿瘤细胞转移及潜在的抗艾滋病病毒等多种药用活性。针对上述现状,目前研究存在以下几个问题:1.具有良好作用的家畜疯草中毒解毒药物仍处于空白。2.疯草中有效活性成分苦马豆素抗病毒作用机制不明确。本研究从疯草中毒机理出发,通过调节苦马豆素与α-甘露糖苷酶的量化关系,研制出1种疯草中毒治疗药物—速康解毒口服溶液,并对其分别进行了药效和临床治疗试验。为了阐明苦马豆素对牛病毒性腹泻病毒的抗病毒作用机制,及其对免疫细胞因子的调节作用影响,进行了以下实验研究,并得出一定的结论。(1)通过建立茎直黄芪饲喂试验家兔中毒模型,利用临床观察、体重称量、α-甘露糖苷酶活性测定和组织病理学观察等方法研究了速康解毒口服溶液对茎直黄芪中毒的药效作用,得出以下结论:攻毒组、攻毒解毒治疗组实验家兔均在18天左右出现中毒症状,表明人工中毒实验家兔模型建立成功。试验中速康解毒口服溶液能显著性升高因中毒造成的体重下降和α-甘露糖苷酶活性降低(P0.05),解毒治疗组较攻毒组的病理学变化轻微,且在试验期内未出现家兔死亡,表明速康解毒口服溶液具有较好的解毒作用。(2)对一起突发疑似疯草中毒的小尾寒羊共计131只,通过临床给予速康解毒口服溶液进行解毒治疗得出以下结论:经过诊断、询问,确定此次突发疑似疯草中毒为黄花棘豆中毒,速康解毒口服溶液有效率达到77.86%,表明速康解毒口服溶液具有良好的解毒效果,可以应用于疯草中毒的临床治疗。(3)利用细胞培养技术,采用CPE观察法和MTT比色法相结合的方法,检测不同作用方式下,不同浓度苦马豆素对BVDV增殖的抑制作用和综合杀灭作用,得出以下结论:在体外随苦马豆素质量浓度的增加,其对BVDV的增殖抑制和综合杀灭作用都具有很好的量效关系,比较两种作用方式下的治疗指数2.5和3.2,说明苦马豆素对BVDV具有良好的综合杀灭作用,且对抑制BVDV病毒增殖具有一定的作用,表明苦马豆素可能对侵入细胞的BVDV的复制增殖产生抑制以及直接杀灭游离BVDV粒子而发挥作用。(4)通过建立BVDV感染BALB/c小鼠模型,利用不同剂量苦马豆素对其进行抗病毒治疗,得出以下结论:高、低剂量苦马豆素药物组(1/5LD50)给药24~48h后对小鼠腹泻症状较病毒组有显著减轻或减慢作用,对各实验组BALB/c小鼠的肝、肾、心、肺、脾组织的病理切片观察结果综合比较发现,各实验组受病毒感染侵害严重程度依次分别为:病毒感染组(B2)苦马豆素低剂量药物组(1/5LD50)(B4)苦马豆素高剂量组(1/5LD50)(B3)≥病毒唑药物组(B5),说明苦马豆素在体内也具有一定的抗病毒作用。(5)采用双抗夹心ELISA法检测苦马豆素对BVDV感染BALB/c小鼠中IFN-α、IFN-β表达水平的影响,得出以下结论:苦马豆素对病毒感染引起的小鼠IFN-α、IFN-β表达量下降具有显著的上调作用,表明苦马豆素可能通过促进IFN-α、IFN-β表达水平诱导细胞合成抗病毒蛋白,发挥间接抗病毒的作用,也可能通过调控活化BVDV易感细胞,如T细胞、B细胞等,提高MHC分子的表达,促使CD8+T细胞反应形成,进而杀伤感染病毒的靶细胞来影响机体的抗病毒免疫应答反应。(6)采用双抗夹心ELISA法检测BVDV感染BALB/c小鼠中共刺激分子CD80和CD86表达水平的影响,得出以下结论:苦马豆素对病毒感染引起的小鼠共刺激分子CD80和CD86表达量下降均具有显著的上调作用,表明苦马豆素可能通过上调共刺激分子CD80和CD86表达水平,刺激T细胞的活化,产生淋巴因子,导致靶细胞发生溶解、破坏,增强机体的抗病毒免疫应答。(7)采用双抗夹心ELISA法检测BVDV感染BALB/c小鼠中IL-1、IL-4、IL-15、IL-12、IL-6和IL-10表达水平的影响,得出以下结论:苦马豆素对病毒感染引起的小鼠IL-1、IL-4、IL-15、IL-12、IL-6表达量下降均具有显著的上调作用,表明苦马豆素能够通过上调小鼠IL-1、IL-4、IL-15、IL-12、IL-6的表达水平,刺激、促进B细胞和T细胞活化增殖,增强机体细胞免疫应答,并通过对感染靶细胞杀伤功能来影响机体的抗病毒免疫调节作用。另苦马豆素对病毒感染引起的小鼠IL-10表达量上升具有显著的下调作用,表明苦马豆素可能通过降低IL-10的表达水平,产生对Th2体液免疫应答的抑制调节,影响机体的抗病毒免疫作用。并推测苦马豆素诱导的机体抗病毒免疫应答以细胞免疫应答的方式为主。综上所述,针对疯草中毒引起的家畜中毒研制的治疗药物速康解毒口服溶液具有良好的解毒效果;从疯草中提取分离获得的生物活性成分苦马豆素在体内、体外均对BVDV具有良好的抗病毒作用,其抗病毒作用机制可能是苦马豆素能进入动物机体细胞内抑制BVDV的复制增殖或直接灭活游离BVDV粒子,并能通过促进、刺激相关细胞因子表达量的上调或下调,诱导机体自身免疫应答,协同发挥抗病毒作用。
[Abstract]:"Locoweed" is a general name for the poisonous plants of the genus Oxytropis, Astragalus and snoomalin, and the animal's long-term food intake can cause poisoning. It has been found that indolithiidine alkaloids are the main toxic substances. Potential anti AIDS virus and other medicinal activities. Aiming at the above situation, there are several problems as follows: 1. the antidetoxification drugs of domestic animal mad grass poisoning are still in the blank of the blank.2. mad grass, the mechanism of the antiviral action of bitter horse bean is not clear. The quantitative relationship between equinoxin and alpha mannase was quantified, and 1 kinds of medications for the treatment of mad grass poisoning - ktak detoxification oral solution were developed, and their efficacy and clinical trials were carried out respectively. In order to clarify the mechanism of the antiviral action of sac on bovine viral diarrhea virus and its effect on the regulation of immune cell factors, The following experimental studies, and draw certain conclusions. (1) through the establishment of a rabbit model of direct Astragalus feeding test, using clinical observation, weight weighing, alpha mannase activity determination and histopathological observation methods, the effect of ktak detoxification oral solution on stem direct yellow Astragalus poisoning was studied. The experimental rabbits in the drug detoxification group all had the poisoning symptoms about 18 days, which showed that the experimental rabbit model of artificial poisoning was established successfully. In the experiment, the body weight loss and the decrease of the activity of alpha mannase (P0.05) were significantly increased in the test, and the pathological changes of the detoxified treatment group were less than that of the attack group. No rabbits died during the period of examination, indicating that the oral solution of jiykang detoxification has good detoxification effect. (2) a total of 131 Small Tail Han sheep, who are suspected of being poisoned by the mad grass poisoning, have been treated by the clinical treatment of ktak detoxification oral solution for detoxification. The rate of Oxytropis poisoning and ktak detoxification oral solution reached 77.86%, indicating that the ktak detoxification oral solution has good detoxification effect and can be used in the clinical treatment of mad grass poisoning. (3) using the cell culture technique, the CPE observation method and the MTT colorimetric method are combined to detect different concentrations of bitter horse beans to BVDV The following conclusions were concluded: with the increase of the mass concentration of bitterin in vitro, it has a good dose effect relationship to the proliferation inhibition and comprehensive killing effect of BVDV, and compared the therapeutic index of 2.5 and 3.2 under the two modes of action, indicating that the kopsine has a good comprehensive killing effect on BVDV. It has a certain effect on inhibiting the proliferation of BVDV virus, which indicates that sac may inhibit the replication and proliferation of BVDV in invasive cells and directly kill free BVDV particles. (4) the following conclusions are obtained by establishing BVDV infected BALB/c mice model and using different doses of bitter horse bean in antiviral treatment. After the dose of 1/5LD50 (24~48h), the symptoms of diarrhea in mice were significantly reduced or slower than that of the virus group. The pathological sections of the liver, kidney, heart, lung and spleen of the BALB/c mice in the experimental groups were compared. The severity of the virus infection in the experimental groups was respectively: the virus infection group (B2), respectively. The low dose medicament group (1/5LD50) (B4) of high dose group (1/5LD50) (B3) or bavirazole (B5) in the high dose group (B3), indicating that the bitter horse bean has some antiviral effect in the body. (5) the effects of the double anti sandwich ELISA on the expression of IFN- A and IFN- beta in BVDV infected BALB/ c mice were detected by the double anti sandwich ELISA method. The following conclusion was drawn: bitter horse The decrease of IFN- alpha and IFN- beta expression in mice caused by virus infection has a significant up-regulation effect. It indicates that Marin may induce the synthesis of antiviral proteins by promoting IFN- alpha and IFN- beta expression level, exerting the effect of indirect antiviral activity. It may also improve the activation of MHC molecules by regulating the activation of BVDV susceptible cells, such as T cells, B cells and so on. The expression promoted the formation of CD8+T cell response and then killed the target cells of the infected virus to influence the immune response of the body. (6) the effect of the expression level of CD80 and CD86 in the BVDV infected BALB/c mice was detected by the double anti sandwich ELISA method. The following conclusion was drawn: the common spiny of the mice caused by the virus infection by the bitter horse bean The decrease of the expression of CD80 and CD86 has a significant up-regulation effect. It indicates that sac may increase the expression level of CD80 and CD86 of CO stimulators, stimulate the activation of T cells, produce lymphatic factors, cause dissolving and destruction of the target cells, and enhance the immune response of the body. (7) BVDV sense is detected by the double anti sandwich ELISA method. The effects of the expression of IL-1, IL-4, IL-15, IL-12, IL-6 and IL-10 in the BALB/c mice were studied. The following conclusions were drawn: the effect of bitter equine on mice IL-1, IL-4, IL-15, IL-12, IL-6 expression decreased significantly. Promote the proliferation of B and T cells, enhance the cellular immune response of the body, and influence the immune regulation of the body by killing the target cells, and the increase of the expression of IL-10 in mice caused by virus infection has a significant downregulation effect. It is shown that sac may reduce the expression of IL-10 by reducing the expression of water. The inhibitory regulation of the immune response to the body's humoral immune response and the antiviral immunity of the body are produced, and it is conjectured that the antiviral response of the body is mainly in the way of cellular immune response. To sum up, the antidote solution of the therapeutic drug, which is developed for the domestic animal poisoning caused by the mad grass poisoning, has good detoxification. Results: the bioactive ingredient, bitter horse bean, extracted from the mad grass in the body, has a good antiviral effect on BVDV in vitro. The mechanism of its antiviral action may be that the bitter horse bean can enter the cell of the animal body to inhibit the replication and proliferation of BVDV or directly inactivate the free BVDV particles, and can stimulate the related cell causes by promoting. Up regulation or down regulation of the expression level can induce the body's autoimmunity and play an antiviral role.
【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S856.9

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