番茄红素对赭曲霉毒素A诱导犬肾小管上皮细胞毒性损伤的缓解作用

Relieving effect of lycopene on ochratoxin A-induced cytotoxicity damage in Madin-Daby canine kidney cells

[目的]本试验以犬肾小管上皮细胞(Madin-Daby canine kidney cell,MDCK)为模型,研究赭曲霉毒素A(OTA)对MDCK的毒性作用以及番茄红素(lycopene,LYC)对细胞损伤的缓解效应。[方法]将对数生长期MDCK,随机分为对照组(无添加)、OTA组(15μmol·L^(-1) OTA)、LYC组(55μmol·L^(-1) LYC)、O+L组(15μmol·L^(-1) OTA+55μmol·L^(-1) LYC),处理36 h,研究OTA对MDCK细胞结构、细胞凋亡、肾功能相关指标、氧化与抗氧化、炎性因子等指标的影响,并探讨LYC在OTA致MDCK损伤中的缓解作用。[结果]OTA作用导致细胞发生明显皱缩,细胞数量骤减;电镜结果显示OTA能够导致细胞膜破裂、细胞器出现不同程度损伤,MDCK中乳酸脱氢酶(LDH)、活性氧(ROS)水平与丙二醛(MDA)含量极显著增加(P<0.01),超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)活性极显著降低(P<0.01);细胞中肾功能相关指标N-乙酰-β-D-氨基葡萄糖苷酶(NAG)、β2-微球蛋白(β2-MG)、肝脏型脂肪酸结合蛋白(L-FABP)、肾损伤分子1(KIM-1)、视黄醇结合蛋白(RBP)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)均极显著升高(P<0.01);同时极显著增加了MDCK的肿瘤坏死因子α(TNF-α)、干扰素6(IL-6)、干扰素18(IL-18)及干扰素1β(IL-1β)的水平(P<0.01);OTA可诱导MDCK凋亡率极显著升高(P<0.01);而LYC能够有效减缓OTA造成的损伤,LYC组与OTA处理组相比形态较饱满,细胞间衔接紧密,细胞凋亡率显著下降;LDH、ROS、MDA水平(含量)降低,SOD、CAT、GSH-Px活性升高,并降低炎性细胞因子的表达。[结论]OTA能够抑制MDCK活性,改变细胞形态,导致肾功能指标、氧化与抗氧化功能与细胞炎性因子异常,加快细胞凋亡;LYC能够显著缓解OTA对MDCK的损伤。

[Objectives]With Madin-Daby canine kidney cell(MDCK)as the model,the toxicity of ochratoxin A(OTA)on MDCK and the protective effect of lycopene(LYC)on cell injury were studied.[Methods]MDCK at logarithmic growth stage was randomly divided into control group(additive free),OTA group(15μmol·L^(-1) OTA),LYC group(55μmol·L^(-1) LYC),and O+L group(15μmol·L^(-1) OTA+55μmol·L^(-1) LYC),and treatment for 36 h.The effects of OTA on MDCK cell structure,apoptosis,renal function-related indexes,oxidation and antioxidant,inflammatory factors and other indicators were studied,and the mitigation effect of LYC on OTA-induced MDCK injury was investigated.[Results]OTA induced obvious shrinkage of cells and sharp decrease of cell number,and the results of electron microscopy showed that OTA could lead to cell membrane rupture and organelles damage to varying degrees,and the release levels of lactate dehydrogenase(LDH),reactive oxygen species(ROS)and the content of malondialdehyde(MDA)in MDCK significantly increased(P<0.01),and the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)significantly decreased(P<0.01).But LYC could effectively alleviate the damage caused by OTA.The levels of renal function related indexes including N-acetyl-β-D-glucosaminidase(NAG),β2-microglobulin(β2-MG),hepatic fatty acid binding protein(L-FABP),renal injury molecule-1(KIM-1),retinol binding protein(RBP)and neutrophil gelatinase-associated lipocalin(NGAL)significantly increased(P<0.01).At the same time,the levels of tumor necrosis factorα(TNF-α),interferon 6(IL-6),interferon 18(IL-18)and interferon 1β(IL-1β)in MDCK significantly increased(P<0.01).Apoptosis rate of MDCK was significantly increased by OTA(P<0.01),and LYC could effectively alleviate the damage caused by OTA.Compared with the OTA treatment group,the morphology of MDCK was more full,with tight intercellular connection and significantly lower apoptosis rate.The level or contents of LDH,ROS and MDA decreased,SOD,CAT and GSH-Px activities increased,and the expression of inflammatory cytokines decreased.[Conclusions]OTA could inhibit the cellular activity of MDCK,change the cell morphology,lead to renal function index,oxidation and antioxidant function and cell inflammatory factors abnormalities,and accelerate cell apoptosis.LYC could significantly alleviate the damage caused by OTA exposed to MDCK and have a certain mitigation ability.