为明确表没食子儿茶素没食子酸酯（Epigallocatechin gallate,EGCG）在体内外对汉滩病毒（Hantaan virus,HTNV）的抑制成效,开展以下试验：（1）运用细胞活力检测法测定EGCG的细胞毒性;（2）在感染HTNV的不同时段添加EGCG进行处理,采用蛋白免疫印迹、实时荧光定量PCR、间接免疫荧光及斑点形成试验（Focus formation assay,FFA）检测HTNV在A549细胞内24、48 h的病毒表达量,同时使用Autodock vina软件进行分子对接,确定EGCG抗HTNV的潜在作用靶点;（3）通过灌胃给药方式给予攻毒裸鼠高中低剂量的EGCG,并检测其体重及生存率。结果表明,使用100 µmol·L-1 EGCG体外有效抑制HTNV感染,且该抑制效果主要作用于HTNV的吸附阶段;分子对接试验显示,EGCG可与HTNV的Gn、Gc相互作用,结合能分别为﹣9.0、﹣7.1 kcal·mol-1;高中低剂量EGCG（50.0、25.0、12.5 mg·kg-1）治疗感染裸鼠后,有效降低了攻毒裸鼠死亡率,裸鼠体重下降幅度均明显减少。
The purpose of this study was to investigate the inhibitory effect of epigallocatechin gallate (EGCG) on the Hantaan virus (HTNV) both in vitro and in vivo, and to identify the potential targets of EGCG against HTNV using molecular docking. First, the cytotoxicity of EGCG was determined using a cell viability assay. Then, EGCG was administered for treatment at different time points during HTNV infection, and the viral expression of HTNV in A549 cells was detected by western blotting, real-time PCR, indirect immunofluorescence, and focus formation assay (FFA) at 24 h and 48 h. Molecular docking was conducted using Autodock Vina software. Finally, the challenged nude mice were administered high, medium, and low doses of EGCG via gavage, and their body weight and survival rates were measured. The results indicate that the administration of 100 μmol·L-1 EGCG effectively inhibited HTNV infection in vitro, primarily affecting the adsorption stage of HTNV. The molecular docking results demonstrate that EGCG could interact with HTNV Gn and Gc, with binding energies of ﹣9.0 kcal·mol-1 and ﹣7.1 kcal·mol-1, respectively. High, medium, and low doses of EGCG (50.0, 25.0, 12.5 mg·kg-1) effectively reduced the mortality rate of challenged nude mice, and significantly mitigated weight loss in these mice.
[1] Zhao Y, Zhao N, Cai Y, et al.An algal lectin griffithsin inhibits Hantaan virus infection in vitro and in vivo[J]. Front Cell Infect Microbiol, 2022, 12: 881083. doi:103389/fcimb.2022.881083.
[2] Demirev A V, Lee S, Park S, et al.Exploring the genetic diversity and molecular evolution of seoul and hantaan orthohantaviruses[J]. Viruses, 2024, 16(1): 105-107.
[3] Afzal S, Ali L, Batool A, et al.Hantavirus: an overview and advancements in therapeutic approaches for infection[J]. Front Microbiol, 2023, 14: 1233433. doi:10.3389/fmicb. 2023.1343080.
[4] Linderholm M, Sandström T, Rinnström O, et al.Impaired pulmonary function in patients with hemorrhagic fever with renal syndrome[J]. Clinical Infectious Diseases, 1997, 25(5): 1084-1089.
[5] 丁文辉, 张鑫雨, 董阳超, 等. 汉滩病毒微基因组的构建与评价[J]. 微生物学免疫学进展, 2020, 48(1): 8-13.
Ding W H, Zhang X Y, Dong Y C.Construction and evaluation of a minigenome system for Hantaan virus[J]. Progress in microbiology and immunology, 2020, 48(1): 8-13.
[6] Sinclair S M, Jones J K, Miller R K, et al.The ribavirin pregnancy registry: an Interim analysis of potential teratogenicity at the mid-point of enrollment[J]. Drug Safety, 2017, 40(12): 1205-1218.
[7] Tatar A, Ozkurt Z, Kiki I.Genotoxic effect of ribavirin in patients with Crimean-Congo hemorrhagic fever[J]. Japanese Journal of Infectious Diseases, 2005, 58(5): 313-315.
[8] 尹敏. 茶叶提取物的药用价值[J]. 福建茶叶, 2024, 46(6): 18-20.
Yin M.Medicinal value of tea extract[J]. Tea in Fujian, 2024, 46(6): 18-20.
[9] Raekiansyah M, Buerano C C, Luz M A D, et al. Inhibitory effect of the green tea molecule EGCG against dengue virus infection[J]. Archives of Virology, 2018, 163(6): 1649-1655.
[10] Mekky R Y, El-Ekiaby N, El Sobky S A, et al. Epigallocatechin gallate (EGCG) and miR-548m reduce HCV entry through repression of CD81 receptor in HCV cell models[J]. Archives of Virology, 2019, 164(6): 1587-1595.
[11] Dinda B, Dinda S, Dinda M.Therapeutic potential of green tea catechin, (-)-epigallocatechin-3-O-gallate (EGCG) in SARS-CoV-2 infection:Major interactions with host/virus proteases[J]. Phytomed Plus, 2023, 3(1): 100402. doi: 10.1016/j.phyplu.2022.100402.
[12] He M, Chu T, Wang Z, et al.Inhibition of macrophages inflammasome activation via autophagic degradation of HMGB1 by EGCG ameliorates HBV-induced liver injury and fibrosis[J]. Fronttiers Immunology, 2023, 14: 1147379. doi:10.3389/fimmu.2023.1147379.
[13] Sabanayagam R, Krishnamoorthy S, Gnanagurusamy J, et al.EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells[J]. Medical Oncology, 2023, 40(9): 261. doi: 10.1007/
s12032-023-02135-1.
[14] Krzyzowska M, Janicka M, Chodkowski M, et al.Epigallocatechin gallate-modified silver nanoparticles show antiviral activity against herpes simplex type 1 and 2[J]. Viruses, 2023, 15(10): 2024. doi: 10.3390/v15102024.
[15] Mavor J, Engler O, Rothenberger S.Antiviral efficacy of ribavirin and favipiravir against hantaan virus[J]. Microorganisms, 2021, 9(6): 1306. doi: 10.3390/microorganisms9061306.
[16] Chapman L E, Ellis B A, Koster F T, et al.Discriminators between hantavirus infected and uninfected persons enrolled in a trial of intravenous ribavirin for presumptive hantavirus pulmonary syndrome[J]. Clinical Infectious Diseases, 2002, 34(3): 293-304.
[17] Yu X B, Hao K, Li J, et al.Effects of moroxydine hydro-chloride and ribavirin on the cellular growth and immune responses by inhibition of GCRV proliferation[J]. Research in Veterinary Science, 2018, 117: 37-44.
[18] 王蕊, 王聪聪, 李晓亮, 等. 柯萨奇病毒A组6型对利巴韦林耐药性的初步研究[J]. 病毒学报, 2023, 39(2): 384-392.
Wang R, Wang C C, Li X L, et al.Preliminary study on resistance of Coxsackievirus A 6 to ribavirin[J]. Chinese Journal of Virology, 2023, 39(2): 384-392.
[19] 邬时民. 注意利巴韦林的生殖毒性和溶血性贫血[J]. 家庭医学, 2021(11): 24.
Wu S M.Note the reproductive toxicity and hemolytic anemia of ribavirin[J]. Family Medicine, 2021(11): 24.
[20] 刘金彪. 绿茶多酚EGCG抑制HIV感染的机制研究[D]. 武汉: 武汉大学, 2016.
Liu J B.Research on the mechanism of green tea polyphenol EGCG in inhibiting HIV infection [D]. Wuhan: Wuhan University, 2016.
[21] 高振兴. 绿茶提取物EGCG抗乙型肝炎病毒的分子机理研究[D]. 郑州: 河南农业大学, 2011.
Gao Z X.Molecular mechanism study of green tea extract EGCG against Hepatitis B virus [D]. Zhengzhou: Henan Agricultural University, 2011.
[22] Jang M, Park Y I, Cha Y E, et al.Tea polyphenols EGCG and theaflavin inhibit the activity of SARS-CoV-2 3CL-Protease in vitro[J]. Evidence Based Complementary and Alternative Medicine, 2020, 16: 5630838. doi: 10.1155/2020/5630838.
[23] 应旗康. 汉滩病毒HTNV Gn-CT利用宿主ESCRT介导病毒出芽机制的研究[D]. 西安: 中国人民解放军空军军医大学, 2018.
Ying Q K.Identification of Interaction between Hantaanvirus Gn Cytoplasma Tail and Host ESCRT in Virus Budding [D]. Xi'an: Air Force Medical University, 2018.
[24] Wang K, Ma H, Liu H, et al.The glycoprotein and nucleocapsid protein of hantaviruses manipulate autophagy flux to restrain host innate immune responses[J]. Cell Reports, 2019, 27(7): 2075-2091.
[25] Gavrilovskaya IN, Shepley M, Shaw R, et al.Beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure[J]. PNAS, 1998, 95(12): 7074-7079.
[26] Graille M, Wild P, Sauvain J J, et al.Urinary 8-OHdG as a biomarker for oxidative stress: a systematic literature review and meta-analysis[J]. International Journal Molecular Sciences, 2020, 21(11): 3743. doi: 10.3390/ijms21113743.
[27] Khan N, Mukhtar H.Tea polyphenols in promotion of human health[J]. Nutrients, 2018, 11(1): 39. doi: 10.3390/nu11010039.
[28] Fujiki H, Watanabe T, Sueoka E, et al.Cancer prevention with green tea and its principal constituent, EGCG: from early investigations to current focus on human cancer stem cells[J]. Molecules and Cells, 2018, 41(2): 73-82.
[29] Cheong Y, Kim M, Ahn J, et al.Epigallocatechin-3-gallate as a novel vaccine adjuvant[J]. Frontiers Immunology, 2021, 12: 769088. doi: 10.3389/fmmu.2021.769088.
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