辣蓼黄酮正丁醇部位对PRV感染3D4/2细胞氧化应激相关因子水平的影响

doi:10.16431/j.cnki.1671-7236.2023.01.035

摘要/Abstract

摘要： 【目的】探究辣蓼黄酮正丁醇部位（N-butanol fraction of Polygonum hydropiper flavonoids，FNB）对猪伪狂犬病毒（Pseudoabies virus，PRV）体外感染猪肺泡巨噬细胞（3D4/2细胞）氧化应激相关因子的影响，旨在初步分析FNB的抗氧化效果。【方法】采用10^-1至10^-10浓度PRV体外感染猪肾细胞（PK15细胞），计算病毒半数组织培养感染剂量（TCID₅₀）；将3D4/2细胞分为BC组、DMSO组、PRV组、FNB 1、FNB2和FNB3组，BC组用DMEM培养液处理，其余各组用感染复数（multiplicity of infection，MOI）=0.1 RPV处理，孵育2 h后，PRV组添加DMEM培养液，DMSO组及FNB1、FNB2、FNB3组分别添加0.05% DMSO及12.5、25、50 μg/mL FNB的DMEM培养液。培养4、8、12、24 h后，分别收取各组细胞培养液上清和细胞，通过试剂盒测定细胞培养液上清中一氧化氮（NO）的含量、细胞内活性氧（ROS）、诱导型一氧化氮合酶（iNOS）、黄嘌呤氧化酶（XOD）、髓过氧化物酶（MPO）的活性和丙二醛（MDA）的含量。【结果】PRV的TCID₅₀为10^-8.25/0.1 mL；与BC组相比，PRV组上清中NO水平在4、8 h时极显著降低（P<0.01），在12、24 h时极显著升高（P<0.01），细胞内ROS在4 h时显著降低（P<0.05），8~24 h时显著或极显著升高（P<0.05；P<0.01），iNOS和XOD活性在8~24 h时显著或极显著升高（P<0.05；P<0.01），MPO活性在4~24 h时显著升高（P<0.05），MDA含量在4 h时显著降低（P<0.05），12~24 h时显著升高（P<0.05）。与PRV组相比，FNB2组NO含量在4~8 h时显著或极显著升高（P<0.05；P<0.01），在12 h时显著降低（P<0.05），FNB3组在24 h时极显著升高（P<0.01）；细胞内ROS水平在4 h时极显著升高（P<0.01），8~24 h时极显著降低（P<0.01）；FNB1组iNOS活性在4 h时显著升高（P<0.05），3个FNB组iNOS活性在8~24 h时显著或极显著升高（P<0.05；P<0.01），MPO和XOD活性在4~24 h时显著或极显著降低（P<0.05；P<0.01），MDA含量在4 h时升高，8 h时FNB2、FNB3组MDA含量显著降低（P<0.05），24 h时FNB1、FNB2组MDA含量显著降低（P<0.05）。【结论】FNB可以通过干预氧化应激相关因子的分泌调节细胞氧化应激水平，维持氧化与抗氧化的平衡，结果可为辣蓼黄酮正丁醇部位的开发应用提供理论依据。

关键词: 辣蓼黄酮正丁醇部位(FNB); 伪狂犬病毒(PRV); 3D4/2细胞; 氧化应激

Abstract: 【Objective】 This study was carried out to explore the intervention effect of N-butanol fraction of Polygonum hydropiper flavonoids (FNB) on oxidative stress in porcine lung-cell alveolar macrophages (3D4/2 cells) infected with Pseudoabies virus (PRV) in vitro, aiming to preliminarily explore and analyze the antioxidant effect and mechanism of FNB.【Method】 Porcine kidney cells (PK15 cells) were infected in vitro with PRV from 10^-10 to 10^-1 concentrations, and TCID₅₀ was calculated.The 3D4/2 cells were divided into BC, DMSO, PRV, FNB1, FNB2 and FNB3 groups. BC group was treated with DMEM medium only.The remain groups were treated with PRV which multiplicity of infection (MOI) was 0.1, after incubation for 2 h, DMEM medium was added to PRV group. DMSO group was supplemented with 0.05% DMSO, and FNB1, FNB2 and FNB3 groups were supplemented with 12.5, 25 and 50 μg/mL FNB, respectively.After cultured in vitro for 4, 8, 12 and 24 h, supernatants and cells were collected from each group separately, and then the content of nitric oxide (NO) in the supernatants, intracellular activity of reactive oxygen species (ROS), inducible nitric oxide synthase (iNOS), xanthine oxidase (XOD), myeloperoxidase (MPO) and intracellular content of malonaldehyde (MDA) at each time were determined by kits.【Result】 The results showed that the TCID₅₀ of PRV was 10^-8.25/0.1 mL.Compared with BC group, the level of NO in the supernatant of PRV group was extremely significantly decreased at 4 and 8 h and extremely significantly increased at 12 and 24 h (P<0.01), the content of intracellular ROS was significantly reduced at 4 h (P<0.05), and significantly or extremely significantly increased at 8-24 h (P<0.05 or P<0.01).The activities of iNOS and XOD were significantly or extremely significantly increased at 8-24 h (P<0.05 or P<0.01).The activity of MPO was significantly increased at 4-24 h (P<0.05).The content of MDA was significantly decreased at 4 h and significantly increased at 12-24 h (P<0.05).Compared with PRV group, the content of NO in FNB2 group was increased significantly or extremely significantly at 4-8 h (P<0.05 or P<0.01), and significantly reduced at 12 h (P<0.05), and was extremely significantly increased at 24 h in FNB3 group (P<0.01).The level of intracellular ROS was extremely significantly increased at 4 h (P<0.01), and extremely significantly decreased at 8-24 h (P<0.01).The activity of iNOS in FNB1 group was significantly increased at 4 h (P<0.05), and were significantly or extremely significantly increased at 8-24 h in the three FNB groups (P<0.05 or P<0.01).The activities of MPO and XOD were significantly or extremely significantly reduced at 4-24 h (P<0.05 or P<0.01).The content of MDA was significantly increased at 4 h and significantly increased at 8 h in FNB2 and FNB3 groups (P<0.05).The content of MDA were significantly reduced at 24 h in FNB1 and FNB2 groups (P<0.05).【Conclusion】 FNB could regulate the level of oxidative stress by interfering the secretion level of oxidative stress-related factors, and maintain the balance between oxidation and antioxidant.The results might provide a theoretical basis for the development and application of FNB.

Key words: N-butanol fraction of Polygonum hydropiper flavonoids (FNB); Pseudoabies virus (PRV); 3D4/2 cells; oxidative stress

中图分类号:

S853.75

缪卫媛, 张思琪, 黄昌巧, 赵怡, 陈奇, 谢小东, 韦英益, 于美玲, 胡庭俊. 辣蓼黄酮正丁醇部位对PRV感染3D4/2细胞氧化应激相关因子水平的影响[J]. 中国畜牧兽医, 2023, 50(1): 349-358.

MIAO Weiyuan, ZHANG Siqi, HUANG Changqiao, ZHAO Yi, CHEN Qi, XIE Xiaodong, WEI Yingyi, YU Meiling, HU Tingjun. Effect of N-butanol Fraction of Polygonum hydropiper Flavonoids on Levels Oxidative Stress-related Factors in 3D4/2 Cells Infected with PRV[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(1): 349-358.

参考文献

[1] GOU H, BIAN Z, CAI R, et al.RIPK3-dependent necroptosis limits PRV replication in PK-15 cells[J].Frontiers Microbiology, 2021, 12:664353.
[2] 任玉鹏, 纳吉, 向华, 等.一种猪伪狂犬病病毒gE基因TaqMan荧光定量PCR检测方法的建立[J].养猪, 2021, 6:109-113. REN Y P, NA J, XIANG H, et al.Development of a TaqMan fluorescent quantitative PCR detection method for porcine pseudorabies virus gE gene[J].Swine Production, 2021, 6:109-113.(in Chinese)
[3] ZHANG C, GUO L, JIA X, et al.Construction of a triple gene-deleted Chinese Pseudorabies virus variant and its efficacy study as a vaccine candidate on suckling piglets[J].Vaccine, 2015, 33(21):2432-2437.
[4] HUAN C, ZHOU Z, YAO J, et al.The antiviral effect of Panax notoginseng polysaccharides by inhibiting PRV adsorption and replication in vitro[J].Molecules, 2022, 27(4):1254.
[5] LIU Q, WANG X, XIE C, et al.A novel human acute encephalitis caused by Pseudorabies virus variant strain[J].Clinical Infectious Disease, 2021, 73(11):e3690-e3700.
[6] 吴裕红, 彭新宇, 张嘉慧, 等.枫蓼提取物成分分析及其主要成分黄酮对肠平滑肌的药效学研究[J].黑龙江畜牧兽医, 2021, 2:128-136. WU Y H, PENG X Y, ZHANG J H, et al.The analysis of Fengliao extracts components and pharmacodynamics research on its main component flavonoids to intestinal smooth muscle[J].Heilongjiang Animal Science and Veterinary, 2021, 2:128-136.(in Chinese)
[7] 沈冰冰, 王敏, 罗娟, 等.蓼属药用植物的化学成分及其药理活性研究进展[J].湖南中医药大学学报, 2015, 35(7):63-70. SHEN B B, WANG M, LUO J, et al.Research progress of chemical constituents and its pharmacological activities from the medicinal plants of Genus Polygonum[J].Journal of Hunan University of Chinese Medicine, 2015, 35(7):63-70.(in Chinese)
[8] 钟建青, 李波, 贾琦, 等.天然黄酮类化合物及其衍生物的构效关系研究进展[J].药学学报, 2011, 46(6):622-630. ZHONG J Q, LI B, JIA Q, et al.Advances in the structure-activity relationship study of natural flavonoids and its derivatives[J].Acta Pharmaceutica Sinica, 2011, 46(6):622-630.(in Chinese)
[9] 雷杰豪, 许爱娥.中草药黄酮类提取物对白癜风抗氧化应激作用机制的研究进展[J].中国中西医结合皮肤性病学杂志, 2018, 17(3):281-284. LEI J H, XU A E.Research progress on the antioxidative mechanism of flavonoids extracts about Chinese herbal medicine in vitiligo[J]. Chinese Journal of Dermatovenereology of Integrated Traditional and Western Medicine, 2018, 17(3):281-284.(in Chinese)
[10] 李化, 张明晓, 白羽琦, 等.基于黄酮类成分含量构成特征和体外抗病毒活性的不同产地黄芩的质-效评价研究[J].中国药房, 2020, 31(10):1166-1171. LI H, ZHANG M X, BAI Y Q, et al.Study on quality-efficiency evaluation of Scutellaria baicalensis from different origins based on content constituent characteristics of flavonoids and in vitro antiviral activity[J].China Pharmacy, 2020, 31(10):1166-1171.(in Chinese)
[11] 蔡孟成, 金永生.中药辣蓼的生物活性实验研究进展[J].药学服务与研究, 2020, 20(6):442-445. CAI M C, JIN Y S.Research progress in bioactivities of Polygonum flaccidum Meism[J].Pharmaceutical Care and Research, 2020, 20(6):442-445.(in Chinese)
[12] 罗文涓, 陶俊宇, 杨剑, 等.辣蓼黄酮对脂多糖诱导下RAW264.7细胞活性氧及炎性因子分泌的影响[J].动物医学进展, 2017, 38(8):1-6. LUO W J, TAO J Y, YANG J, et al.Effects of flavonoids from Polygonum hydropiper on levels of reactive oxygen species and inflammatory factors in RAW264.7 cells induced by lipopolysaccharide[J].Progress in Veterinary Medicine, 2017, 38(8):1-6.(in Chinese)
[13] 王良刚, 成静, 谢小东, 等.辣蓼黄酮提取物对南美白对虾血淋巴细胞内免疫相关活性的影响[J].现代畜牧兽医, 2021, 7:55-60. WANG L G, CHENG J, XIE X D, et al.Effect of flavone extract of Polygonum hydropiper L.on activities of immune related enzymes in blood lymphocytes of Penaeus vannamei[J]. Modern Journal of Animal Husbandry and Veterinary Medicine, 2021, 7:55-60.(in Chinese)
[14] 匡娜, 陶俊宇, 韦英益, 等.辣蓼黄酮的分离提取及抗炎作用的分子机制研究[A].中国畜牧兽医学会兽医药理毒理学分会第十五次学术讨论会论文集[C].2019. KUANG N, TAO J Y, WEI Y Y, et al.Isolation and extraction of Polygonum hydropiper L.and molecular mechanism of anti-inflammatory effect[A].Proceedings of the 15th Academic Symposium of Veterinary Pharmacological and Toxicology Branch of Chinese Animal Husbandry and Veterinary Society[C].2019.(in Chinese)
[15] 于美玲, 缪卫媛, 吴苑滢, 等.猪伪狂犬病病毒诱导猪肺泡巨噬细胞氧化应激和炎症反应研究[J].动物医学进展, 2022, 43(6):13-21. YU M L, MIAO W Y, WU Y Y, et al.Oxidative stress and inflammatoin induced by Pseudorabies virus infection in 3D4/2 cells[J]. Progress in Veterinary Medicine, 2022, 43(6):13-21.(in Chinese)
[16] WANG Q, XIE X, CHEN Q, et al.Effects of quercitrin on PRV-induced secretion of reactive oxygen species and prediction of lncRNA regulatory targets in 3D4/2 cells[J].Antioxidants (Basel), 2022, 11(4):631.
[17] LI T, PE T.Traditional Chinese herbal medicine as a source of molecules with antiviral activity[J].Antiviral Research, 2013, 97(1):1-9.
[18] 王爽, 刘星尧, 贾昊天, 等.硫化氢对顺铂致犬肾脏氧化损伤及炎症的缓解作用[J].中国畜牧兽医, 2022, 49(6):2376-2384. WANG S, LIU X Y, JIA H T, et al.Alleviative effect of hydrogen sulfide on cisplatin-induced kidney oxidative damage and inflammation in dogs[J].China Animal Husbandry & Veterinary Medicine, 2022, 49(6):2376-2384.(in Chinese)
[19] APELL K, HIRT H.Reactive oxygen species:Metabolism, oxidative stress, and signal transduction[J]. Annual Review of Plant Biology, 2004, 55:373-399.
[20] SANDER W J, FOURIE C, SABIU S, et al.Reactive oxygen species as potential antiviral targets[J].Review in Medical Virology, 2022, 32(1):2240.
[21] 陈泽琪, 傅佳民, 杨娣, 等.炎性细胞因子对滑膜细胞诱导型一氧化氮合酶和一氧化氮表达的作用及其机制研究[J].浙江医学, 2019, 41(17):1816-1820. CHEN Z Q, FU J M, YANG D, et al.Effects of inflammatory cytokines on expression of inducible nitric oxide synthase and nitric oxide in synovial cells and its mechanism[J].Zhejiang Medical Journal, 2019, 41(17):1816-1820.(in Chinese)
[22] GONG G, QIN Y, HUANG W, et al.Protective effects of diosgenin in the hyperlipidemic rat model and in human vascular endothelial cells against hydrogen peroxide-induced apoptosis[J].Chemico-Biological Interactions, 2010, 184(3):366-375.
[23] FANG J, MA L, ZHANG S, et al.Association of myeloperoxidase gene variation with carotid atherosclerosis in patients with essential hypertension[J].Molecular Medicine Reports, 2013, 7(1):313-317.
[24] 贾妮娜, 曹迷霞, 杨剑, 等.山豆根多糖对猪圆环病毒Ⅱ型感染猪肺泡巨噬细胞炎性因子水平的影响[J].中国畜牧兽医, 2021, 48(5):1825-1831. JIA N N, CAO M X, YANG J, et al.Effect of Sophora subprosrate polysaccharide on the levels of inflammatory factors in 3D4/2 cells infected with Porcine circovirus virus Ⅱ type[J].China Animal Husbandry & Veterinary Medicine, 2021, 48(5):1825-1831.(in Chinese)
[25] 尹丹, 谭红连, 郝祝兵, 等.猪圆环病毒2型诱导3D4/2细胞氧化应激模型的建立[J].动物医学进展, 2017, 38(3):16-21. YIN D, TAN H L, HAO Z B, et al.Establishment of oxidative stress model of 3D4/2 cells induced by PCV2 in vitro[J].Progress in Veterinary Medicine, 2017, 38(3):16-21.(in Chinese)
[26] 郑明, 陈征.影响药物作用的因素[J].中国兽药杂志, 2009, 43(5):57-58. ZHENG M, CHEN Z.Factors influencing the action of the drugs[J]. Chinese Journal of Veterinary Drug, 2009, 43(5):57-58.(in Chinese)
[27] LIU C W, LIN H W, YANG D J, et al.Luteolin inhibits viral-induced inflammatory response in RAW264.7 cells via suppression of STAT1/3 dependent NF-κB and activation of HO-1[J].Free Radical Biology Medicine, 2016, 95:180-189.
[28] 真国辉.菟丝子中黄酮类提取物的神经保护活性研究[D].大连:大连理工大学, 2006. ZHEN G H.Studies on neuroprotective activities of flavonoids extracts from Cuscuta chinensis[D].Dalian:Dalian Technology University, 2006.(in Chinese)
[29] 柏慧敏.沙棘黄酮对大鼠抗氧化与免疫功能作用的研究[D].哈尔滨:东北农业大学, 2009. BAI H M.Study of Seabuckthorn flavonoids on antioxidation and immune adjustment function of rats[D].Harbin:Northeastern Agricultural University, 2009.(in Chinese)
[30] 付远妨, 赵尉丹, 杨潇, 等.鸡血藤总黄酮对猪圆环病毒2型感染小鼠脾脏氧化应激的影响[J].中国畜牧兽医, 2017, 44(5):1526-1532. FU Y F, ZHAO W D, YANG X, et al.Effect of total flavonoids of Spatholobus suberectus Dunn on oxidative stress in mice spleen induced by PCV2[J].China Animal Husbandry & Veterinary Medicine, 2017, 44(5):1526-1532.(in Chinese)
[31] 姜恩平, 于春荣, 李贺, 等.北五味子总木脂素对PC12细胞氧化应激损伤的保护作用及其抑制NF-κB/iNOS/NO信号通路的机制[J].吉林大学学报(医学版), 2015, 41(3):532-536. JIANG E P, YU C R, LI H, et al.Protective effects of Schisandra Chinensis lignans on oxidative stress injury of PC12 cells and its mechanism of inhibition on NF-κB/iNOS/NO signaling pathway[J].Journal of Jilin University(Medicine Edition), 2015, 41(3):532-536.(in Chinese)
[32] 敬美莲, 伍卫红, 米健国, 等.不同品种黄花菜中黄酮类化合物的提取工艺及抗氧化活性研究[J].广东化工, 2022, 49(14):16-18. JING M L, WU W H, MI J G, et al.Study on extraction technology and antioxidant activity of flavonoids from different varieties of Hemerocallis fulva[J].Guangdong Chemical Industry, 2022, 49(14):16-18.(in Chinese)