石斛碱抑制甲型流感病毒复制的活性研究
发布时间：2019-05-27 05:18
【摘要】：研究目的:流感是严重危害人类健康的传染性疾病。历史上已出现多次的流感大流行,波及范围广,死亡率高。由于流感病毒抗原性转变和抗原性漂移的存在,流感病毒具有极高的变异性,新型流感病毒的出现已对人类的生存造成了严重的影响。流感的防治,尤其是药物治疗,迫在眉睫。迄今为止,在全球临床应用的抗流感药物主要有神经氨酸酶抑制剂和M2离子通道抑制剂两类,但药物耐药性及毒副作用等问题一直困扰着这些药物的发展和应用。目前用于流感病毒感染预防和治疗的化学合成药物十分有限。因此,从天然产物中寻找低毒有效的抗流感病毒中药显得尤为重要。《时病论》清热保津法曾收载复方石斛汤可治温热有汗,风热化火,热病伤津等,其中石斛具有广泛的生物学活性,包括益胃生津,润肺止咳、滋阴清热等,常用于口干烦渴、热病伤津、病后虚热等多种病症。石斛碱一种从金钗石斛中提取出来的主要生物碱。本项目通过研究石斛碱抗甲型流感病毒活性及其作用机制,揭示其作用靶点,以期充分挖掘石斛的药用价值,为开发具有自主知识产权的新型抗流感药物奠定基础,为流感的防治提供新的思路。研究方法:1.MTT法与空斑实验检测石斛碱的细胞毒性及抗甲型流感病毒的活性。2.Western blotting、免疫荧光和qRT-PCR检测石斛碱对流感病毒复制的影响。3.血凝抑制实验、SPR实验、H5N1假病毒体系、神经氨酸酶抑制实验和time-of-addition实验研究石斛碱作用机制。4.Mini-replicon实验、定点突变技术、免疫共聚焦和计算机模拟分子对接技术研究石斛碱的作用靶点。实验结果:1.石斛碱能够有效地抑制多种甲型流感病毒的感染,包括A/Aichi/2/68(H3N2),A/FM1/47(H1N1)和 A/Puerto Rico/8/34 H274Y(H1N1),IC50 分别是 5.32±1.68,3.39±0.32,2.16±0.91 μg/ml。2.石斛碱可抑制甲型流感病毒的多轮复制,且抗病毒作用可达72h。3.石斛碱对流感病毒的进入无抑制作用。4.石斛碱在病毒感染细胞后的0-4 h能够明显抑制流感病毒HA的表达。5.石斛碱可降低vRNP的活性,能够与NP蛋白结合,且能抑制NP的出核,降低NP的核内表达。6.石斛碱可通过R267、V270、F338和E339与NP蛋白结合,且4个位点的保守性大于99%。实验结论:1.石斛碱具有抗甲型流感病毒的活性,且能抑制病毒的多轮复制。2.石斛碱有效地抑制流感病毒的早期复制。3.石斛碱抗流感病毒的作用靶点为NP蛋白。4.石斛碱与NP蛋白结合的位点是NP蛋白的保守功能区域。5.石斛碱通过与NP蛋白的结合抑制其出核以及寡聚化,从而阻碍流感病毒的复制。
[Abstract]:Objective: influenza is an infectious disease that seriously endangers human health. There have been many influenza pandemics in history, with a wide range of spread and high mortality. Because of the antigenic transformation and antigenic drift of influenza virus, influenza virus has a high variability. The emergence of new influenza virus has had a serious impact on human survival. The prevention and treatment of influenza, especially drug treatment, is urgent. Up to now, there are mainly two kinds of anti-influenza drugs used in the world: neurosinase inhibitor and M2 ion channel inhibitor, but drug resistance and toxic and side effects have been perplexing the development and application of these drugs. At present, the chemical synthetic drugs used in the prevention and treatment of influenza virus infection are very limited. Therefore, it is particularly important to find low toxic and effective anti-influenza virus traditional Chinese medicine from natural products. "time disease theory" Qingre Baojin method once included compound Dendrobium decoction to treat warm heat and sweat, wind heat and heat, heat injury, etc. Among them, Dendrobium has a wide range of biological activities, including tonifying stomach, relieving cough, nourishing yin and clearing heat, etc., which are often used in many diseases, such as dry thirst, febrile injury, deficiency and heat after disease and so on. A major alkaloid extracted from Dendrobium nobile. By studying the activity and mechanism of Dendrobium alkaloid against influenza A virus, this project reveals its target in order to fully excavate the medicinal value of Dendrobium candidum and lay a foundation for the development of new anti-influenza drugs with independent intellectual property rights. It provides a new way of thinking for the prevention and treatment of influenza. Methods: 1.MTT assay and plaque test were used to detect the cytotoxicity and anti-influenza A virus activity of Dendrobium. 2. Western blotting, immunofluorescence and qRT-PCR were used to detect the effect of Dendrobium on influenza virus replication. Hemagglutination inhibition test, SPR test, H5N1 pseudovirus system, neuraminase inhibition test and time-of-addition experiment were used to study the mechanism of Dendrobium alkaloid action. 4.Mini-replicon test, site-directed mutation technique, Immunoconfocal and computer simulated molecular docking techniques were used to study the target of Dendrobium alkaloid. The experimental results are as follows: 1. Dendrobium can effectively inhibit the infection of a variety of influenza A viruses, including A/Aichi/2/68 (H3N2), A/FM1/47 (H1N1) and A/Puerto Rico/8/34 H274Y (H1N1). IC50 was 5.32 卤1.68, 3.39 卤0.32, 2.16 卤0.91 渭 g 鈮，
本文编号：2485923
[Abstract]:Objective: influenza is an infectious disease that seriously endangers human health. There have been many influenza pandemics in history, with a wide range of spread and high mortality. Because of the antigenic transformation and antigenic drift of influenza virus, influenza virus has a high variability. The emergence of new influenza virus has had a serious impact on human survival. The prevention and treatment of influenza, especially drug treatment, is urgent. Up to now, there are mainly two kinds of anti-influenza drugs used in the world: neurosinase inhibitor and M2 ion channel inhibitor, but drug resistance and toxic and side effects have been perplexing the development and application of these drugs. At present, the chemical synthetic drugs used in the prevention and treatment of influenza virus infection are very limited. Therefore, it is particularly important to find low toxic and effective anti-influenza virus traditional Chinese medicine from natural products. "time disease theory" Qingre Baojin method once included compound Dendrobium decoction to treat warm heat and sweat, wind heat and heat, heat injury, etc. Among them, Dendrobium has a wide range of biological activities, including tonifying stomach, relieving cough, nourishing yin and clearing heat, etc., which are often used in many diseases, such as dry thirst, febrile injury, deficiency and heat after disease and so on. A major alkaloid extracted from Dendrobium nobile. By studying the activity and mechanism of Dendrobium alkaloid against influenza A virus, this project reveals its target in order to fully excavate the medicinal value of Dendrobium candidum and lay a foundation for the development of new anti-influenza drugs with independent intellectual property rights. It provides a new way of thinking for the prevention and treatment of influenza. Methods: 1.MTT assay and plaque test were used to detect the cytotoxicity and anti-influenza A virus activity of Dendrobium. 2. Western blotting, immunofluorescence and qRT-PCR were used to detect the effect of Dendrobium on influenza virus replication. Hemagglutination inhibition test, SPR test, H5N1 pseudovirus system, neuraminase inhibition test and time-of-addition experiment were used to study the mechanism of Dendrobium alkaloid action. 4.Mini-replicon test, site-directed mutation technique, Immunoconfocal and computer simulated molecular docking techniques were used to study the target of Dendrobium alkaloid. The experimental results are as follows: 1. Dendrobium can effectively inhibit the infection of a variety of influenza A viruses, including A/Aichi/2/68 (H3N2), A/FM1/47 (H1N1) and A/Puerto Rico/8/34 H274Y (H1N1). IC50 was 5.32 卤1.68, 3.39 卤0.32, 2.16 卤0.91 渭 g 鈮，
本文编号：2485923
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