银翘散对呼吸道合胞病毒感染小鼠呼吸系统模型的作用研究

发布时间：2018-06-05 09:48

本文选题：银翘散 + 小鼠　；参考：《广州中医药大学》2016年博士论文

【摘要】：呼吸道合胞病毒(respiratory syncytial virus, RSV)是世界范围的一个主要公共健康问题。RSV感染是导致婴幼儿下呼吸道感染、细支气管炎和肺炎的最主要的病原体,也是世界范围内儿童流行性呼吸道疾病的主要感染因素之一。RSV感染目前仍没有令人信服的安全的针对该病毒的疫苗,也没有行之有效的抗病毒治疗手段。目前可供临床选用治疗RSV的药物较少,一直以来,西药方面治疗RSV感染的药物主要有抗病毒药物、支气管扩张剂、激素类抗炎药和免疫调节剂等。但是这些药物都存在比较严重的毒副作用,而疫苗又难以奏效。与西药相比,中药的毒副作用小,药源丰富,防治RSV感染具有的独特效果。中药防治RSV感染的优势在于通过调整机体的免疫状态,来增强机体抵抗流感病毒的免疫力。因此,在中医药宝库中寻找有效防止RSV感染的药物就成为一项有重大意义的工作。银翘散为中药复方,是中医辛凉解表法的代表方,是临床辨证治疗风热型流感的主要方剂;对流行性感冒、肺炎、支气管炎、扁桃腺炎、腮腺炎等属于中医温热病范畴的病毒性感染疾病等有切实疗效;其配方由连翘,金银花,苦桔梗,薄荷,竹叶,生甘草,荆芥穗,淡豆豉,牛蒡子,芦根等10味药物组成;其中,金银花、连翘既有辛凉透邪、清热之功,又具芳香辟秽解毒之效;薄荷、牛蒡子为辛凉之性疏风清热而利咽喉。荆芥穗、淡豆豉为辛温之性助君药开皮毛而逐邪,芳香辟秽。竹叶可清上焦热；芦根可清热生津；桔梗可宣肺止咳。甘草既可调和诸药,护胃安中,又可合桔梗清利咽喉。研究表明银翘散中各组成分均能够可能发挥抑制RSV感染的作用。本研究建立体内RSV感染小鼠呼吸系统模型和体外RSV感染小鼠上皮细胞TC-1模型,采用低、中、高剂量银翘散(分别为150 mg/kg、300 mg/kg和600mg/kg)和利巴韦林(150mg/kg)进行治疗,通过对肺指数进行测定,HE染色观察肺组织和鼻黏膜组织炎性浸润等病理形态学改变,检测炎性因子IL-1β、TNF-α和IL-6的表达,从动物和细胞水平来观察银翘散对RSV所致的呼吸系统感染是否具有改善的作用。RSV感染常常导致免疫损伤和炎症的出现。在活化炎症因子的过程中,NALP3炎性体有不可或缺的作用。NALP3炎性体是核苷酸结构域样受体(NALPs)家族成员,是体内免疫系统对病原体的感受器,可识别病原体并产生相应的免疫应答反应。NALP3炎性体由NOD样受体家族成员3 (NALP3)、调亡相关斑点样蛋白(ASC)、以及半胱天冬酶caspase-1组成。当配体与NALP3结合后促进炎症小体的形成,并对caspase-1进行自身激活,导致IL-1β和IL-18等各种炎性因子的成熟及分泌,引起各种免疫炎症反应。因此本研究建立RSV感染呼吸系统的体内外模型并给予银翘散干预后,采用qPCR和western blot检测NALP3炎性体相关蛋白NALP3、ASC和caspase-1的表达,探讨银翘散治疗RSV感染作用与NALP3炎性体激活的关系,以期深入揭示银翘散抗RSV感染效应的免疫应答信号机制,为RSV感染的临床应用进一步提供理论依据。研究目的探讨银翘散对呼吸道合胞病毒感染小鼠系统模型的治疗效果以及作用机制。研究方法第一部分：建立呼吸道合胞病毒感染小鼠呼吸系统模型；采用低、中、高剂量银翘散(分别为150mg/kg、300mg/kg和600mg/kg)与利巴韦林阳性对照药(150mg/kg)进行治疗；通过称量小鼠体重和肺重量计算肺指数。HE染色检测小鼠鼻粘膜和肺组织炎性细胞浸润等病理形态学的改变。酶联免疫吸附法测定检测小鼠肺泡灌洗液中炎症因子IL-1β、TNF-α以及IL-6的表达水平。qPCR和western blot检测小鼠肺组织中NALP3炎性体的相关因子NALP3、ASC和caspase-1的表达。第二部分：建立呼吸道合胞病毒感染小鼠肺上皮细胞TC-1的体外模型,采用银翘散与利巴韦林含药血清进行干预,或者通过转染NALP3小干扰RNA抑制NALP3表达；酶联免疫吸附法测定检测TC-1细胞培养上清中炎症因子IL-1β、TNF-α以及IL-6的表达；qPCR和western blot检测各组细胞NALP3、ASC和caspase-1的表达。研究结果第一部分：体内实验表明采用银翘散治疗呼吸道合胞病毒感染的小鼠呼吸系统模型可以降低小鼠肺指数,减轻鼻黏膜上皮细胞和肺组织的损伤。并抑制NALP3炎性体相关蛋白NALP3、ASC和caspase-1的表达水平,降低炎性因子IL-1 β、TNF-α、IL-6的表达水平。且银翘散对RSV感染小鼠呼吸系统的改善作用与利巴韦林阳性对照药的改善作用相当。第二部分：体外细胞实验进一步证实银翘散对RSV感染的改善作用,及其作用机制与NALP3炎性体激活相关。此外,给予NALP3小干扰RNA后发现其可逆转RSV感染TC-1细胞所致的IL-1β、TNF-α和IL-6及NALP3炎性体相关蛋白NALP3、ASC和caspase-1的表达水平升高。研究结论银翘散在体内外实验中均能够改善呼吸道合胞病毒感染所致的呼吸道系统炎症,其作用机制与抑制NALP3炎性体的激活和炎症因子的分泌相关。通过观察NALP3小分子干扰组对RSV感染的改善作用,进一步揭示银翘散治疗RSV感染作用与NALP3炎性体激活的关系,也为下一步分子靶向治疗RSV感染的研究提供了实验基础和理论依据。
[Abstract]:Respiratory syncytial virus (respiratory syncytial virus, RSV) is a major public health problem worldwide,.RSV infection is the leading cause of lower respiratory tract infection, bronchiolitis and pneumonia in infants and children, and is also one of the major infectious factors of respiratory diseases in children worldwide,.RSV infection is still not available. There are convincing and safe vaccines against the virus, and there are no effective antiviral treatments. There are fewer drugs for the treatment of RSV. The drugs for RSV infection in western medicine have been mainly antiviral drugs, bronchodilators, irritable antiinflammatory drugs and immunomodulators. There are serious toxic and side effects, and the vaccine is difficult to work. Compared with western medicine, the toxic and side effects of traditional Chinese medicine are small, the source of medicine is rich and the unique effect of preventing RSV infection. The advantage of traditional Chinese medicine to prevent RSV infection is to enhance the immunity of the body by adjusting the immune state of the body. Therefore, in the treasure house of Chinese medicine, the Chinese medicine is in the treasure house of Chinese medicine. Finding effective drugs to prevent RSV infection is of great significance. Yinqiao powder is a traditional Chinese medicine compound. It is the representative of Chinese medicine Xin Liang solution. It is the main prescription for the treatment of wind heat influenza, and the virus infection of influenza, pneumonia, bronchitis, tonsillitis, mumps and so on is the category of warm disease of traditional Chinese medicine. Disease and other effective effects; its formula consists of Forsythia forsythia, honeysuckle, bitter orange stem, mint, bamboo leaves, raw Glycyrrhiza, Jing Jiesui, black bean, burdock, root of reed. The fermented soya bean has the nature to help the gentleman to drive the skin to drive the evil, and the aroma is unclean. The bamboo leaves can clear the heat of the coke; the reed root can clear the hot water; the Platycodon grandiflorum can promote the lung to cough. The glycyrrhiza can reconcile the various drugs, protect the stomach and make the throat. The study shows that all the components of the yinqiao powder can play the role of inhibiting RSV infection. This study established the study. The respiratory system model of mice infected with RSV and the TC-1 model of RSV infected mice epithelial cells in vitro were treated with low, medium and high dose Yinqiao Powder (150 mg/kg, 300 mg/kg and 600mg/kg) and Leigh Bhave Lin (150mg/kg). The pulmonary index was measured, and the pathological morphology of lung tissue and nasal mucosa tissue was observed by HE staining. Changes, detection of the expression of inflammatory factors IL-1 beta, TNF- alpha and IL-6, from animal and cell level to observe the effect of Yinqiao Powder on RSV induced respiratory infection,.RSV infection often leads to the emergence of immune injury and inflammation. In the process of activating inflammatory factors, NALP3 inflammatory bodies have an indispensable role in the.NALP3 inflammatory body. A member of the nucleotide structural domain like receptor (NALPs) family, the receptor of the immune system in the body, which recognizes pathogens and produces corresponding immune responses,.NALP3 inflammatory bodies are composed of NOD like receptor family members 3 (NALP3), apoptosis related speckle like protein (ASC), and cysteine aspartic enzyme caspase-1. When ligands are combined with NALP3, the ligand is combined with NALP3. It promotes the formation of inflammatory bodies and activates the caspase-1 itself, which leads to the maturation and secretion of various inflammatory factors such as IL-1 beta and IL-18, and causes various immune inflammatory responses. Therefore, this study established the model of RSV infected respiratory system in vivo and in vitro and gave the prognosis of Yinqiao Powder. QPCR and Western blot were used to detect the N associated protein N in NALP3. The expression of ALP3, ASC and caspase-1, to explore the relationship between the effect of Yinqiao Powder on RSV infection and the activation of NALP3 inflammatory body, in order to further reveal the immune response signal mechanism of the anti RSV infection effect of Yinqiao Powder, and to provide the theoretical basis for the clinical application of RSV infection. Therapeutic effect and mechanism of action. Part 1: establish respiratory system model of respiratory syncytial virus infection in mice; use low, medium and high dose Yinqiao Powder (150mg/kg, 300mg/kg and 600mg/kg) and Leigh Bhave Lin positive control medicine (150mg/kg); calculate the lung finger by weighing the weight and lung weight of the mice. .HE staining was used to detect the pathological changes of inflammatory cells in the nasal mucosa and lung tissue of mice. The enzyme linked immunosorbent assay was used to detect the expression level of inflammatory factors IL-1 beta, TNF- A and IL-6 in the alveolar lavage fluid of mice,.QPCR and Western blot to detect the related factors of NALP3 inflammatory in lung tissues of mice, NALP3, ASC and caspase-1. The second part: establish an in vitro model of respiratory syncytial virus (RSV) infected mice lung epithelial cell TC-1, intervention by yinqiao powder and ribavirin containing serum, or NALP3 expression by transfection of NALP3 small interference RNA, and enzyme linked immunosorbent assay for the determination of the inflammatory factors IL-1 beta, TNF- A and IL-6 in the cultured supernatant of TC-1 cells. QPCR and Western blot were used to detect the expression of NALP3, ASC and caspase-1 in each group. Part 1: in vivo experiments showed that the mice respiratory system model using Yinqiao Powder in the treatment of respiratory syncytial virus infection could reduce the lung index of mice, reduce the damage of the nasal epithelium and lung tissue, and inhibit the NALP3 inflammatory body. The expression level of related protein NALP3, ASC and caspase-1 decreased the expression level of inflammatory factor IL-1 beta, TNF- alpha and IL-6, and the improvement effect of Yinqiao Powder on the respiratory system of RSV infected mice was similar to that of Leigh Bhave Lin positive control. The second part: in vitro cell experiment confirmed the effect of Yinqiao Powder on the improvement of RSV infection, and The mechanism of its action was related to the activation of NALP3 inflammatory body. In addition, after the NALP3 small interference RNA was given, the expression of IL-1 beta, TNF- A and IL-6 and NALP3 inflammatory related protein NALP3, ASC and caspase-1 were increased after the small interference of NALP3 RNA. Conclusion yinqiao powder can improve the respiratory syncytial virus in both in vivo and in vivo. The mechanism of respiratory tract inflammation caused by dyeing is related to the inhibition of the activation of NALP3 inflammatory body and the secretion of inflammatory factors. By observing the effect of NALP3 small molecule interference group on RSV infection, it further reveals the relationship between the effect of Yinqiao Powder on RSV infection and the activation of NALP3 inflammatory body, and the next molecular targeting treatment of RSV infection. The research provides the experimental basis and theoretical basis.
【学位授予单位】：广州中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R285.5;R-332

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