芒果苷抗DHAV-1致鸭胚肝细胞炎性损伤的研究

doi:10.16431/j.cnki.1671-7236.2022.11.040

摘要/Abstract

摘要： 【目的】探究芒果苷（mangiferin，Man）对鸭甲型肝炎病毒1型（Duck hepatitis A virus-1，DHAV-1）致鸭胚肝细胞（DEHCs）炎性损伤的影响。【方法】从14日龄SPF级麻鸭鸭胚肝脏组织分离原代DEHCs，用含不同浓度芒果苷（0、5、10、20、40、80和160 μmol/L）的培养液培养48 h后，通过CCK-8法检测其安全浓度范围；用安全浓度的芒果苷培养DEHCs 12、24和48 h后，检测培养液上清中乳酸脱氢酶（LDH）的活力，判定芒果苷对DEHCs的毒性作用。将DEHCs分为对照组（Mock）、模型组（Model）、芒果苷组（Man10、Man20和Man40）和利巴韦林组（Rib），每组3个重复，所有组细胞血清饥饿培养12 h后，Mock组加入含10%胎牛血清（FBS）的DMEM培养基，Model、Man10、Man20、Man40和Rib组用DHAV-1（MOI=1.0）攻毒2 h后，Model组更换为含10% FBS的DMEM培养基，Man10、Man20、Man40培养基中分别添加10、20和40 μmol/L芒果苷，Rib组添加1 μmol/L利巴韦林。48 h后收集各组细胞，用比色法检测丙二醛（MDA）含量及过氧化氢酶（CAT）、总抗氧化能力（T-AOC）、超氧化物歧化酶（SOD）、总一氧化氮合酶（T-NOS）和谷胱甘肽过氧化物酶（GSH-Px）的活性；免疫荧光（IF）法检测DHAV-1在各组DEHCs中的分布情况；Western blotting法检测NLRP3/Pro-Caspase1/IL-1β通路蛋白的表达情况。提取2日龄SPF麻鸭的新鲜全血，分离鸭外周血单个核细胞（duPBMCs），duPBMCs分组及处理同DEHCs，ELISA法检测各组duPBMCs上清液中白介素8（IL-8）和IL-1β的含量。【结果】与0 μmol/L芒果苷组相比，80和160 μmol/L芒果苷组细胞的增殖能力显著降低（P<0.05），因此5~40 μmol/L芒果苷为处理DEHCs的安全给药浓度。20和40 μmol/L芒果苷处理48 h细胞上清液中LDH活力显著低于处理12和24 h （P<0.05），20和40 μmol/L芒果苷处理12和24 h细胞上清液中LDH活力显著高于5和10 μmol/L芒果苷处理（P<0.05），各浓度芒果苷处理48 h细胞上清液中LDH活力均差异不显著（P>0.05），故48 h为最适处理时间。与Mock组相比，Model组DEHCs中CAT、T-AOC、SOD和GSH-Px活性均显著降低（P<0.05），DEHCs中MDA含量、T-NOS活性、DHAV-1拷贝数、DHAV-1阳性率、NLRP3表达量及duPBMCs中IL-8、IL-1β分泌量均显著升高（P<0.05）。与Model组相比，Man10、Man20和Man40组DEHCs中CAT、T-AOC、SOD和GSH-Px活性均显著提高（P<0.05），DEHCs中MDA含量、T-NOS活性、DHAV-1拷贝数、DHAV-1阳性率、NLRP3表达量及duPBMCs中IL-8、IL-1β分泌量均显著降低（P<0.05）。【结论】芒果苷可通过增加抗氧化能力、降低DHAV-1的复制水平、抑制NLRP3通路的激活、降低促炎细胞因子的释放抗DHAV-1感染DEHCs造成的炎性损伤。

关键词: 芒果苷; 鸭甲型肝炎病毒1型(DHAV-1); 鸭胚肝细胞; 鸭外周血单个核细胞

Abstract: 【Objective】 The aim of this study was to explore the effect of mangiferin (Man) on the inflammatory injury of duck embryo hepatocytes (DEHCs) induced by Duck hepatitis A virus-1 (DHAV-1).【Method】 The primary DEHCs were isolated from the liver tissues of 14-day-old SPF duck embryos, and cultured for 48 h with different concentrations of mangiferin (0, 5, 10, 20, 40, 80 and 160 μmol/L).Then, the safe concentration range of mangiferin was detected by CCK-8 method.The toxicity of mangiferin to DEHCs was determined by detecting the activity of lactate dehydrogenase (LDH) in the supernatant of DEHCs cultured with safe concentration of mangiferin for 12, 24 and 48 h.The DEHCs were divided into control group (Mock), model group (Model), mangiferin groups (Man10, Man20 and Man40) and ribavirin group (Rib), with three replicates in each group, and cells in all groups were cultured with serum starvation for 12 h.After that, Mock group was added with DMEM medium containing 10% FBS, Model, Man10, Man20, Man40 and Rib groups were challenged with DHAV-1 (MOI=1.0) for 2 h, and then Model group was replaced with DMEM medium containing 10% FBS, the culture medium of Man10, Man20, Man40 groups was supplemented with 10, 20 and 40 μmol/L mangiferin, respectively, and 1 μmol/L ribavirin was added to Rib group.After 48 h, cells in each group were collected, and the content of malondialdehyde (MDA) and the activities of catalase (CAT), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), total nitric oxide synthase (T-NOS) and glutathione peroxidase (GSH-Px) were detected by colorimetric method.The distribution of DHAV-1 in DEHCs cells in each group was detected by immunofluorescence (IF).Expression of NLRP3/Pro-Caspase1/IL-1β pathway proteins were detected by Western blotting method.The fresh whole blood of 2-day-old SPF ducks was extracted, and the peripheral blood mononuclear cells (duPBMCs) of the ducks were isolated.The grouping and processing of duPBMCs were the same as DEHCs.ELISA assay was used to detect the content of IL-8 and IL-1β in the supernatant of duPBMCs.【Result】 Compared with 0 μmol/L mangiferin group, the cell proliferation ability of 80 and 160 μmol/L mangiferin was significantly reduced (P<0.05), so 5-40 μmol/L mangiferin was a safe administration concentration for DEHCs.The activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 48 h was significantly lower than that of cells treated with 12 and 24 h (P<0.05), the activity of LDH in the supernatant of cells treated with 20 and 40 μmol/L mangiferin for 12 and 24 h was significantly higher than that treated with 5 and 10 μmol/L mangiferin (P<0.05), there was no significant difference in LDH activity in the supernatant of cells treated with mangiferin at different concentrations for 48 h (P>0.05), so 48 h was the optimum treatment time.Compared with the Mock group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of the Model group were significantly decreased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly increased (P<0.05).Compared with Model group, the activities of CAT, T-AOC, SOD and GSH-Px in DEHCs of Man10, Man20 and Man40 groups were significantly increased (P<0.05), the content of MDA, the activity of T-NOS, DHAV-1 copy number, DHAV-1 positive rate, the expression of NLRP3 in DEHCs and the secretion levels of IL-8 and IL-1β in duPBMCs were significantly decreased (P<0.05).【Conclusion】 Mangiferin could resist the inflammatory damage caused by DHAV-1 infection of DEHCs by increasing the antioxidant capacity, reducing the replication level of DHAV-1, inhibiting the activation of NLRP3 pathway and reducing the release of pro-inflammatory cytokines.

Key words: mangiferin; Duck hepatitis A virus-1 (DHAV-1); duck embryonic hepatocytes; duck peripheral blood mononuclear cells

中图分类号:

R285.5

孙伟翔, 秦枫, 袁亚梅, 卜潇, 张力, 吴双, 林梦舟, 吴植, 朱善元. 芒果苷抗DHAV-1致鸭胚肝细胞炎性损伤的研究[J]. 中国畜牧兽医, 2022, 49(11): 4485-4494.

SUN Weixiang, QIN Feng, YUAN Yamei, BU Xiao, ZHANG Li, WU Shuang, LIN Mengzhou, WU Zhi, ZHU Shanyuan. Effect of Mangiferin on the Inflammatory Injury of Duck Embryonic Hepatocytes Induced by Duck Hepatitis A Virus-1[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(11): 4485-4494.

参考文献

[1] OU X, MAO S, CAO J, et al.Comparative analysis of virus-host interactions caused by a virulent and an attenuated Duck hepatitis A virus genotype 1[J].PLoS One, 2017, 12(6):e0178993.
[2] YUGO D M, HAUCK R, SHIVAPRASAD H L, et al.Hepatitis virus infections in poultry[J].Avian Diseases, 2016, 60(3):576-588.
[3] YEHIA N, ERFAN A M, OMAR S E, et al.Dual circulation of Duck hepatitis A virus genotypes 1 and 3 in Egypt[J].Avian Diseases, 2021, 65(1):1-9.
[4] SHA H, ZENG H, ZHAO J, et al.Mangiferin ameliorates gestational diabetes mellitus-induced placental oxidative stress, inflammation and endoplasmic reticulum stress and improves fetal outcomes in mice[J].European Journal of Pharmacology, 2019, 859:172522.
[5] LIN Y S, TSAI K L, CHEN J N, et al.Mangiferin inhibits lipopolysaccharide-induced epithelial-mesenchymal transition (EMT) and enhances the expression of tumor suppressor gene PER1 in non-small cell lung cancer cells[J].Environmental Toxicology, 2020, 35(10):1070-1081.
[6] RECHENCHOSKI D Z, AGOSTINHO K F, FACCIN-GALHARDI L C, et al.Mangiferin:A promising natural xanthone from Mangifera indica for the control of acyclovir-resistant herpes simplex virus 1 infection[J].Bioorganic & Medicinal Chemistry, 2020, 28(4):115304.
[7] YUN C, CHANG M, HOU G, et al.Mangiferin suppresses allergic asthma symptoms by decreased Th9 and Th17 responses and increased Treg response[J].Molecular Immunology, 2019, 114:233-242.
[8] PIAO C H, FAN Y J, NGUYAN T V, et al.Mangiferin alleviates ovalbumin-induced allergic rhinitis via Nrf2/HO-1/NF-κB signaling pathways[J].International Journal of Molecular Sciences, 2020, 21(10):3415.
[9] LI N, XIONG R, HE R, et al.Mangiferin mitigates lipopolysaccharide-induced lung injury by inhibiting NLRP3 inflammasome activation[J].Journal of Inflammation Research, 2021, 14:2289-2300.
[10] JIA L, SUN P, GAO H, et al.Mangiferin attenuates bleomycin-induced pulmonary fibrosis in mice through inhibiting TLR4/p65 and TGF-β1/Smad2/3 pathway[J].Journal of Pharmacy and Pharmacology, 2019, 71(6):1017-1028.
[11] ZHANG C, YUAN Y, OU M.Mangiferin attenuates cigarette smoke-induced chronic obstructive pulmonary disease in male albino rats[J].Microvascular Research, 2021, 138:104208.
[12] ZHANG D, HAN S, ZHOU Y, et al.Therapeutic effects of mangiferin on sepsis-associated acute lung and kidney injuries via the downregulation of vascular permeability and protection of inflammatory and oxidative damages[J].European Journal of Pharmaceutical Sciences, 2020, 152:105400.
[13] WANG H, ZHU Y Y, WANG L, et al.Mangiferin ameliorates fatty liver via modulation of autophagy and inflammation in high-fat-diet induced mice[J].Biomedicine & Pharmacotherapy, 2017, 96:328-335.
[14] HOU J, ZHENG D, XIAO W, et al.Mangiferin enhanced autophagy via inhibiting mTORC1 pathway to prevent high glucose-induced cardiomyocyte injury[J]. Frontiers in Pharmacology, 2018, 9:383.
[15] SUCHAL K, MALIK S, GAMAD N, et al.Mangiferin protect myocardial insults through modulation of MAPK/TGF-β pathways[J]. European Journal of Pharmacology, 2016, 776:34-43.
[16] LIU Y W, ZHU X, ZHANG L, et al.Up-regulation of glyoxalase 1 by mangiferin prevents diabetic nephropathy progression in streptozotocin-induced diabetic rats[J].European Journal of Pharmacology, 2013, 721(1-3):355-364.
[17] DING L Z, TENG X, ZHANG Z B, et al.Mangiferin inhibits apoptosis and oxidative stress via BMP2/Smad-1 signaling in dexamethasone-induced MC3T3-E1 cells[J].International Journal of Molecular Medicine, 2018, 41(5):2517-2526.
[18] WANG Y, CHEN Y, DU H, et al.Comparison of the anti-Duck hepatitis A virus activities of phosphorylated and sulfated Astragalus polysaccharides[J].Experimental Biology and Medicine, 2017, 242(3):344-353.
[19] SIES H.Oxidative stress:A concept in redox biology and medicine[J].Redox Biology, 2015, 4:180-183.
[20] VAIRETTI M, DI PASQUA L G, CAGNA M, et al.Changes in glutathione content in liver diseases:An update[J].Antioxidants (Basel, Switzerland), 2021, 10(3):364.
[21] IRSHAD M, CHAUDHURI P S, JOSHI Y K.Superoxide dismutase and total anti-oxidant levels in various forms of liver diseases[J]. Hepatology Research, 2002, 23(3):178-184.
[22] MAO S, OU X, WANG M, et al.Duck hepatitis A virus 1 has lymphoid tissue tropism altering the organic immune responses of mature ducks[J]. Transboundary and Emerging Diseases, 2021, 68(6):3588-3600.
[23] ABDERRAZAK A, SYROVETS T, COUCHIE D, et al.NLRP3 inflammasome:From a danger signal sensor to a regulatory node of oxidative stress and inflammatory diseases[J]. Redox Biology, 2015, 4:296-307.
[24] NISHIKAWA T, ARAKI E.Impact of mitochondrial ROS production in the pathogenesis of diabetes mellitus and its complications[J]. Antioxidants & Redox Signaling, 2007, 9:343-353.
[25] LI R, LIN J, HOU X, et al.Characterization and roles of Cherry Valley duck NLRP3 in innate immunity during avian pathogenic Escherichia coli infection[J]. Frontiers in Immunology, 2018, 9:2300.
[26] SUTTERWALA F S, OGURA Y, SZCZEPANIK M, et al.Critical role for NALP3/CIAS1/cryopyrin in innate and adaptive immunity through its regulation of Caspase-1[J].Immunity, 2006, 24(3):317-327.
[27] CRAVEN R R, GAO X, ALLEN I C, et al.Staphylococcus aureus α-hemolysin activates the NLRP3-inflammasome in human and mouse monocytic cells[J].PLoS One, 2009, 4(10):e7446.
[28] YEN H, SUGIMOTO N, TOBE T.Enteropathogenic Escherichia coli uses NleA to inhibit NLRP3 inflammasome activation[J].PLoS Pathogens, 2015, 11(9):e1005121.
[29] ZHOU R B, YAZDI A S, MENU P, et al.A role for mitochondria in NLRP3 inflammasome activation[J]. Nature, 2011, 469(7329):221-225.
[30] 张蕾, 赵国发, 李楠, 等.芒果苷通过作用于NLRP3炎症小体发挥抗抑郁和神经保护作用[J].河北大学学报(自然科学版), 2021, 41(3):265-272. ZHANG L, ZHAO G F, LI N, et al.Mangiferin reduces stress induced depressive-like behavior and exerts a neuroprotective effect by inhibition of the NLRP3 inflammasome[J].Journal of Hebei University (Natural Science Edition), 2021, 41(3):265-272.(in Chinese)
[31] 党双双, 易甫, 武锋, 等.芒果苷通过PI3K/Akt/mTOR通路抑制缺氧缺血性脑损伤大鼠神经细胞凋亡及炎症反应[J].现代生物医学进展, 2020, 20(10):1820-1823. DANG S S, YI F, WU F, et al.Mangiferin inhibits neuronal apoptosis and inflammatory response in rats with hypoxic-ischemic brain injury via PI3K/Akt/mTOR pathway[J].Progress in Modern Biomedicine, 2020, 20(10):1820-1823.(in Chinese)