银花平感颗粒和三拗汤体内体外结合对甲型流感病毒的影响
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摘要
目的通过银花平感颗粒和三拗汤两方药体内外对甲型H1N1流感病毒影响的实验研究,探讨不同方剂配伍对流感病毒的抑制作用。方法体内实验:采用滴鼻感染建立小鼠流感病毒肺炎模型,按体重分层随机分为正常对照组、病毒模型组、达菲组[21. 63 mg/(kg·d)]、银花平感颗粒组[12 g/(kg·d)]和三拗汤组[9 g/(kg·d)],灌胃给予相应药物,每天1次,连续5天。灌胃结束后,摘取肺、脾及胸腺组织,分别称重,计算各组小鼠肺指数、肺组织抑制率及免疫脏器指数;采用实时荧光定量PCR法检测各组小鼠肺组织病毒载量表达,苏木精-伊红(HE)染色法检测各组小鼠肺组织的病理改变。体外实验:将银花平感颗粒和三拗汤两方药作用于感染甲型H1N1流感病毒的犬肾细胞(MDCK),采用MTT法检测两方药对甲型H1N1流感病毒的抗病毒有效率及显微镜观察其引起的细胞病变效应(CPE)。结果体内实验中,与正常对照组比较,病毒模型组小鼠肺指数升高,脾指数、胸腺指数均降低,多处肺实变,炎症细胞浸润,肺组织内病毒载量高表达(P <0. 01),与病毒模型组比较,银花平感颗粒和三拗汤均降低流感病毒感染小鼠肺指数(P <0. 01),升高脾指数、胸腺指数(P <0. 05,P <0. 01),并明显减轻流感病毒肺炎小鼠肺组织病理变化,降低肺组织病毒载量(P <0. 05,P <0. 01);体外实验中,银花平感颗粒和三拗汤可以明显抑制流感病毒引起的CPE,对病毒感染也有明显的保护作用。结论银花平感颗粒和三拗汤两方药在体内外均有良好的病毒抑制作用,其抗病毒作用可能与抑制流感病毒复制、保护免疫器官、减轻肺部炎症损伤及抑制病毒引起的CPE有关,且在体内外对流感病毒的抑制方面,银花平感颗粒比三拗汤配伍更全面,疗效更好。
Objective To evaluate the effects of Yinhua Pinggan Granule and San-ao Decoction on inhibition influenza A/H1 N1 virusin vitro andin vivo,and to investigate the inhibitory effect of different compatibility of formula on the influenza A/H1 N1 virus. Methods In vivo experiments,the model of pneumonia by nasal dropping influenza virus was established in mice. According to the weight stratification,then they were divided randomly into five groups: normal control group,influenza virus model group,oseltamivir control group( 21. 63 mg/kg per day),Yinhua Pinggan Granule group( 12 g/kg per day),and San-ao Decotion group( 9 g/kg per day). The animals were received gavage administration with corresponding drugs,once per day for 5 successive days. After the gavage administration,lungs,spleens and thymuses were removed and weighed,respectively. Lung index,inhibition ratio of lung index and the immune organ index were calculated. The expressions of viral load in lung tissue of each group were detected via Real-time fluorescent quantitative PCR. The pathological change of lung tissue in each group were detected by hematoxylin-eosin staining( HE) staining. In vitro experiments,Yinhua Pinggan Granule and San-ao Decoction were used to treat Madin-Darby Canine Kidney( MDCK) cells infected with influenza A/H1 N1 virus. The antiviral efficiacy of two formulas against influenza A/H1 N1 virus were detected by MTT method and the cytopathic effects( CPE) that were induced by influenza virus were observed by Microscopy. Results In vivo experiments showed that compared with the normal control group,lung index increased,spleen index and thymus index decreased,multiple lung consolidation,inflammatory cell infiltration,and high expression of viral load in lung tissue of influenza virus model group(P < 0. 01). Compared with influenza virus model group,Yinhua Pinggan Granule and San-ao Decoction significantly decreased lung index,increased spleen index and thymus index in mice infected with influenza virus(P < 0. 05,P < 0. 01),and significantly alleviated pathological change of lung tissue in mice with influenza viral pneumonia,and significantly decreased virus load in lung tissue(P < 0. 05,P < 0. 01). In vitro experiments also showed Yinhua Pinggan Granule and Sanao Decoction significantly inhibited cytopathic effect caused by influenza virus,and also had a obvious protective effect on influenza virus infection. Conclusions The viral inhibitory effects of Yinhua Pinggan Granule and San-ao Decoction were good in vivo andvitro experiments. Antiviral effects may be associated with inhibition the replication of influenza virus,protection immune organs,alleviation lung inflammation damage and inhibition the cytopathic effects caused by influenza virus. Moreover,Yinhua Pinggan granule was more comprehensive and effective than San-ao Decoction in the aspect of inhibiting influenza virusin vivo andin vitro.
Objective To evaluate the effects of Yinhua Pinggan Granule and San-ao Decoction on inhibition influenza A/H1 N1 virusin vitro andin vivo,and to investigate the inhibitory effect of different compatibility of formula on the influenza A/H1 N1 virus. Methods In vivo experiments,the model of pneumonia by nasal dropping influenza virus was established in mice. According to the weight stratification,then they were divided randomly into five groups: normal control group,influenza virus model group,oseltamivir control group( 21. 63 mg/kg per day),Yinhua Pinggan Granule group( 12 g/kg per day),and San-ao Decotion group( 9 g/kg per day). The animals were received gavage administration with corresponding drugs,once per day for 5 successive days. After the gavage administration,lungs,spleens and thymuses were removed and weighed,respectively. Lung index,inhibition ratio of lung index and the immune organ index were calculated. The expressions of viral load in lung tissue of each group were detected via Real-time fluorescent quantitative PCR. The pathological change of lung tissue in each group were detected by hematoxylin-eosin staining( HE) staining. In vitro experiments,Yinhua Pinggan Granule and San-ao Decoction were used to treat Madin-Darby Canine Kidney( MDCK) cells infected with influenza A/H1 N1 virus. The antiviral efficiacy of two formulas against influenza A/H1 N1 virus were detected by MTT method and the cytopathic effects( CPE) that were induced by influenza virus were observed by Microscopy. Results In vivo experiments showed that compared with the normal control group,lung index increased,spleen index and thymus index decreased,multiple lung consolidation,inflammatory cell infiltration,and high expression of viral load in lung tissue of influenza virus model group(P < 0. 01). Compared with influenza virus model group,Yinhua Pinggan Granule and San-ao Decoction significantly decreased lung index,increased spleen index and thymus index in mice infected with influenza virus(P < 0. 05,P < 0. 01),and significantly alleviated pathological change of lung tissue in mice with influenza viral pneumonia,and significantly decreased virus load in lung tissue(P < 0. 05,P < 0. 01). In vitro experiments also showed Yinhua Pinggan Granule and Sanao Decoction significantly inhibited cytopathic effect caused by influenza virus,and also had a obvious protective effect on influenza virus infection. Conclusions The viral inhibitory effects of Yinhua Pinggan Granule and San-ao Decoction were good in vivo andvitro experiments. Antiviral effects may be associated with inhibition the replication of influenza virus,protection immune organs,alleviation lung inflammation damage and inhibition the cytopathic effects caused by influenza virus. Moreover,Yinhua Pinggan granule was more comprehensive and effective than San-ao Decoction in the aspect of inhibiting influenza virusin vivo andin vitro.
引文
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[13]Dai WP,Li G,Li X,et al.The roots of Ilex asprella extract lessens acute respiratory distress syndrome in mice induced by influenza virus[J].J Ethnopharmacol,2014,155(3):1575-1582.
[14]Li YC,Peng SZ,Chen HM,et al.Oral administration of patchouli alcohol isolated from Pogostemonis Herba augments protection against influenza viral infection in mice[J].Int Immunopharmacol,2012,12(1):294-301.
[15]Imai Y,Kuba K,Neely GG,et al.Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury[J].Cell,2008,133(2):235-249.
[16]刘颖,时宇静,时瀚,等.银翘解毒软胶囊对流感病毒感染小鼠肺炎模型肺组织病毒载量及M1蛋白表达的影响[J].药学学报,2011,46(6):650-655.
[17]南淑玲,徐顺富,陈许,等.升降散对流感病毒鼠肺适应株FM1感染小鼠免疫功能的影响[J].中药药理与临床,2016,32(5):8-13.
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[19]Pau MG,Ophorst C,Koldijk MH,et al.The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines[J].Vaccine,2001,19(17):2716-2721.
[20]Katz JM,Webster RG.Efficacy of inactivated influenza Avirus(H3N2)vaccines grown in mammalian cells or embryonated eggs[J].J Infect Dis,1989,160(2):191-198.
[21]Kistner O,Barrett PN,Mundt W,et al.Development of a mammalian cell(Vero)derived candidate influenza virus vaccine[J].Vaccine,1998,16(9-10):960-968.
[22]Yang Y,Jiang Z,Song W,et al.Glucosylated caffeoylquinic acid derivatives from the flower buds of Lonicera japonica[J].Acta Pharm Sin B,2015,5(3):210-214.
[23]陈兴文.葛根的药理作用研究论述[J].内蒙古中医药,2014,33(27):93.
[24]Lin CJ,Lin HJ,Chen TH,et al.Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression[J].PLo S One,2015,10(2):e0117602.
[2]姚振民,李志勇,姜文娴,等.中药及复方抗病毒最新研究进展[J].山东化工,2017,46(3):48-49.
[3]何昱,余道军,张宇燕,等.银花平感颗粒镇咳作用的实验研究[J].中华中医药学刊,2014,32(9):2060-2061.
[4]彭慧琴,徐哲明,万海同,等.金平感颗粒对流感病毒的抑制作用[J].浙江中医杂志,2005,40(2):87.
[5]宋丹,武孔云,梁光义.经方“三拗汤”的研究进展[J].贵阳中医学院学报,2004,26(4):10-12.
[6]温瑞书,宋翠淼,邓国兴,等.苓桂术甘合三拗汤止咳、化痰、平喘作用的实验研究[J].河北中医药学报,2015,30(3):4-7.
[7]杜海霞,周惠芬,何昱,等.银花平感颗粒和三拗汤体内抗甲型流感病毒的免疫机制研究[J].中国中药杂志,2018,43(05):1028-1033.
[8]邵传森,周丽萍主编.医学免疫学[M].浙江大学出版社,2003,68-72.
[9]王镓,董竞成.中医药防治呼吸道病毒感染研究综述[J].中国中西医结合杂志,2014,34(5):633-636.
[10]王志强,朱毅.中药抗甲型H1N1流感病毒的研究概况[J].现代中西医结合杂志,2011,20(14):1812-1814.
[11]史鹏辉,曲新艳,周喆,等.中药复方抗流感病毒的研究进展[J].中华中医药杂志,2017,32(3):1172-1175.
[12]Yu CH,Yan YL,Wu XN,et al.Anti-influenza virus effects of the aqueous extract from Mosla scabra[J].J Ethnopharmacol,2010,127(2):280-285.
[13]Dai WP,Li G,Li X,et al.The roots of Ilex asprella extract lessens acute respiratory distress syndrome in mice induced by influenza virus[J].J Ethnopharmacol,2014,155(3):1575-1582.
[14]Li YC,Peng SZ,Chen HM,et al.Oral administration of patchouli alcohol isolated from Pogostemonis Herba augments protection against influenza viral infection in mice[J].Int Immunopharmacol,2012,12(1):294-301.
[15]Imai Y,Kuba K,Neely GG,et al.Identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury[J].Cell,2008,133(2):235-249.
[16]刘颖,时宇静,时瀚,等.银翘解毒软胶囊对流感病毒感染小鼠肺炎模型肺组织病毒载量及M1蛋白表达的影响[J].药学学报,2011,46(6):650-655.
[17]南淑玲,徐顺富,陈许,等.升降散对流感病毒鼠肺适应株FM1感染小鼠免疫功能的影响[J].中药药理与临床,2016,32(5):8-13.
[18]Listed N.Influenza virus vaccine live intranasal-MedImmune vaccines:CAIV-T,influenza vaccine live intranasal[J].Drugs R D,2003,4(5):312-319.
[19]Pau MG,Ophorst C,Koldijk MH,et al.The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines[J].Vaccine,2001,19(17):2716-2721.
[20]Katz JM,Webster RG.Efficacy of inactivated influenza Avirus(H3N2)vaccines grown in mammalian cells or embryonated eggs[J].J Infect Dis,1989,160(2):191-198.
[21]Kistner O,Barrett PN,Mundt W,et al.Development of a mammalian cell(Vero)derived candidate influenza virus vaccine[J].Vaccine,1998,16(9-10):960-968.
[22]Yang Y,Jiang Z,Song W,et al.Glucosylated caffeoylquinic acid derivatives from the flower buds of Lonicera japonica[J].Acta Pharm Sin B,2015,5(3):210-214.
[23]陈兴文.葛根的药理作用研究论述[J].内蒙古中医药,2014,33(27):93.
[24]Lin CJ,Lin HJ,Chen TH,et al.Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression[J].PLo S One,2015,10(2):e0117602.