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

Abstract

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

CLC Number:

S853.74

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.

References

[1] 张多敏.高致病性禽流感的研究与防范[J].农家参谋,2022,24:122-124.ZHANG D M.Research and prevention of highly pathogenic avian influenza[J]. The Farmers Consultant,2018,24:122-124.(in Chinese)
[2] 麦燕娜,杨淑媚,黄焕聪.鸡禽流感的发病特点与防控研究[J].畜牧业环境,2023,4:58-60.MAI Y N,YANG S M,HUANG H C.Research on the characteristics and prevention and control of avian influenza in chickens[J].Animal Industry and Environment,2023,4:58-60.(in Chinese)
[3] 李晗琪.禽流感病毒的传播与流行进化特点分析[J].农业与技术,2018,38(4):143.LI H Q.Analysis of the transmission and evolutionary characteristics of Avian influenza virus[J]. Agriculture and Technology,2018,38(4):143.(in Chinese)
[4] 唐宁馨,陈聪洁,韩玲玉,等.H9N2亚型禽流感病毒流行特征及疫苗研究进展[J].中国人兽共患病学报,2024,40(4):369-376.TANG N X,CHEN C J,HAN L Y,et al.Research progress in epidemiology and vaccine development of the H9N2 Avian influenza virus[J].Chinese Journal of Zoonoses,2024,40(4):369-376.(in Chinese)
[5] 李春娜,刘洋洋,李鹏收,等.中药复方双黄连剂型研究[J].中国中医药科技,2015,22(1):115-117.LI C N,LIU Y Y,LI P S,et al.Research on the formulation of traditional Chinese medicine compound Shuanghuanglian[J].Chinese Journal of Traditional Medical Science and Technology,2015,22(1):115-117.(in Chinese)
[6] 王建俊,李卫鹏,姚红艳,等.黄芩苷对H6N6禽流感感染小鼠肺损伤的影响[J].经济动物学报,2022,26(1):11-18.WANG J J,LI W P,YAO H Y,et al.Therapeutic effect of baicalin on lung injury of mice infected by H6N6 Avian influenza virus[J].Journal of Economic Animal,2022,26(1):11-18.(in Chinese)
[7] 罗旋,王薇,胡本祥,等.金银花抗病毒化学成分及作用研究进展[J].陕西中医药大学学报,2024,47(2):138-143.LUO X,WANG W,HU B X,et al.Research progress on antiviral chemical components and effects of honeysuckle[J].Journal of Shaanxi University of Chinese Medicine,2024,47(2):138-143.(in Chinese)
[8] 盛可心,王昊冉,徐凌川,等.贯叶连翘药理作用及其机制研究新进展[J].中药药理与临床,2023,39(3):117-122.SHENG K X,WANG H R,XU L C,et al.Advances in the pharmacological effects and mechanisms of Hypericum perforatum L.[J].Pharmacology and Clinics of Chinese Materia Medica,2023,39(3):117-122.(in Chinese)
[9] 丘国清,杜秋伶,江海明,等.双黄连雾化吸入对小鼠H9N2禽流感病毒感染的疗效研究[J].实用临床医药杂志,2021,25(20):1-5.QIU G Q,DU Q L,JIANG H M,et al.Efficacy of Shuanghuanglian aerosol inhalation in treatment of mice with H9N2 Avian influenza virus infection[J].Journal of Clinical Medicine in Practice,2021,25(20):1-5.(in Chinese)
[10] YOUNG M R.Endothelial cells in the eyes of an immunologist[J].Cancer Immunology,Immunotherapy,2012,61(10):1609-1616.
[11] TEIJARO J R,WALSH K B,CAHALAN S,et al.Endothelial cells are central orchestrators of cytokine amplification during Influenza virus infection[J].Cell,2011,146(6):980-991.
[12] ZENG H,PAPPAS C,BELSER J A,et al.Human pulmonary microvascular endothelial cells support productive replication of highly pathogenic Avian influenza viruses:Possible involvement in the pathogenesis of human H5N1 virus infection[J].Journal of Virology, 2012,86(2):667-678.
[13] 程邓芳,梅晨,刘娟,等.中药复方对H9N2亚型禽流感病毒在MDCK细胞增殖的作用[J].中国畜牧兽医,2021,48(2):650-657.CHENG D F,MEI C,LIU J,et al.Effect of Chinese herbal prescription on the proliferation of H9N2 in MDCK cells[J].China Animal Husbandry & Veterinary Medicine 2021,48(2):650-657.(in Chinese)
[14] NOZHAT Z,KHALAJI M S,HEDAYATI M,et al.Different methods for cell viability and proliferation assay:Essential tools in pharmaceutical studies[J].Anti-cancer Agents in Medicinal Chemistry,2020,22(4):703-712.
[15] 李岩.内毒素耐受致中性粒细胞跨内皮迁移障碍及潜在机制研究[D].上海：中国人民解放军海军军医大学,2019.LI Y.The neutrophil trans endothelial migration suppressed by endotoxin tolerance and underlying mechanisms[D].Shanghai:Naval Medical University,2019.(in Chinese)
[16] SHORT K R,KASPER J,VAN DER AA S,et al.Influenza virus damages the alveolar barrier by disrupting epithelial cell tight junctions[J].The European Respiratory Journal,2016,47(3):954-966.
[17] YU W Y,LI L,WU F,et al. Moslea herba flavonoids alleviated Influenza A virus-induced pulmonary endothelial barrier disruption via suppressing NOX4/NF-κB/MLCK pathway[J]. Journal of Ethnopharmacology,2020，253:112641.
[18] 褚皓天,赵鹏,田燕歌,等.MTT法用于中药成分细胞毒性和增殖检测的干扰因素及控制措施[J].中国现代中药,2024,26(4):749-756.CHU H T,ZHAO P,TIAN Y G,et al.Interference factors and countermeasures in MTT assay for detection of Chinese herbal medicine components with cytotoxicity and influencing cell proliferation[J].Modern Chinese Medicine,2024,26(4):749-756.(in Chinese)
[19] 任周新.MTT法在中草药成分干预细胞增殖及活性中的应用[J].中医学报,2023,38(10):2043-2049.REN Z X.Application of MTT method in intervention of Chinese medicine ingredients in cell proliferation and activity[J].Acta Chinese Medicine,2023,38(10):2043-2049.(in Chinese)
[20] 杨宇航,王文玉,刘红娜,等.细胞活力及细胞免疫力检测方法的研究进展[J].中国畜牧兽医,2013,40(6):142-145.YANG Y H,WANG W Y,LIU H N,et al.Research progress on the detection method of the cell vitality and cell immunity[J].China Animal Husbandry & Veterinary Medicine,2013,40(6):142-145.(in Chinese)
[21] 赵世云,赵新新,苏华荔,等.猪外周血T淋巴细胞增殖反应MTT检测方法的建立[J].中国畜牧兽医,2010,37(12):35-38.ZHAO S Y,ZHAO X X,SU H L,et al.Development of MTT assay for the detection of peripheral blood T cell proliferation of swine[J].China Animal Husbandry & Veterinary Medicine,2010,37(12):35-38.(in Chinese)
[22] 梁巩,张强,郭旭川,等.不同来源血清对体外培养驴巨噬细胞的影响[J].江苏农业科学,2020,48(13):72-75.LIANG G,ZHANG Q,GUO X C,et al.Effects of serum from different sources on donkey macrophage cultured in vitro[J].Jiangsu Agricultural Sciences,2020,48(13):72-75.(in Chinese)
[23] 刘雪莉,郭庆,王静,等.细胞贴壁效应是评价胎牛血清质量的重要因素[J].农业与技术,2021,41(22):1-4.LIU X L,GUO Q,WANG J,et al.Cell adhesion effect is an important factor in evaluating the quality of fetal bovine serum[J].Agriculture and Technology,2021,41(22):1-4.(in Chinese)
[24] CHAN M C,CHAN R W,YU W C,et al.Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells[J].Respiratory Research,2009,10(1):102.
[25] ZENG H,BELSER J A,GOLDSMITH C S,et al.A(H7N9) virus results in early induction of proinflammatory cytokine responses in both human lung epithelial and endothelial cells and shows increased human adaptation compared with Avian H5N1 virus[J].Journal of Virology,2015,89(8):4655-4667.
[26] STEINBERG B E,GOLDENBERG N M,LEE W L.Do viral infections mimic bacterial sepsis? The role of microvascular permeability:A review of mechanisms and methods[J].Antiviral Research,2012,93(1):2-15.
[27] LEI Y,SUN Y,WU W,et al.Influenza H7N9 virus disrupts the monolayer human brain microvascular endothelial cells barrier in vitro[J].Virology Journal,2023,20(1):219.
[28] AMERSFOORT J,EELEN G,CARMELIET P.Immunomodulation by endothelial cells-partnering up with the immune system?[J].Nature Reviews.Immunology,2022,22(9):576-588.
[29] CARMAN V C,EMARTINELLI R.T lymphocyte-endothelial interactions:Emerging understanding of trafficking and antigen-specific immunity[J].Frontiers in Immunology,2015,6:603.
[30] CANNA K,MCARDLE P A,MCMILLAN D C,et al.The relationship between tumour T-lymphocyte infiltration,the systemic inflammatory response and survival in patients undergoing curative resection for colorectal cancer[J].British Journal of Cancer,2005,92(4):651-654.
[31] PIAO W,XIONG Y,LI L,et al.Regulatory T cells condition lymphatic endothelia for enhanced transendothelial migration[J].Cell Reports,2020,30(4):1052-1062.e5.

Study on the Regulation of Cross-endothelial Lymphocytes of Shuanghuanglian Against Avian Influenza Virus

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