本文選題：銀翹散 + 小鼠　； 參考：《廣州中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：呼吸道合胞病毒(respiratory syncytial virus, RSV)是世界范圍的一個(gè)主要公共健康問題。RSV感染是導(dǎo)致嬰幼兒下呼吸道感染、細(xì)支氣管炎和肺炎的最主要的病原體,也是世界范圍內(nèi)兒童流行性呼吸道疾病的主要感染因素之一。RSV感染目前仍沒有令人信服的安全的針對該病毒的疫苗,也沒有行之有效的抗病毒治療手段。目前可供臨床選用治療RSV的藥物較少,一直以來,西藥方面治療RSV感染的藥物主要有抗病毒藥物、支氣管擴(kuò)張劑、激素類抗炎藥和免疫調(diào)節(jié)劑等。但是這些藥物都存在比較嚴(yán)重的毒副作用,而疫苗又難以奏效。與西藥相比,中藥的毒副作用小,藥源豐富,防治RSV感染具有的獨(dú)特效果。中藥防治RSV感染的優(yōu)勢在于通過調(diào)整機(jī)體的免疫狀態(tài),來增強(qiáng)機(jī)體抵抗流感病毒的免疫力。因此,在中醫(yī)藥寶庫中尋找有效防止RSV感染的藥物就成為一項(xiàng)有重大意義的工作。銀翹散為中藥復(fù)方,是中醫(yī)辛涼解表法的代表方,是臨床辨證治療風(fēng)熱型流感的主要方劑;對流行性感冒、肺炎、支氣管炎、扁桃腺炎、腮腺炎等屬于中醫(yī)溫?zé)岵》懂牭牟《拘愿腥炯膊〉扔星袑?shí)療效;其配方由連翹,金銀花,苦桔梗,薄荷,竹葉,生甘草,荊芥穗,淡豆豉,牛蒡子,蘆根等10味藥物組成;其中,金銀花、連翹既有辛涼透邪、清熱之功,又具芳香辟穢解毒之效;薄荷、牛蒡子為辛涼之性疏風(fēng)清熱而利咽喉。荊芥穗、淡豆豉為辛溫之性助君藥開皮毛而逐邪,芳香辟穢。竹葉可清上焦熱；蘆根可清熱生津；桔梗可宣肺止咳。甘草既可調(diào)和諸藥,護(hù)胃安中,又可合桔梗清利咽喉。研究表明銀翹散中各組成分均能夠可能發(fā)揮抑制RSV感染的作用。本研究建立體內(nèi)RSV感染小鼠呼吸系統(tǒng)模型和體外RSV感染小鼠上皮細(xì)胞TC-1模型,采用低、中、高劑量銀翹散(分別為150 mg/kg、300 mg/kg和600mg/kg)和利巴韋林(150mg/kg)進(jìn)行治療,通過對肺指數(shù)進(jìn)行測定,HE染色觀察肺組織和鼻黏膜組織炎性浸潤等病理形態(tài)學(xué)改變,檢測炎性因子IL-1β、TNF-α和IL-6的表達(dá),從動物和細(xì)胞水平來觀察銀翹散對RSV所致的呼吸系統(tǒng)感染是否具有改善的作用。RSV感染常常導(dǎo)致免疫損傷和炎癥的出現(xiàn)。在活化炎癥因子的過程中,NALP3炎性體有不可或缺的作用。NALP3炎性體是核苷酸結(jié)構(gòu)域樣受體(NALPs)家族成員,是體內(nèi)免疫系統(tǒng)對病原體的感受器,可識別病原體并產(chǎn)生相應(yīng)的免疫應(yīng)答反應(yīng)。NALP3炎性體由NOD樣受體家族成員3 (NALP3)、調(diào)亡相關(guān)斑點(diǎn)樣蛋白(ASC)、以及半胱天冬酶caspase-1組成。當(dāng)配體與NALP3結(jié)合后促進(jìn)炎癥小體的形成,并對caspase-1進(jìn)行自身激活,導(dǎo)致IL-1β和IL-18等各種炎性因子的成熟及分泌,引起各種免疫炎癥反應(yīng)。因此本研究建立RSV感染呼吸系統(tǒng)的體內(nèi)外模型并給予銀翹散干預(yù)后,采用qPCR和western blot檢測NALP3炎性體相關(guān)蛋白NALP3、ASC和caspase-1的表達(dá),探討銀翹散治療RSV感染作用與NALP3炎性體激活的關(guān)系,以期深入揭示銀翹散抗RSV感染效應(yīng)的免疫應(yīng)答信號機(jī)制,為RSV感染的臨床應(yīng)用進(jìn)一步提供理論依據(jù)。研究目的探討銀翹散對呼吸道合胞病毒感染小鼠系統(tǒng)模型的治療效果以及作用機(jī)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統(tǒng)模型；采用低、中、高劑量銀翹散(分別為150mg/kg、300mg/kg和600mg/kg)與利巴韋林陽性對照藥(150mg/kg)進(jìn)行治療；通過稱量小鼠體重和肺重量計(jì)算肺指數(shù)。HE染色檢測小鼠鼻粘膜和肺組織炎性細(xì)胞浸潤等病理形態(tài)學(xué)的改變。酶聯(lián)免疫吸附法測定檢測小鼠肺泡灌洗液中炎癥因子IL-1β、TNF-α以及IL-6的表達(dá)水平。qPCR和western blot檢測小鼠肺組織中NALP3炎性體的相關(guān)因子NALP3、ASC和caspase-1的表達(dá)。第二部分：建立呼吸道合胞病毒感染小鼠肺上皮細(xì)胞TC-1的體外模型,采用銀翹散與利巴韋林含藥血清進(jìn)行干預(yù),或者通過轉(zhuǎn)染NALP3小干擾RNA抑制NALP3表達(dá)；酶聯(lián)免疫吸附法測定檢測TC-1細(xì)胞培養(yǎng)上清中炎癥因子IL-1β、TNF-α以及IL-6的表達(dá)；qPCR和western blot檢測各組細(xì)胞NALP3、ASC和caspase-1的表達(dá)。研究結(jié)果第一部分：體內(nèi)實(shí)驗(yàn)表明采用銀翹散治療呼吸道合胞病毒感染的小鼠呼吸系統(tǒng)模型可以降低小鼠肺指數(shù),減輕鼻黏膜上皮細(xì)胞和肺組織的損傷。并抑制NALP3炎性體相關(guān)蛋白NALP3、ASC和caspase-1的表達(dá)水平,降低炎性因子IL-1 β、TNF-α、IL-6的表達(dá)水平。且銀翹散對RSV感染小鼠呼吸系統(tǒng)的改善作用與利巴韋林陽性對照藥的改善作用相當(dāng)。第二部分：體外細(xì)胞實(shí)驗(yàn)進(jìn)一步證實(shí)銀翹散對RSV感染的改善作用,及其作用機(jī)制與NALP3炎性體激活相關(guān)。此外,給予NALP3小干擾RNA后發(fā)現(xiàn)其可逆轉(zhuǎn)RSV感染TC-1細(xì)胞所致的IL-1β、TNF-α和IL-6及NALP3炎性體相關(guān)蛋白NALP3、ASC和caspase-1的表達(dá)水平升高。研究結(jié)論銀翹散在體內(nèi)外實(shí)驗(yàn)中均能夠改善呼吸道合胞病毒感染所致的呼吸道系統(tǒng)炎癥,其作用機(jī)制與抑制NALP3炎性體的激活和炎癥因子的分泌相關(guān)。通過觀察NALP3小分子干擾組對RSV感染的改善作用,進(jìn)一步揭示銀翹散治療RSV感染作用與NALP3炎性體激活的關(guān)系,也為下一步分子靶向治療RSV感染的研究提供了實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R285.5;R-332

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3 吳振起;銀翹散及其性味分組提取物對流感病毒感染誘導(dǎo)細(xì)胞凋亡作用的實(shí)驗(yàn)研究[D];遼寧中醫(yī)藥大學(xué);2008年

4 陳俏妍;銀翹散治療急性病毒性咽炎的臨床和基礎(chǔ)研究[D];廣州中醫(yī)藥大學(xué);2013年

5 謝慧s，

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