双黄连调控跨内皮淋巴细胞抗禽流感病毒的研究

doi:10.16431/j.cnki.1671-7236.2025.07.042

摘要/Abstract

摘要： 【目的】探究双黄连(Shuanghuanglian，SHL)通过调控跨内皮淋巴细胞抗H9N2亚型抗禽流感病毒(Avian influenza virus,AIV)感染的作用机制，为抗流感病毒中药的研发提供新思路和理论依据。【方法】采用MTT法检测不同浓度和不同作用时间含SHL培养基对大鼠肺微血管内皮细胞(rat pulmonary microvascular endothelial cells,rPMVECs)的药物毒性，筛选合适的药物作用浓度和时间。探究SHL对H9N2亚型AIV在rPMVECs上复制动力学的影响。对影响大鼠外周淋巴细胞增殖能力的各项指标(淋巴细胞浓度、FBS浓度和培养时间)进行优化。在此基础上建立rPMVECs与淋巴细胞的Transwell共培养体系，将rPMVECs接种上室，分为6组：活病毒组、灭活病毒组、活病毒加中药组、灭活病毒加中药组、内皮组及空白组，接种淋巴细胞。H9N2亚型AIV与跨内皮淋巴细胞孵育后接种MDCK细胞，通过MDCK细胞死亡数和上清液中的活病毒量评估SHL的抗病毒效果。【结果】浓度≤100 μg/mL的SHL与rPMVECs共培养24 h后显微镜下未观察到明显的细胞病变。在rPMVECs完整的情况下，当SHL浓度为100 μg/mL时，其与被H9N2亚型AIV感染的rPMVECs共作用24 h时对H9N2亚型AIV增殖的抑制作用最强。灭活病毒组的MDCK死亡细胞数和活病毒量显著低于活病毒组(P＜0.05)；活病毒加中药组的MDCK死亡细胞数和活病毒量显著低于内皮组和活病毒组(P＜0.05)。【结论】 SHL在保护宿主抗H9N2亚型AIV的过程中发挥着重要作用，其是通过保护内皮细胞的完整性，激活内皮细胞募集淋巴细胞从而发挥免疫作用，进而降低病毒载量，起到抗病毒的作用。

关键词: 双黄连; H9N2亚型禽流感病毒; 大鼠肺微血管内皮细胞; 淋巴细胞

Abstract: 【Objective】 The experiment aimed to explore the mechanism of Shuanghuanglian (SHL) in regulating cross-endothelial lymphocytes against H9N2 subtype Avian influenza virus (AIV) infection,and provide new ideas and theoretical basis for the research and development of traditional Chinese medicines against Influenza virus. 【Method】 The drug toxicity of SHL-containing medium with different concentrations and action time on rat pulmonary microvascular endothelial cells (rPMVECs) was detected by MTT assay,and appropriate drug concentrations and time were screened. The effect of SHL on the replication dynamics of the H9N2 subtype avian influenza virus (H9N2 subtype AIV) in rPMVECs was investigated.The various indicators of rat peripheral lymphocyte proliferation capacity,including lymphocyte concentration,FBS concentration,and incubation time were optimized.On this basis,a Transwell co-culture system was established between rPMVECs and lymphocytes.rPMVECs were inoculated into the upper chamber and divided into six groups:Virus group,Particle group,Virus+SHL group,Particle+SHL group,EC group,and Blank group,and lymphocytes were inoculated.After incubation of H9N2 subtype AIV with cross-endothelial lymphocytes and subsequent inoculation on MDCK cells,the antiviral effect of SHL was evaluated by assessing the number of dead MDCK cells and the amount of live virus in the supernatant. 【Result】 No significant cytopathic effects were observed in rPMVECs co-cultured with ≤100 μg/mL concentration of SHL for 24 h.SHL exhibited strong inhibitory effects on H9N2 AIV proliferation when co-cultured with virus-infected rPMVECs at a concentration of 100 μg/mL for 24 h.The number of dead MDCK cells and the amount of live virus in the Particle group were significantly lower than those in Virus group (P＜0.05).The number of dead MDCK cells and the amount of live virus in the Virus+SHL group were significantly lower than those in EC group and Virus group(P＜0.05). 【Conclusion】 SHL played a crucial role in protecting the host from H9N2 subtype AIV by maintaining the integrity of endothelial cells and activating their recruitment of lymphocytes to exert immune effects,thereby reducing viral load and exerting antiviral effects.

Key words: Shanghuanglian (SHL); H9N2 subtype AIV; rPMVECs; lymphocytes

中图分类号:

S853.74

张朝晖, 孙志刚, 穆祥, 冯波, 梁宏伟, 刘晓晔, 张倩. 双黄连调控跨内皮淋巴细胞抗禽流感病毒的研究[J]. 中国畜牧兽医, 2025, 52(7): 3440-3448.

ZHANG Zhaohui, SUN Zhigang, MU Xiang, FENG Bo, LIANG Hongwei, LIU Xiaoye, ZHANG Qian. Study on the Regulation of Cross-endothelial Lymphocytes of Shuanghuanglian Against Avian Influenza Virus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3440-3448.

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