T-2毒素致猪肾细胞损伤及氧化应激相关机理的研究

doi:10.16431/j.cnki.1671-7236.2021.12.039

摘要/Abstract

摘要： 试验旨在研究T-2毒素对猪肾细胞(PK15)的毒性作用及其氧化应激反应。用不同浓度的T-2毒素处理细胞,通过MTT法检测细胞活性、测定细胞内乳酸脱氢酶(LDH)释放率及苏木素伊红(HE)染色观察细胞形态变化评估T-2毒素对细胞的毒性作用;通过检测细胞内的活性氧(ROS)水平、丙二醛(MDA)含量及谷胱甘肽(GSH)含量、谷胱甘肽过氧化物酶(GPx)活性,评估不同剂量T-2毒素对细胞氧化应激水平的影响;检测Nrf2-ARE信号通路相关基因Nrf2、Keap1、GPx-1、Nqo1及Hmox1 mRNA的表达水平,分析T-2毒素对该信号通路的影响。结果表明,PK15细胞活性呈毒素剂量依赖性下降(P<0.05),LDH释放率呈毒素剂量依赖性上调(P<0.05);随T-2毒素剂量升高,细胞间隙逐渐增加,细胞固缩,细胞数量也随之减少。细胞内ROS阳性细胞率呈剂量依赖性升高,且T-2毒素为100 nmol/L时,ROS阳性细胞率显著高于其他剂量组(P<0.05);细胞内MDA含量与GPx活性呈毒素剂量依赖性升高(P<0.05),而GSH含量呈毒素剂量依赖性下降(P<0.05)。20、50及100 nmol/L的T-2毒素可显著上调Keap1、Gpx-1及Nqo1基因mRNA的相对表达量(P<0.05),且50 nmol/L的相对表达量最高。T-2毒素对K15细胞有毒性作用,可诱导其氧化损伤,且该氧化应激过程受Nrf2-AER信号通路调控。

关键词: T-2毒素; PK15细胞; 毒性作用; 氧化应激; Nrf2-ARE通路

Abstract: The purpose of this test was to study the toxic effect of T-2 toxin on porcine kidney cells (PK15) and its oxidative stress response.The cells were treated with different concentrations of T-2 toxin.The cytotoxicity of the toxin was evaluated by MTT assay, intracellular lactate dehydrogenase (LDH) release rate and HE staining.The effects of different doses of T-2 toxin on the level of oxidative stress were evaluated by detecting the levels of reactive oxygen species (ROS), malondialdehyde (MDA), total glutathione (GSH) and total glutathione peroxidase (GPx).The expression levels of Nrf2, Keap1, GPx-1, Nqo1 and Hmox1 genes mRNA related to Nrf2-ARE signal pathway were detected, and the effect of T-2 toxin on this signal pathway was analyzed.The results showed that the activity of PK15 cells decreased in a toxin dose-dependent manner (P<0.05), and the release rate of LDH increased in a toxin dose-dependent manner (P<0.05).With the increase of T-2 toxin dose, the cell gap gradually increased, the cell pyknosis and the number of cells decreased.The rate of ROS positive cells increased in a dose-dependent manner, and when T-2 toxin was 100 nmol/L, the rate of ROS positive cells was significantly higher than that in other dose groups (P<0.05).The intracellular MDA content and GPx activity increased in a toxin dose-dependent manner (P<0.05), while the GSH content decreased in a toxin dose-dependent manner (P<0.05).20, 50 and 100 nmol/L T-2 toxin could significantly up regulate the relative expression of Keap1, GPx-1 and Nqo1 genes mRNA (P<0.05), and the relative expression of 50 nmol/L was the highest.T-2 toxin caused toxic effects on PK15 cells and induced oxidative damage, and the oxidative stress process was regulated by Nrf2-AER signal pathway.

Key words: T-2 toxin; PK15 cells; toxicity; oxidative stress; Nrf2/ARE pathway

中图分类号:

S859.87

熊款款, 吴映欣, 易思亮, 杨凌宸. T-2毒素致猪肾细胞损伤及氧化应激相关机理的研究[J]. 中国畜牧兽医, 2021, 48(12): 4710-4717.

XIONG Kuankuan, WU Yingxin, YI Siliang, YANG Lingchen. Mechanism of T-2 Toxin Induced Injury and Oxidative Stress in PK15 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4710-4717.

参考文献

[1] KALANTARI H,MOOSAVI M.Review on T-2 toxin[J].Jundishapur Journal of Natural Pharmaceutical Products,2018,5(1):26-38.
[2] 窦彩霞,王倩,安建勇,等.Keap1-Nrf2/ARE信号通路及其在畜禽抗氧化中的研究进展[J].中国畜牧兽医,2019,46(9):2567-2574. DOU C X,WANG Q,AN J Y,et al.The reasearch progress of Keap1-Nrf2/ARE signaling pathway and its role in the antioxidation of livestock and poultry[J].China Animal Husbandry & Veterinary Medicine,2019,46(9):2567-2574.(in Chinese)
[3] 黄珂,温晓鹿,王丽,等.霉菌毒素对畜禽的危害及削减方法研究进展[J].中国畜牧兽医,2019,46(9):2600-2607. HUANG K,WEN X L,WANG L,et al.Progress research on the hazard of mycotoxins to livestock and poultry and the methods of reduction[J].China Animal Husbandry & Veterinary Medicine,2019,46(9):2600-2607.(in Chinese)
[4] YANG L,TU D,ZHAO Z,et al.Cytotoxicity and apoptosis induced by mixed mycotoxins (T-2 and HT-2 toxin) on primary hepatocytes of broilers in vitro[J].Toxicon,2017,129(3):1-10.
[5] LI Y,WANG Z,BEIER R C,et al.T-2 toxin,a trichothecene mycotoxin:Review of toxicity,metabolism,and analytical methods[J].Journal of Agricultural and Food Chemistry,2011,59(8):3441-3453.
[6] SARDARO N,DELLA F,INCALZA M,et al.Oxidative stress and oral mucosal diseases:An overview[J].In Vivo,2019,33(2):289-296.
[7] KOSURU R,KANDULA V,RAI U,et al.Pterostilbene decreases cardiac oxidative stress and inflammation via activation of AMPK/Nrf2/HO-1 pathway in fructose-fed diabetic rats[J].Cardiovascular Drugs and Therapy,2018,32(2):147-163.
[8] JIA Z,LI S,QIAO W,et al.Ebselen protects mitochondrial function and oxidative stress while inhibiting the mitochondrial apoptosis pathway after acute spinal cord injury[J].Neuroscience Letters,2018,678(2):110-117.
[9] DAI C,XIAO X,SUN F,et al.T-2 toxin neurotoxicity:Role of oxidative stress and mitochondrial dysfunction[J].Archives of Toxicology,2019,93(11):3041-3056.
[10] EL GOLLI E,HASSEN W,BOUSLIMI A,et al.Induction of Hsp 70 in Vero cells in response to mycotoxins cytoprotection by sub-lethal heat shock and by vitamin E[J].Toxicology Letters,2006,166(2):122-130.
[11] ZHANG Y F,YANG J Y,LI Y K,et al.Toxicity and oxidative stress induced by T-2 toxin in cultured mouse Leydig cells[J].Toxicology Mechanisms and Methods,2017,27(2):100-106.
[12] CHEN Y, HAN S, WANG Y,et al.Oxidative stress and apoptotic changes in broiler chicken splenocytes exposed to T-2 toxin[J].BioMed Research International,2019,2019(2):5493870.
[13] YANG L,ZHAO Z,WU A,et al.Determination of trichothecenes A (T-2 toxin,HT-2 toxin,and diacetoxyscirpenol) in the tissues of broilers using liquid chromatography coupled to tandem mass spectrometry[J].Journal of Chromatography B,2013,942(2):88-97.
[14] 孙暾,王迎,代重山,等.T2毒素对N2a细胞的细胞毒性作用及其毒作用机制研究[A].中国毒理学会第九次全国毒理学大会论文集[C].2019. SUN T,WANG Y,DAI Z S,et al.The cytotoxic effect of T2 toxin on N2a cells and its mechanism[A].Proceedings of the 9th National Congress of Toxicology of the Chinese Society of Toxicology[C].2019.(in Chinese)
[15] 晋圣坤.白藜芦醇稳定性及与T2毒素对HepG2细胞作用的研究[D].重庆:西南大学,2012. JIN S K.Studies on the stability of resveratrol and the effect of T2 toxin on HepG2 cells[D].Chongqing:Southwest University,2012.(in Chinese)
[16] WANG Y,LIU S,ZHENG H,et al.T-2 toxin,zearalenone and fumonisin B₁ in feedstuffs from China[J].Food Additives & Contaminants Part B,Surveillance,2013,6(2):116-122.
[17] 杨杰,王克维.T-2毒素和黄绿青霉素的研究进展[J].中华地方病学杂志,2021,40(1):81-86. YANG J,WANG K W.Research progress on toxic effect and toxic mechanism of T-2 toxin[J].Chinese Journal of Endemiology,2021,40(1):81-86.(in Chinese)
[18] HUANG D,CUI L,SAJID A,et al.The epigenetic mechanisms in Fusarium mycotoxins induced toxicities[J].Food and Chemical Toxicology,2019,12(3):595-601.
[19] ABASSI H,AYED I,SHIRLEY S,et al.Ochratoxin a and T-2 toxin induce clonogenicity and cell migration in human colon carcinoma and fetal lung fibroblast cell lines[J].Journal of Biochemical and Molecular Toxicology,2016,30(3):128-135.
[20] MAKOWSKA K,OBREMSKI K,GONKOWSKI S.The impact of T-2 toxin on vasoactive intestinal polypeptide-like immunoreactive (VIP-LI) nerve structures in the wall of the porcine stomach and duodenum[J].Toxins,2018,10(4):123-129.
[21] TARONCHER M,YELKO R C,RUIZ M.Interactions between T-2 toxin and its metabolites in HepG2 cells and in silico approach[J].Food and Chemical Toxicology,2020,148:111942.
[22] RAKKESTAD K E,SKAAR I,ANSTEINSSON V E,et al.DNA damage and DNA damage responses in THP-1 monocytes after exposure to spores of either Stachybotrys chartarum or Aspergillus versicolor or to T-2 toxin[J].Toxicological Sciences,2010,115(1):140-194.
[23] HE S J,HOU J F,DAI Y Y,et al.N-acetyl-cysteine protects chicken growth plate chondrocytes from T-2 toxin-induced oxidative stress[J].Journal of Applied Toxicology,2012,32(12):980-985.
[24] BOUAZIZ C,ABID-ESSEFI S,BOUSLIMI A,et al.Cytotoxicity and related effects of T-2 toxin on cultured Vero cells[J].Toxicon,2006,48(3):343-352.
[25] YANG L,TU D,WANG N,et al.The protective effects of DL-selenomethionine against T-2/HT-2 toxins-induced cytotoxicity and oxidative stress in broiler hepatocytes[J].Toxicology in Vitro,2019,5(4):137-146.
[26] CHAUDHARI M,JAYARAJ R,BHASKAR A S B,et al.Oxidative stress induction by T-2 toxin causes DNA damage and triggers apoptosis via caspase pathway in human cervical cancer cells[J].Toxicology,2009,262(2):153-161.
[27] AW T Y.Intestinal glutathione:Determinant of mucosal peroxide transport,metabolism,and oxidative susceptibility[J].Toxicology and Applied Pharmacology,2005,204(3):320-327.
[28] TSUTSUI H,KINUGAWA S,MATSUSHIMA S.Mitochondrial oxidative stress and dysfunction in myocardial remodelling[J].Cardiovascular Research,2009,81(3):449-504.
[29] MA Q.Role of nrf2 in oxidative stress and toxicity[J].Annual Review of Pharmacology and Toxicology,2013,53(8):401-426.
[30] 姚琦,郑男,郭志欣,等.水生生物Nrf2-Keap1/Are信号通路的研究进展[J].中国畜牧兽医,2021,48(2):492-500. YAO Q,ZHENG N,GUO Z X,et al.Advances in the Nrf2-Keap1/Are signaling pathway in aquatic organism[J].China Animal Husbandry & Veterinary Medicine,2021,48(2):492-500.(in Chinese)
[31] STOCKER R,GLAZER A N,AMES B N.Antioxidant activity of albumin-bound bilirubin[J].Proceedings of the National Academy of Sciences of the United States of America,1987,84(16):5918-5922.