干扰素诱导跨膜蛋白抗小儿流感的效果研究

目的：探讨干扰素诱导跨膜蛋白抗小儿流感的效果。方法采用实时荧光定量 PCR法、Western blot 检测流感NS蛋白对人肺癌A549细胞中IFITM1、2、3表达水平的变化。结果 IFITM1、IFITM2、IFITM3蛋白能够抑制流感病毒NS蛋白的表达。结论 IFITM1、IFITM2、IFITM3蛋白能够抑制流感病毒NS蛋白的表达,值得临床进行更深入的研究。     

甲型H1N1流感病毒非结构蛋白NS1与人CPSF30结合拮抗剂筛选模型的建立及药物筛选

利用酵母双杂交技术研究甲型H1N1流感病毒非结构蛋白NS1和人切割与多聚腺苷酸化特异因子30 kDa 亚基 (CPSF30) 的相互作用, 构建了一个酵母模型用于筛选NS1和CPSF30相互作用的拮抗剂, 从而为筛选抗甲型H1N1流感病毒药物奠定基础。采用连续重叠PCR技术克隆得到H1N1流感病毒NS1基因。提取人HeLa细胞RNA, 通过RT-PCR克隆得到人CPSF30基因。将NS1基因克隆到表达载体pGBKT7中获得诱饵载体pGBKNS1, 将CPSF30基因克隆到载体pGADT7中获得捕获载体pGADCPSF; 将pGBKNS1和pGADCPSF共转入酿酒酵母AH109, 获得重组酿酒酵母AH109[pGADCPSF+pGBKNS1]。利用该模型筛选了30余种中成药, 发现双黄连口服液等4种中成药能抑制NS1和CPSF30之间的相互作用。     

复方抗病毒制剂体外抗流感病毒和呼吸道合胞病毒作用及毒性研究

目的 测定复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒的抗病毒作用. 方法 用鸡红细胞血凝素滴定法测定不同浓度复方抗病毒制剂在狗肾传代细胞（MDCK）中对流感病毒的抑制作用;用中和实验测定复方抗病毒制剂在Hep 2细胞中对呼吸道合胞病毒的活性. 结果 复方抗病毒制剂浓度＜1.5 mg•mL^-1时对MDCK细胞及Hep 2细胞无明显毒性. 浓度＞0.15 mg•mL^-1能抑制流感病毒的血凝活性和呼吸道合胞病毒对靶细胞的攻击作用. 结论 复方抗病毒制剂体外对流感病毒和呼吸道合胞病毒具有较强的抑制作用,且有较大的安全性.     

银翘散对FM1诱导肺炎小鼠肺组织中JAK/STAT信号转导通路及NS1蛋白、Mx A蛋白表达的影响

摘要：目的:研究银翘散对甲型流感病毒FM1诱导肺炎小鼠肺组织中非结构蛋白1（NS1）、黏病毒抗性蛋白A（Mx A）蛋白表达的影响及Janus激酶/信号转导与转录激活因子（JAK/STAT）信号传导机制。方法:将72只昆明小鼠随机分为6组:正常组、模型组、利巴韦林组(100 g·kg-1·d-1)及银翘散提取物高、中、低剂量组(17,6. 8,3. 4 g·kg-1·d-1),每组12只。除正常组外,其余各组建立FM1滴鼻感染的小鼠流感病毒性肺炎模型。感染后24 h,正常组和模型组给予双蒸水灌胃,治疗组分别予各药物治疗,连续3 d。以苏木素-伊红（HE）染色观察病理;免疫组化法测定肺组织中NS1、Mx A蛋白含量; Westen blot检测肺组织NS1、STAT1蛋白水平。结果:与正常组相比,其余各组的NS1、STAT1、Mx A蛋白表达差异均有统计学意义（P<0. 05）;模型组小鼠肺组织病变严重,NS1、STAT1蛋白水平明显升高（P<0. 05）;与模型组比较,利巴韦林组和银翘散各剂量组小鼠肺组织病变明显改善;小鼠肺组织中NS1、高剂量组STAT1蛋白表达明显降低,Mx A蛋白表达明显升高（P<0. 05）。银翘散治疗病毒性肺炎存在一定的量-效关系。结论:在小鼠甲型流感病毒肺炎发病过程中,JAK/STAT信号通路处于激活状态,STAT1蛋白表达增强,机体NS1表达增强;银翘散治疗FM1肺炎有显著疗效,其机制可能是抑制流感病毒NS1和JAK/STAT信号通路的活化,刺激Mx A蛋白表达,从而达到减少肺组织损伤的治疗目的。     

NS-398对高糖诱导的系膜细胞结缔组织生长因子表达的影响

［摘要］目的研究选择性环氧化酶 2抑制药NS 398对高糖诱导的系膜细胞刺激结缔组织生长因子（CTGF）表达的影响。 方法20 μmol&#8226;L-1 NS 398加入30 mmol&#8226;L-1高糖培养的大鼠肾小球系膜细胞中，分别培养0，12，24，48，72 h，应用逆转录聚合酶链反应的方法评价CTGF mRNA的表达改变，应用免疫细胞化学方法评价CTGF蛋白的表达水平。结果未加高糖刺激的肾小球系膜细胞CTGF mRNA和蛋白表达水平较低，高糖刺激后肾小球系膜细胞CTGF mRNA和蛋白表达水平增加，6 h活性已渐增高，12 h达高峰，18 h开始下降，24 h降至较低水平。用NS 398干预30 min后再用30 mmol&#8226;L-1高糖刺激，未见 CTGF mRNA和蛋白表达水平增加（P＞0.05）。 结论高糖能上调肾小球系膜细胞CTGF mRNA和蛋白的表达，NS 398能抑制高糖诱导系膜细胞CTGF的表达。     

miRNA在开发抗流感病毒药物中的研究进展

目的:为开发新型抗流感病毒药物提供参考。方法:以"流感""抗病毒""micro RNA(mi RNA)""Influenza""Anti-virus"等为关键词,组合查询2001年1月-2016年3月在Pub Med、中国知网、万方、维普等数据库中有关mi RNA在抗流感病毒方面的文献,对内源性mi RNA和外源性植物mi RNA的抗流感病毒作用及其机制进行综述。结果与结论:共检索到相关文献265篇,其中有效文献37篇。抗流感病毒的内源性mi RNA和外源性植物mi RNA对病毒基因的表达具有调控作用。这些mi RNA能靶向流感病毒的基因,如PB1、PB2、NP、M1、NS1等,抑制相应蛋白的表达,影响病毒的复制与转录,从而发挥抗病毒作用。mi RNA在抗流感病毒中发挥着重要作用,但存在很多未知领域,更多的抗病毒mi RNA有待发现。为进一步了解mi RNA在病毒与宿主间所充当的角色以及开发新型抗流感病毒药物,还需深入研究mi RNA的作用机制、调控途径以及生物学功能。     

前列腺素E1抑制血管平滑肌细胞增殖的作用

目的 从细胞周期蛋白和凋亡相关基因探讨前列腺素E₁(PGE₁)对血管平滑肌细胞(VSMC)增殖的调控作用。方法 采用培养的新生牛主动脉VSMC，以胎牛血清及白介素1(IL-1)分别作为促增殖剂，测定不同浓度PGE₁对VSMC增殖、细胞内周期蛋白d₁ mRNA表达、p53及Bcl-2基因表达和培养液内NO含量的影响。结果 PGE₁浓度依赖性抑制胎牛血清促VSMC增殖作用；显著减少细胞周期蛋白d₁ mRNA含量；浓度依赖性诱导IL 1孵育下VSMC产生大量NO, 同时抑制VSMC增殖；并促进p53基因表达，下调bcl-2基因，诱导细胞凋亡。结论 PGE₁具有抑制VSMC增殖的作用，其机制可能与其阻滞VSMC生长有关；在炎性因子存在情况下，PGE₁可诱导VSMC内大量产生NO，抑制VSMC增殖，并加速VSMC凋亡。     

β-榄香烯对人肺腺癌A549细胞株裸鼠移植瘤放疗增敏作用的研究★

目的 建立人肺腺癌A549细胞株裸鼠移植瘤模型，用增敏剂量的β-榄香烯联合放疗对移植瘤进行干预，探讨放疗增敏机制是否与抑制葡萄糖转运蛋白1（GLUT-1）表达有关。方法 采用细胞悬液接种法建立裸鼠移植瘤模型，将肿瘤体积达到75 mm³左右的20只裸鼠随机分为空白对照组（NS组）、β-榄香烯单药组（ELE组）、β-榄香烯联合放疗组（ELE+RAD组）和单纯放疗组（RAD组）。监测干预后各组裸鼠肿瘤体积的变化，获得各组裸鼠的肿瘤生长曲线。通过计算增敏系数，检测45 mg·kg^-1 β-榄香烯是否发挥增敏作用，采用Real-Time PCR、Western blotting及免疫组化染色检测各组移植瘤中GLUT-1的表达水平。结果 成功建立A549细胞株裸鼠移植瘤模型，依据各组移植瘤体积变化绘制生长曲线。测得增敏系数（EF）为2.44，提示β-榄香烯的剂量已达到增敏效果。与NS组相比，实验组移植瘤GLUT-1 mRNA及蛋白表达水平均显著降低（P<0.01）；ELE组与RAD组GLUT-1 mRNA及蛋白表达水平无显著差异（P>0.05）；与ELE组、RAD组相比，ELE+RAD组GLUT-1 mRNA及蛋白表达水平均显著降低（P<0.01）。免疫组化结果显示，与NS组比较，实验组GLUT-1的表达均被显著抑制（P<0.05）；其中ELE组和RAD组GLUT-1的表达抑制作用相对较弱，RAD组的抑制作用略强于ELE组，但两组差异无统计学意义（P>0.05）；而ELE+RAD组GLUT-1的表达抑制作用较强（P<0.05）。结论 增敏剂量的β-榄香烯与放疗联合应用可显著抑制裸鼠移植瘤中GLUT-1 mRNA及蛋白表达，明显增强放疗对肺癌裸鼠移植瘤的生长抑制效果，提示β-榄香烯可能通过抑制GLUT-1表达发挥放疗增敏作用。     

千金藤素抗病毒作用的免疫学机制研究

目的 探索千金藤素体内外的抗病毒作用,并基于抗病毒天然免疫通路探究其抗病毒作用的分子机制。方法 CCK-8 法检测千金藤素（0.062 5～64.000 0 μmol·L^-1）对 A549 细胞活力的影响;利用表达绿色荧光蛋白的水疱性口炎病毒（VSV-GFP）感染 A549 细胞模型,结合流式细胞术检测千金藤素对病毒复制的影响并探究预处理、吸附过程及吸附后加药对 VSV-GFP 病毒复制的影响;实时荧光定量 PCR（qRT-PCR）检测千金藤素对甲型流感病毒（H1N1）、脑心肌炎病毒（EMCV）和单纯疱疹病毒 I 型（HSV-1）复制的影响;构建 VSV 感染小鼠模型探究千金藤素的体内抗病毒作用;A549细胞中利用生物信息学方法探究其抗病毒机制;qRT-PCR 检测药物处理 A549 和原代胚胎成纤维细胞（MEF）后 IFNB1 及干扰素刺激基因 （ISGs） 表达变化;免疫印迹法 （Immunoblotting） 检测人源单核细胞白血病细胞（THP-1）中 TBK1 和STAT1 的磷酸化水平。结果 与模型组相比,千金藤素在 A549 细胞中显著抑制 VSV、H1N1、EMCV 和 HSV-1 复制;千金藤素不影响 VSV 的吸附过程,而预处理或吸附后给药可以显著抑制病毒复制;千金藤素提高 VSV 感染小鼠的存活率;千金藤素激活基于IFN-I通路的抗病毒天然免疫应答。结论 千金藤素通过激活基于IFN-I通路的抗病毒天然免疫发挥体内外抗病毒作用。     

西替利嗪对皮肤细胞炎症中单核趋化蛋白-1表达的干预作用

目的考察西替利嗪(CET)对皮肤细胞炎症模型中单核趋化蛋白-1(MCP-1)表达的干预作用。方法组胺(HIS)和IFN-γ刺激真皮成纤维细胞(DF)和人角质形成细胞株HaCaT细胞，采用RT-PCR和ELISA法考察两种细胞MCP-1 mRNA和蛋白表达水平。结果与对照组比较，HIS(10 μmol·L^-1)和IFN-γ(20 ng·mL^-1)组显著上调两种细胞(DF和HaCaT) MCP-1 mRNA表达，同时分别使DF的MCP-1蛋白分泌量增加3.5倍和8.4倍，对HaCaT细胞也有相似的影响趋势； CET (1和10 μmol·L^-1) 显著地抑制了它们对细胞MCP-1蛋白产生的增强作用。结论CET可能通过抑制MCP-1的表达而发挥抗皮肤炎症作用。     

The influenza virus NS1 protein: inhibitor of innate and adaptive immunity

The influenza virus NS1 protein has been shown to be a multifunctional immune modulator and a virulence factor for this virus. Among its multiple functions are the inhibition of the type I interferon (IFN) system in infected cells, the binding and sequestration of dsRNA, the interference with the host mRNA processing, the facilitation of preferential viral mRNA translation, and the inhibition of dendritic cell (DC) activation. The combination of all these functions makes the NS1 protein a very potent inhibitor of immunity and allows influenza virus to efficiently escape the immune surveillance and to establish infection in the host. There are different domains in the NS1 protein that are required for specific functions, which provides several potential targets for the action of antiviral drugs. Additionally, the crystal structure of both the N-terminal RNA binding domain and the C-terminal effector domain of the NS1 protein have been resolved, potentially allowing for better antiviral drug design. Recent advances in the understanding how viruses are detected by infected cells are unveiling the mechanisms by which the NS1 protein can perform some of its multiple immune modulating activities. In this review the multiple functions of the NS1 protein are discussed and several possible options for drug targets within the influenza virus NS1 protein will be explored. Such drugs could make influenza viruses less efficient at evading the immune system in the host.     

Inhibition of miR-93 promotes interferon effector signaling to suppress influenza A infection by upregulating JAK1

Type I interferons play a critical role in host defense against influenza virus infection. Interferon cascade induces the expression of interferon-stimulated genes then subsequently promotes antiviral immune responses. The microRNAs are important regulators of innate immunity, but microRNAs-mediated regulation of interferon cascade during influenza infection remains to be fully identified. Here we found influenza A virus (IAV) infection significantly inhibited miR-93 expression in alveolar epithelial type II cells through RIG-I/JNK pathway. IAV-induced downregulation of miR-93 was found to upregulate JAK1, the target of miR-93, and then feedback promote antiviral innate response by facilitating IFN effector signaling. Importantly, in vivo administration of miR-93 antagomiR markedly suppressed IAV infection, protecting mice form IAVs -associated death. Hence, the inducible downregulation of miR-93 feedback suppress IAV infection by strengthening IFN-JAK-STAT pathway via JAK1 upregulation, and in vivo inhibition of miR-93 bears considerable therapeutic potential for suppressing IAV infection.     

Type II transmembrane serine proteases as potential target for anti-influenza drug discovery

Introduction: The outbreak of an influenza pandemic as well as the continued circulation of seasonal influenza highlights the need for effective antiviral therapies. The emergence of drug-resistant strains further necessitates the development of novel antivirals that target the host factors crucial for viral replication.

Area covered: This review summarizes the current understanding of the structural and functional properties of type II transmembrane serine proteases (TTSPs) as a proteolytic activator of influenza virus infection and discusses their potential as antiviral targets. It also explores the experimental evidence accumulated for inhibitors of TTSPs as novel, broad-spectrum antivirals against various influenza virus subtypes. The review also provides an overview of the properties of small molecules, proteins, and peptides that efficiently inhibit the proteolytic activation of the influenza virus.

Expert opinion: TTSPs activate a wide range of influenza virus subtypes including avian influenza viruses, both in vitro and in vivo, via proteolytic cleavage of influenza hemagglutinin (HA) into infection-competent fusogenic conformation. Other viruses such as SARS-, MERS-coronaviruses and human metapneumoviruses may use the same host cell proteases for activation, implying that TTSP inhibition might be a novel strategy for developing broad-spectrum antiviral agents for respiratory viral infections.     

Recruitment of an interferon molecular signaling complex to the mitochondrial membrane: disruption by hepatitis C virus NS3-4A protease

Recent advances in the understanding of the signaling pathways leading to the host antiviral response to hepatitis C virus (HCV), the mechanisms used by HCV to evade the immune response, and the development of small molecule inhibitors of HCV have generated optimism that novel therapeutic approaches to control HCV disease may soon be available. HCV infection is detected by the cytoplasmic, RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently the adapter molecule that links RIG-I sensing of incoming viral RNA to downstream signaling and gene activation events was characterized by four different groups: MAVS/IPS-1-1/VISA/Cardif contains an amino-terminal CARD domain and carboxyl-terminal mitochondrial transmembrane sequence that localizes to the mitochondrial membrane. Furthermore, the hepatitis C virus NS3-4A protease complex specifically targets MAVS/IPS-1/VISA/Cardif for cleavage as part of its immune evasion strategy. Using a combination of biochemical analysis, subcellular fractionation and confocal microscopy, we demonstrate that: (1) NS3-4A cleavage of MAVS/IPS-1/VISA/Cardif causes relocation from the mitochondrial membrane to the cytosolic fraction, resulting in disruption of signaling to the antiviral immune response; (2) disruption requires a function NS3-4A protease; (3) a point mutant of MAVS/IPS-1/VISA/Cardif (Cys508Ala) is not cleaved from the mitochondria by active protease; and (4) the virus-induced IKK epsilon kinase, but not TBK1, co-localizes strongly with MAVS at the mitochondrial membrane and the localization of both molecules is disrupted by NS3-4A expression. These observations provide an outline of the mechanism by which HCV evades the IFN antiviral response.     

In vitro evaluation of the antiviral activity of SP-303, an euphorbiaceae shrub extract,against a panel of respiratory viruses

The antiviral dn cytotoxic activity of SP-303, a naturally occurring polyphenolic polymer (average M.W. = 2,100 daltons) isolated from a Euphorbiaceae shrub, was evaluated in tissue culture. The compound exhibited selective antiviral activity (ratio IC₅₀/EC₅₀ ≥10) against parainfluenza virus type 1, respiratory syncytial virus, influenza A viruses, and influenza B viruses; marginal selective antiviral activity (IC₅₀/EC₅₀ ratio >2 to <10) against parinfluenza virus type 3; and no selective antiviral activity (IC₅₀/EC₅₀ ratio ≤1) against measles virus or adenovirus type 5. Hemagglutination and other studies suggested that SP-303 may at least partially inactivate virus by direct interaction with virus or host cell lipid membranes. © Wiley-Liss, Inc.     

Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses

The 1918 pandemic H1N1 influenza virus and the recently emerged Southeast Asian H5N1 avian influenza virus are unique among influenza A virus isolates in their high virulence for humans and their lethality for a variety of animal species without prior adaptation. Reverse genetic studies have implicated several viral genes as virulence determinants. For both the 1918 and H5N1 viruses, the hemagglutinin and the polymerase complex contribute to high virulence. Non-structural proteins NS1 and PB1-F2, which block host antiviral responses, also influence pathogenesis. Additionally, recent studies correlate high levels of viral replication and induction of strong proinflammatory responses with the high virulence of these viruses. Defining how individual viral proteins promote enhanced replication, inflammation and severe disease will provide insight into the pathogenesis of severe influenza virus infections and suggest novel therapeutic approaches.     

盐酸阿比朵尔抗流感病毒感染的药效学研究

目的　探讨盐酸阿比朵尔的抗流感病毒作用。方法　建立体内、体外流感病毒模型 ,体外实验为应用一定浓度盐酸阿比朵尔作用于甲型流感病毒H1N1亚型病毒感染宿主细胞 ,通过观察CPE及采用MTT法测定细胞存活率 ;体内实验为应用盐酸阿比朵尔治疗流感病毒感染小鼠模型 ,计算肺指数的变化。结果　盐酸阿比朵尔在体内、体外均具有明显的抗流感病毒作用。结论　盐酸阿比朵尔是一种较好的抗流感病毒药物。     

芦丁对流感病毒的体外抑制作用及其机制研究

目的研究芦丁对流感病毒的体外抑制作用并探讨其机制。方法利用流感病毒感染MDCK细胞模型,观察芦丁对流感病毒的体外抑制作用;结合流感病毒感染前、感染同时及感染后加药的不同方式探索芦丁作用于流感病毒生命周期的具体阶段;采用荧光底物法考察芦丁对流感病毒神经氨酸酶的抑制作用。结果芦丁对A/Puerto Rico/8/1934(H1N1)、A/FM1/1/47(H1N1)、A/Human/Hubei/3/2005(H3N2)、A/Beijing/32/92(H3N2)4株流感病毒均具有体外抑制作用,其中对A/Puerto Rico/8/1934(H1N1)株的半数有效浓度(EC50)最小,选择指数(SI)最大,体外药效最好。芦丁在流感病毒感染后给药的效果最为明显,对流感病毒A/FM1/1/47(H1N1)的IC50最小,对神经氨酸酶活性抑制作用相对最好。结论芦丁抑制了流感病毒神经氨酸酶活性,具有较好的体外抗流感病毒作用。     

Inhibition of influenza A virus replication by a kanamycin derivative

We studied the antiviral activity and the mechanism of action of a new antiviral agent and kanamycin derivative, 1-N-eicosanoyl-3"-N-trifluoroacetyl kanamycin A (ETKA), against influenza A virus. From yield reduction assays with VERO cells, ETKA showed a significant antiviral activity with negligible cytotoxic effect. In the presence of 20 micrograms/ml of ETKA at which VERO cell growth was not inhibited, virus titer was suppressed to 11.2% of control, and at 100 micrograms/ml virus production was suppressed to more than 99%. ETKA markedly inhibited viral protein synthesis when cells were pretreated with the drug before infection, but there was no inhibition when the drug was added 15 min post-infection. ETKA did not inhibit virus adsorption and penetration. Nor did it affect the activity of viral RNA polymerase in vitro. We found that the drug had a direct inactivating effect on influenza A virus under acidic conditions. These results suggest that ETKA exerts its antiviral action mainly in the early stage, prior to uncoating by direct inactivation of the virus due to the acidic environment of the endocytic vesicle. Aerosol treatment with the drug protected mice against a lethal influenza A virus infection.     

Vitisin B inhibits influenza A virus replication by multi-targeting neuraminidase and virus-induced oxidative stress

The development of drug-resistant influenza and new pathogenic virus strains underscores the need for antiviral therapeutics. Currently, neuraminidase(NA)inhibitors are commonly used antiviral drugs approved by the US Food and Drug Administration(FDA)for the prevention and treatment of influenza. Here, we show that vitisin B(VB)inhibits NA activity and suppresses H1N1 viral replication in MDCK and A549 cells. Reactive oxygen species(ROS), which frequently occur during viral infection,increase vi...