苦丁茶紫茎女贞增强T细胞免疫应答抗甲型流感病毒的物质基础及作用机制研究

Ku Ding Cha, one kind of resourceful species, is also an important resource for the medicinal plant. The enhancement of T cell immune response is the footstone of the host to clear virus in body rapidly, and provides an broad-spectrum protective effects. In the preliminary study, our group found total glycosides of Ligustrum purpurascens (LPG) can effectively improve the survival rate of Influenza A virus (IAV) infected mice and alleviate the lung inflammatory reaction; Meanwhile, LPG can significantly increase the numbers of CD4+IFN-γ+ and CD8+IFN-γ+T cells in the spleen of IAV infected mice, accompanying with the inhibition of IAV. It's necessary to find out the exact antiviral material basis of Ligustrum purpurascens through bioassay-guided method with enhancing T lymphocytes response in vitro. Investigating the LPG pharmacological effects of anti-IAV worth highly focused. The mechanism of anti-IAV by activating CD4+ and CD8+T cells response also needs further study from the molecular level. The aim of this study is to provide a theoretical basis for the research of innovative antiviral drug as well as providing a scientific basis for the comprehensive exploitation of Ligustrum purpurascens.

苦丁茶是我国特有的大宗经济物种，也是重要的药用植物资源。T细胞介导的免疫应答是机体清除体内病毒的关键，起着广谱而有效的保护作用。本课题组研究发现苦丁茶紫茎女贞(Ligustrum purpuracens) 总苷（LPG）可有效提高甲型流感病毒感染小鼠的存活率，缓解肺部炎症；同时显著提高脾脏中CD4+IFN-γ+和CD8+IFN-γ+ T细胞数量，并与抗甲型流感病毒活性呈现相关性。本项目拟在以上研究工作基础上，采用活性追踪法，进一步研究苦丁茶紫茎女贞可增强T细胞免疫应答抗甲型流感病毒的物质基础，并重点研究苦丁茶紫茎女贞活性部位或单体抗甲型流感病毒的药理作用，从分子水平上揭示激活体内CD4+和CD8+T细胞在抗甲型流感病毒过程中的作用及机制，为研发基于增强T细胞免疫应答新型天然小分子抗病毒感染药物提供理论基础；为苦丁茶紫茎女贞物种资源的综合开发利用提供科学依据。

紫茎女贞（Ligustrum purpurascens）叶经提取分离得到苯丙素总苷（Ligustrum purpurascens phenylethanoid glycosides, LPG）。运用多种色谱分离手段，从紫茎女贞总苷中分离得到了17个化合物，并鉴定了它们的结构，分别为15个苯乙醇苷类化合物：acteoside (LP-1)、ligupurpuroside J (LP-2)、ligupurpuroside A (LP-3)、syringalide A 3'-α-L-rhamnopyranoside (LP-4)、lipedoside A-I (LP-5)、kankanoside G (LP-6)、lipedoside A-II (LP-7)、isoacteoside (LP-8)、osmanthuside B (LP-9)、ligupurpuroside C (LP-10)、cis-lipedoside A-I (LP-11)、ligurobustoside N (LP-12)、(E)-ligupurpuroside B (LP-13)、angoroside A (LP-14)和(Z)-ligupurpuroside B (LP-15)；1个黄酮苷类化合物：luteilin-7-O-β-D-glucopyranoside (LP-16)；以及1个环烯醚萜苷类化合物：ligupurpuroside K (LP-17)。体外诱导IFN-γ筛选结果表明，化合物LP-1、5、6、10、11、13和15能够显著地促进小鼠脾脏淋巴细胞分泌IFN-γ；体外抗H1N1活性测试结果显示， acteoside (LP-1)、angoroside A (LP-14)、ligupurpuroside A (LP-3)、lipedoside A-I(LP-5)对H1N1流感病毒的治疗指数（TI）大于2，acteoside作用结果最强，TI为40.87。acteoside抗H1N1机制研究表明，acteoside可激活小鼠T淋巴细胞表面受体CD28、MHCII、TCR和转录因子T-bet表达，通过激活MAPK/ERK信号通路促进IFN-γ分泌；另一方面，acteoside直接靶向抑制H1N1的核蛋白（NP），阻断病毒的复制。