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| 从清热解表类中药中筛选神经氨酸酶抑制剂及其抗流感病毒活性的研究
中文摘要
流感是由流感病毒引起的高度传染性的急性呼吸道疾病，它给人类健康和生命造成了极大的伤害，同时给全球经济带来了巨大的损失。它是人类尚未控制的世界传染病之一。
核蛋白（NP）和基质蛋白（M1）的抗原差异使流感病毒可分为A、B和C型。对于A型病毒，不同的亚型基于血凝素（HA）和神经氨酸酶（NA）表面糖蛋白的抗原性。这两种重要的表面糖蛋白，可以催化糖苷键的水解裂解，从而从宿主细胞表面分离出子代病毒。NA在流感病毒的感染，复制和脱壳中起着重要作用，使其成为对抗流感病毒最有效的靶蛋白。目前，已获得美国FDA认证的神经氨酸酶抑制剂药物主要包括扎那米韦，帕拉米韦和他菲（oseltamivir）。但是，流感病毒的NA突变使该病毒对扎那米韦，帕拉米韦和达菲具有抗药性。因此，迫切需要开发抑制NA的新药。
中药在我国具有悠久的抗流感历史。在抗流感药方面，在我国民间中流传着许多古老的处方。近年来，许多研究人员就中药的抗流感作用及其机理进行了研究报告。但是，目前尚无从抑制中药NA活性的角度研究中药抗流感作用的报道。
本实验主要采用NA活性荧光检测法从133种中药水提物中筛选出NAI，采用传统的水煮浸法分别提取各种中药。筛选后，我们得到了莲须、红景天和罗布麻三种抑制效果好的中药。然后测定这三种中药对NA抑制的IC50值。
之后，我们进一步测试了具有NA抑制活性的中药对MDCK细胞感染H1N1病毒的保护作用。我们首先确定了实验药物的剂量和被感染病毒的稀释度。之后，我们将病毒与中药水提取物共同感染MDCK细胞，然后再共同感染MDCK细胞。MTT法检测中药水提取物对H1N1病毒感染的细胞病变作用（CPE）。通过NA活性检测测试了中药对MDCK细胞中病毒复制的保护作用。结果表明，这三种中药可以更好地保护流感病毒对MDCK细胞的CPE作用，并可以剂量依赖的方式减少MDCK细胞中病毒的复制。
最后，从筛选出的中药中通过查阅文献找出其主要含有的成分，并分析其与NA的相互作用。首先从蛋白质数据库中检索两类经典甲型流感病毒NA类型N1的三维晶体结构。通过 Discovery Studio 3.5 软件将所有的 NA 蛋白质 3D 结构去水、去杂原子等以备接下来的实验所用。随后，建立清热解毒解表类中药单体化合物文库。下载其三维构象，组成小分子化合物文库。最后，利用 AutoDock4.2 软件，以 N1为靶点，分别对小分子数据库进行虚拟筛选。确定化合物与 N1结合位点，通过结合能打分排序，获得候选化合物，以用于后续酶学实验。
关键词：
H1N1
Abstract
Influenza is a highly contagious acute respiratory disease caused by influenza viruses.It has caused great harm to human health and life,and at the same time brought huge losses to the global economy. It is one of the world's infectious diseases that humans have not yet controlled.
The antigenic difference between nucleoprotein (NP) and matrix protein (M1) allows influenza viruses to be divided into A, B and C types. For type A viruses, the different subtypes are based on the antigenicity of hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. These two important surface glycoproteins can catalyze the hydrolytic cleavage ofglycosidic bonds, thereby isolating progeny viruses from the surface of host cells. NA plays an important role in influenza virus infection,replication and uncoating, making it the most effective target protein against influenza virus. At present, the neuraminidase inhibitor drugs that have been certified by the US FDA mainly include zanamivir, peramivir and oseltamivir. However, the NA mutation of the influenza virus makesthe virus resistant to zanamivir, peramivir and tamiflu. Therefore, there is an urgent need to develop new drugs that inhibit NA.
Traditional Chinese medicine has a long history of anti-influenza in our country.In terms of anti-flu drugs,many ancient prescriptions are circulating among the people of our country. In recent years, many researchers have conducted research reportson the anti-influenza effect of Chinese medicine and its mechanism. However, there is no report on the anti-influenza effect of traditional Chinese medicinefrom the perspective of inhibiting the activity of traditional Chinese medicine NA.
In this experiment, the NA activity fluorescence detection method was used toscreen out NAI from 133 kinds of Chinese medicine water extracts,and the traditional water boiling method was used to extract various Chinese medicines. After screening, we obtained three Chinese medicines with good inhibitory effects: Stamen Nelumbinis,Rhodiola and Apocynum venetum L. Then the IC50 values of these three traditional Chinesemedicines against NA inhibition were determined.
After that, we further tested the protective effect of traditional Chinesemedicine with NA inhibitory activity on MDCK cells infected with H1N1 virus. We first determined the dose of the experimental drug and the dilution of the infected virus.After that, we co-infect MDCK cells with the virus and the water extract of Chinese medicine, and then co-infect MDCK cells. The MTT method was used to detect the cytopathic effect (CPE) of thewater extract of traditional Chinese medicine on H1N1 virus infection. The protective effect of traditional Chinese medicine on virusreplication in MDCK cells was tested by NA activity detection. The results show that these three traditional Chinese medicines can better protect the CPE effect of influenza virus on MDCK cells,and can reduce virus replication in MDCK cells in a dose-dependent manner.
Finally, from the selected Chinese medicines,the main components are found by consulting the literature, and the interaction between them and NA is analyzed. Firstly, the three-dimensional crystal structure of NA type N1 of two types of classic influenza A viruses was retrieved from the protein database. Through Discovery Studio 3.5 software, all 3D structures of NA protein are removed for water and heteroatoms to prepare for the next experiment. Subsequently, a single compound library of traditional Chinese medicine for clearing away heat, detoxifying and detoxifying was established. Download its three-dimensional conformation to form a library of small molecule compounds.Finally, using AutoDock4.2 software, with N1 as the target, virtual screening of the small molecule database was performed.Determine the binding site of the compound and N1,and rank by binding energy to obtain candidate compounds for subsequent enzymatic experiments.
Key words：
Chinese medicine, influenza virus, neuraminidase, inhibitor, H1N1
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