小花清风藤水提物体内抗流感病毒的研究

目的:研究小花清风藤水提物体内抗流感病毒的活性。方法:采用滴鼻感染流感病毒建立BALB/c小鼠肺炎模型,以小鼠存活率、平均存活时间、肺指数、肺组织中病毒滴度及肺组织病理改变为指标,观察不同剂量小花清风藤水提物体内抗流感病毒的活性。结果:与模型组比较,小花清风藤水提物中剂量(i.g,6.5 g/kg)能提高小鼠生存率30%,延长小鼠存活时间,降低小鼠肺指数和肺组织中病毒滴度,明显改善感染小鼠肺组织炎症病变。结论:小花清风藤水提物在体内具有一定的抗流感病毒的作用。     

基于TLR3/TRIF通路探讨栀子苷抗流感病毒引起的病毒性肺炎的实验研究

中药在预防和治疗流感病毒方面具有独特的优势,栀子苷具有抗炎抗病毒作用,但栀子苷保护流感病毒引起的肺损伤的作用机制尚不明确。为了基于Toll样受体3(Toll-like receptor 3,TLR3)/β干扰素TIR结构域衔接蛋白(TIR-domain-containing adaptor inducing interferon-β,TRIF)信号传导途径探讨栀子苷对流感病毒诱导的肺损伤的调节作用,本研究按照随机数字表法将108只小鼠分为空白对照组、模型组、阳性对照组(50mg/kg利巴韦林)、低剂量栀子苷组(5mg/kg栀子苷)、中剂量栀子苷组(10mg/kg栀子苷)、高剂量栀子苷组(20mg/kg栀子苷)、TLR3激动剂组、TLR3激动剂+阳性药物组、TLR3激动剂+高剂量栀子苷组。采用流感病毒亚甲型鼠肺适应株A/FM/1/47(H1N1)建立流感病毒性肺炎小鼠模型,摘取肺组织,测定小鼠肺指数,HE染色观察肺组织病理学变化,酶联免疫法检测肺组织白介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和干扰素-β(Interferon-β,IFN-β)水平,qRT-PCR检测肺组织TLR3和TRIF mRNA表达,Western Blot检测肺组织TLR3、TRIF和p-NF-κB65蛋白表达。结果显示,与空白对照组相比,模型组小鼠肺指数、IL-6、TNF-α水平显著升高(P0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著升高(P0.05)。与模型组比,中、高剂量栀子苷组和阳性对照组小鼠肺指数、IL-6和TNF-α水平均显著降低(P0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著降低(P0.05)。与阳性对照组比,高剂量栀子苷组小鼠肺指数、IL-6、TNF-α、TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白均无显著差异(P0.05)。与空白对照组相比,TLR3激动剂组小鼠肺指数显著升高(P0.05),与TLR3激动剂组相比,TLR3激动剂+高剂量栀子苷组小鼠肺指数显著降低(P0.05),TLR3激动剂+阳性药物组小鼠肺指数无显著差异(P0.05)。本研究揭示,栀子苷可能通过调节TLR3/TRIF通路介导的p-NF-κB65活性增强机体的抗病毒能力。     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用.结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用.体内实验中0.1 g/kg,0.5g/kg,1.2g/kg 3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间;降低肺炎小鼠的肺指数和血凝滴度(P<0.01).其作用与病毒唑相当.结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂.     

重组人干扰素α2b抗流感病毒的研究

采用鼻腔给药方法,随机选取昆明小鼠60只,每组20只,共分3组,第1组为病毒对照组,第2组为感染前给药组即感染病毒前24h给药,第3组为同时给药组即病毒感染与用药同时进行,小鼠每天滴鼻给药1次,IFNα2b滴鼻剂剂量为10000IU/只,病毒浓度为10个EID50,对照组给予生理盐水,连续给药6d后处死小鼠,观察小鼠肺部病变,并采用血凝滴定法检测鼠肺中流感病毒含量。观察重组人干扰素α2b(IFNα2b)抗流感病毒效果,小鼠肺部病变严重程度依次为1组>3组>2组,血凝滴定结果表明,1、2与3组GMT平均值分别为4.141,2.000,2.070,重组人干扰素α2b滴鼻剂对流感病毒有明显抑制作用。     

清热消炎复方制剂抗流感病毒作用的研究

为评价清热消炎复方制剂(简称AI)的抗流感病毒活性,我们以病毒唑为对照,通过在体外观察病毒致细胞病变效应(CPE)、MTT细胞染色检查病毒抑制率和检测病毒血凝滴度;在体内观察其对染毒小鼠的死亡保护作用,对小鼠流感病毒性肺炎的抑制作用,以及对小鼠肺内病毒增殖的影响,从而判定其抗流感病毒作用。结果发现AI在160ug/mL时能完全抑制流感病毒在MDCK细胞内的增殖复制作用。体内实验中0.1g/kg,0.5g/kg,1.2g/kg3个剂量均能明显降低染毒小鼠的致死率,延长平均存活时间:降低肺炎小鼠的肺指数和血凝滴度(P<0.01)。其作用与病毒唑相当。结论认为清热消炎复方制剂是一种有效的体内、体外抗流感病毒中药复方制剂。     

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白

利用竞争型ELISA法鉴定硒诱导的金属硫蛋白 (MT) ,发现对照组小鼠肝脏MT含量为 (2 .47± 0 .90 )μg/g湿重组织 ,硫酸锌组小鼠肝脏MT含量为 (8.15± 2 .2 0 ) μg/ g湿重组织 ,硒麦芽组小鼠肝脏MT含量为(12 .80± 1.44 ) μg/ g湿重组织。锌组和硒组的MT含量与对照组相比有显著性差异 (P <0 .0 5 ,P <0 .0 5 )。硒组MT含量要显著高于锌组的MT含量 (P <0 .0 5 )。     

茯苓多糖对流感灭活疫苗的免疫增强作用

探讨茯芩多糖作为流感病毒灭活疫苗佐剂的免疫增强作用.将不同剂量(200 μg或1000 μg)的茯苓多糖分别与低剂量(0.015 μg)或高剂量(1.5 μg)流感病毒(A/PR/8)灭活疫苗共同免疫小鼠,以相应剂量灭活疫苗的单独免疫组、灭活疫苗与氢氧化铝(100 μg)共同免疫组、PBS免疫组作为对照组.一次免疫后3周收集血清,ELISA检测血清中IgG、IgG1和IgG2a的抗体水平;并用致死量(40×LD<,50>)流感病毒(A/PR/8)攻击小鼠,通过观察小鼠的体重丢失率、肺部病毒量、存活率来反映佐剂的免疫增强效果和疫苗的保护作用.结果显示,茯苓多糖能显著增加血清抗体水平,并提高小鼠抗致死量流感病毒攻击的能力,其免疫增强效果与氢氧化铝相当.茯苓多糖可作为一种新型的流感病毒灭活疫苗的免疫佐剂.     

探讨接种时间和剂量对小鼠肺炎支原体肺炎模型建立的影响

目的 用不同时间和剂量建立BALB/c小鼠的肺炎支原体肺部感染模型,探索小鼠急性肺炎支原体感染的过程.方法 BALB/c小鼠随机分为Ⅰ、Ⅱ两组,Ⅰ组在0、1、2 d滴鼻接种肺炎支原体菌液3次,Ⅱ组在0 d滴鼻接种小剂量肺炎支原体菌液1次后于8、9 d再接种和Ⅰ组相同的肺炎支原体菌液2次,均设立不同剂量、批次的生理盐水对照组.全部实验动物在3～18 d内分批处死.所有小鼠均取肺组织做病理切片.以组织病理学评分来确定小鼠的肺部炎症反应程度.取肺组织匀浆及肺泡灌洗液(BALF)进行肺炎支原体培养做病原学检测.结果 接种后实验动物全部存活无死亡.实验组动物均出现不同程度的肺炎支原体肺炎样病理改变,组织病理学评分为1.5～14.3分,分别于第5、6天和第11天达到最高,平均分为4.9分;Ⅰ组实验组动物多呈轻、中度改变,Ⅱ组实验组动物多呈中、重度改变.所有实验组动物肺组织匀浆及BALF的肺炎支原体培养在接种后1月内先后出现阳性结果.对照组动物未出现明显肺部炎症改变,组织病理学评分为0～1分,肺炎支原体培养为阴性.结论 成功建立BALB/c小鼠的肺部肺炎支原体感染模型;组织病理学评分方法可用以评价肺部炎症反应的严重程度;不同接种时间和剂量所致的病变程度不同.     

当归对阴虚哮喘小鼠气道黏液高分泌及TNF-α/NF-κB信号通路的影响*

目的: 探讨当归对阴虚哮喘小鼠气道黏液高分泌及TNF-α/NF-κB信号通路的影响。方法: 取KM小鼠随机分为空白对照组、模型对照组、氨溴索组、当归低、中、高剂量(2、4、8 g/kg)组(n=12),采用卵蛋白与甲状腺片复制阴虚哮喘模型,观测当归对小鼠哮喘症状、IgE、TNF-α以及肺组织Muc5ac与NF-κB表达的影响。结果: 2、4、8 g/kg当归能明显缓解阴虚哮喘小鼠的哮喘症状,降低血清IgE与BALF中TNF-α水平,抑制肺组织Muc5ac与NF-κB的过度表达。结论: 当归具有明显的平喘作用,抑制TNF-α/NF-κB信号通路而缓解气道黏液高分泌是其平喘的作用机制之一。     

硒对甲型H1N1流感病毒感染小鼠的保护作用

目的:通过研究硒对流感病毒悬液滴鼻处理的小鼠的体重变化、死亡率、血清硒水平和细胞因子的影响,探讨硒对感染流感病毒小鼠的保护作用。方法:将60只昆明小鼠分为5组,每组12只,分别为缺硒组(0 mg/kg)、正常给硒组(0.2 mg/kg)、补充硒组(0.3 mg/kg)、补充硒组(0.4 mg/kg)、补充硒组(0.5 mg/kg)。给5周龄的小鼠滴鼻接种50μL的A/NWS/33(H1N1)病毒悬液并观察21天,监测每组小鼠的体重变化和死亡率;并在接种病毒后的第3天、第5天,检测小鼠的血清硒、TNF-α和IFN-γ水平。结果:缺硒组小鼠的死亡率高于正常给硒组和补充硒组(P0.05);缺硒组小鼠的血清硒水平明显低于正常给硒组和补充硒组(P0.05);在病毒感染第5天,缺硒组小鼠的TNF-α和IFN-γ含量低于正常给硒组和补充硒组(P0.05),差异均有统计学意义。结论:硒可以提高机体对抗流感病毒的免疫反应。     

Flavonoids from Houttuynia cordata attenuate H1N1-induced acute lung injury in mice via inhibition of influenza virus and Toll-like receptor signalling

BackgroundInfluenza virus is one of the most important human pathogens, causing substantial seasonal and pandemic morbidity and mortality. Houttuynia cordata is a traditionally used medicinal plant for the treatment of pneumonia. Flavonoids are one of the major bioactive constituents of Houttuynia cordata.PurposeThis study was designed to investigate the therapeutic effect and mechanism of flavonoid glycosides from H. cordata on influenza A virus (IAV)-induced acute lung injury (ALI) in mice.MethodsFlavonoids from H. cordata (HCF) were extracted from H. cordata and identified by high-performance liquid chromatography. Mice were infected intranasally with influenza virus H1N1 (A/FM/1/47). HCF (50, 100, or 200 mg/kg) or Ribavirin (100 mg/kg, the positive control) were administered intragastrically. Survival rates, life spans, weight losses, lung indexes, histological changes, inflammatory infiltration, and inflammatory markers in the lungs were measured. Lung virus titers and neuraminidase (NA) activities were detected. The expression of Toll-like receptors (TLRs) and levels of NF-κB p65 phosphorylation (NF-κB p65(p)) in the lungs were analysed. The effects of HCF on viral replication and TLR signalling were further evaluated in cells.ResultsHCF contained 78.5% flavonoid glycosides. The contents of rutin, hyperin, isoquercitrin, and quercitrin in HCF were 8.8%, 26.7%, 9.9% and 31.7%. HCF (50, 100 and 200 mg/kg) increased the survival rate and life span of mice infected with the lethal H1N1 virus. In H1N1-induced ALI, mice treated with HCF (50, 100 and 200 mg/kg) showed lesser weight loss and lower lung index than the model group. The lungs of HCF-treated ALI mice presented more intact lung microstructural morphology, milder inflammatory infiltration, and lower levels of monocyte chemotactic protein 1 (MCP-1), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α) and malondialdehyde (MDA) than in the model group. Further investigation revealed that HCF exerted antiviral and TLR-inhibitory effects in vivo and in vitro. HCF (50, 100 and 200 mg/kg) reduced lung H1N1 virus titers and inhibited viral NA activity in mice. HCF (100 and 200 mg/kg) elevated the levels of interferon-β in lungs. HCF also decreased the expression of TLR3/4/7 and level of NF-κB p65(p) in lung tissues. In vitro experiments showed that HCF (50, 100 and 200 μg/ml) significantly inhibited viral proliferation and suppressed NA activity. In RAW 264.7 cells, TLR3, TLR4, and TLR7 agonist-stimulated cytokine secretion, NF-κB p65 phosphorylation, and nuclear translocation were constrained by HCF treatment. Furthermore, among the four major flavonoid glycosides in HCF, hyperin and quercitrin inhibited both viral replication and TLR signalling in cells.ConclusionHCF significantly alleviated H1N1-induced ALI in mice, which were associated with its dual antiviral and anti-inflammatory effects via inhibiting influenzal NA activity and TLR signalling. among the four major flavonoid glycosides in HCF, hyperin and quercitrin played key roles in the therapeutic effect of HCF.     

清温解毒汤提取物体内抗病毒活性研究

目的:研究清温解毒汤提取物体内对流感病毒的防治作用,阐明该提取物体内抗病毒的作用机制。方法:选用NIH小鼠,于乙醚浅麻醉下经鼻滴入流感病毒,观察清温解毒汤提取物对感染病毒小鼠的保护作用,以及对小鼠肺指数的影响。结果:对感染病毒小鼠的保护作用实验显示,清温解毒汤提取物低、中、高剂量组的小鼠死亡率均较病毒对照组减少;清温解毒颗粒中、高剂量组能明显提高小鼠的平均存活天数(P<0.05)及延长动物的生命率;对小鼠的肺指数的影响实验则显示清温解毒汤提取物组动物的低、中、高剂量组动物肺指数与病毒对照组比较,虽无显著性差异(P>0.05),但存在明显的剂量依赖关系。结论:清温解毒汤提取物具有一定的抗流感病毒作用,其机制可能与其能够延长小鼠存活时间及抑制肺肿胀有关。     

Anti-Influenza A Virus Effect of Hypericum perforatum L. Extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H1N1) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC50) of 40 μg/mL. The mean 50% cytotoxic concentration (CC50) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC50 values of 5.0 μg/mL; the mean CC50 for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin's minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD50 dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza.     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

Antiviral activity of 3,4’-dihydroxyflavone on influenza a virus

Influenza virus infection causes thousands of deaths and millions of hospitalizations worldwide every year and the emergence of resistance to anti-influenza drugs has prompted scientists to seek new natural antiviral materials. In this study, we screened 13 different flavonoids from various flavonoid groups to identify the most potent antiviral flavonoid against human influenza A/PR/8/34 (H1N1). The 3-hydroxyl group flavonoids, including 3,2?dihydroxyflavone (3,2?DHF) and 3,4?dihydroxyflavone (3,4?DHF), showed potent anti-influenza activity. They inhibited viral neuraminidase activity and viral adsorption onto cells. To confirm the anti-influenza activity of these flavonoids, we used an in vivo mouse model. In mice infected with human influenza, oral administration of 3,4?DHF significantly decreased virus titers and pathological changes in the lung and reduced body weight loss and death. Our data suggest that 3-hydroxyl group flavonoids, particularly 3,4?DHF, have potent antiviral activity against human influenza A/PR/8/34 (H1N1) in vitro and in vivo. Further clinical studies are needed to investigate the therapeutic and prophylactic potential of the 3-hydroxyl group flavonoids in treating influenza pandemics.     

Anti-influenza A virus effect of Hypericum perforatum L. extract

To study the antiviral effect of Hypericum perforatum L. extract (HPE) on influenza A virus (IAV) (H₁N₁) in vitro and in vivo. Cytopathic effect (CPE) and neutral red (NR) dye uptake were used to examine the antiviral effect of HPE on Madin Darby Canine Kidney (MDCK) cells which were infected with IAV in vitro. HPE was effective against influenza A virus (IAV) in vitro, with a 50% effective concentration (EC₅₀) of 40 μg/mL. The mean 50% cytotoxic concentration (CC₅₀) in the MDCK used in these experiments was 1.5 mg/mL. Ribavirin was run in parallel with EC₅₀ values of 5.0 μg/mL; the mean CC₅₀ for ribavirin was 520 μg/mL. Oral gavage administrations of HPE or ribavirin to mice infected with the IAV were highly effective in preventing death, slowing the decline of arterial oxygen saturation, inhibiting lung consolidation and reducing lung virus titers. The minimum effective dose of HPE in these studies was 31.25 mg/kg/day, which was administered twice daily for 5 d beginning 4 h prior to virus exposure. Below a dosage of 2000 mg/kg/day, almost all treated mice survived, which suggests that HPE is of low toxicity. Ribavirin’s minimum effective dose was 40 mg/kg/day with the LD50 determined to be 200 mg/kg/day. Delay of the initiation of either HPE or ribavirin therapy, using approximately 1/3 LD₅₀ dose each time, could still be protective as late as 48 h after exposure to the IAV. While both agents appeared to have similar efficacy against IAV infections, HPE was considered to be less toxic and may warrant further evaluation as a possible therapy for influenza. Foundation items: One Hundred Person Project of The Chinese Academy of Sciences (2008-287); The Project of Basic Scientific Research Fund for Central Public-Welfare of Institute of Sciences (BRF070402).     

H9N2亚型猪流感病毒诱导小鼠急性肺损伤中TNF-α、IL-1β、IL-6和IL-10的变化和作用

目的探讨H9N2亚型猪流感病毒诱导小鼠急性肺损伤过程中炎症因子的变化和作用。方法通过滴鼻的方法将H9N2亚型猪流感病毒感染BALB/c小鼠,于感染后2、4、6、10、14 d取小鼠肺组织匀浆,分别测定肺组织匀浆中TNF-α、IL-1β、IL-6和IL-10的浓度。结果实验组肺组织匀浆中TNF-α、IL-1β、IL-6和IL-10在不同时间点浓度均显著高于正常对照组(P<0.05),TNF-α和IL-1β于14 d后趋于正常,而IL-6和IL-10增高持续至14d后。结论 H9N2猪流感病毒诱导小鼠急性肺损伤过程中炎症因子发挥重大作用,TNF-α和IL-1β起致炎作用,IL-6可能和IL-10一样,发挥抗炎作用。     

Antiviral Effect of Methylated Flavonol Isorhamnetin against Influenza

Influenza is an infectious respiratory disease with frequent seasonal epidemics that causes a high rate of mortality and morbidity in humans, poultry, and animals. Influenza is a serious economic concern due to the costly countermeasures it necessitates. In this study, we compared the antiviral activities of several flavonols and other flavonoids with similar, but distinct, hydroxyl or methyl substitution patterns at the 3, 3′, and 4′ positions of the 15-carbon flavonoid skeleton, and found that the strongest antiviral effect was induced by isorhamnetin. Similar to quercetin and kaempferol, isorhamnetin possesses a hydroxyl group on the C ring, but it has a 3′-methyl group on the B ring that is absent in quercetin and kaempferol. Co-treatment and pre-treatment with isorhamnetin produced a strong antiviral effect against the influenza virus A/PR/08/34(H1N1). However, isorhamnetin showed the most potent antiviral potency when administered after viral exposure (post-treatment method) in vitro. Isorhamnetin treatment reduced virus-induced ROS generation and blocked cytoplasmic lysosome acidification and the lipidation of microtubule associated protein1 light chain 3-B (LC3B). Oral administration of isorhamnetin in mice infected with the influenza A virus significantly decreased lung virus titer by 2 folds, increased the survival rate which ranged from 70–80%, and decreased body weight loss by 25%. In addition, isorhamnetin decreased the virus titer in ovo using embryonated chicken eggs. The structure-activity relationship (SAR) of isorhamnetin could explain its strong anti-influenza virus potency; the methyl group located on the B ring of isorhamnetin may contribute to its strong antiviral potency against influenza virus in comparison with other flavonoids.