富二十二碳六烯酸裂殖壶藻藻油对过氧化氢诱导的小肠上皮细胞抗炎和抗氧化系统的影响

Effects of Docosahexaenoic Acid-Enriched Schizochytrium Oil on Anti-Inflammatory and Antioxidant Systems of Hydrogen Peroxide-Induced Intestinal Epithelial Cells

本研究以大鼠小肠上皮细胞(IEC-6细胞)为研究对象,探究二十二碳六烯酸(DHA)和富DHA裂殖壶藻藻油(SZO)预防和缓解过氧化氢(H_(2)O_(2) )诱导的IEC-6细胞氧化损伤的影响。首先添加0、5、10、20、40、80μg/mL的DHA和SZO培养IEC-6细胞24和48 h,通过四氮甲基唑蓝(MTT)细胞活力检测试剂盒检测细胞增殖指数,确定最适添加浓度和培养时间。然后在H_(2)O_(2) 诱导前后以最适添加浓度添加DHA和SZO至细胞培养基中进行培养。培养结束后,收集细胞和培养基上清,检测细胞的抗氧化指标和炎性因子表达情况,以及细胞内核因子E2相关因子2(Nrf2)和转录核因子-κB(NF-κB)信号通路蛋白表达情况。结果显示:DHA最适添加浓度和培养时间分别为40μg/mL和24 h。DHA和SZO处理提高了H_(2)O_(2) 诱导的IEC-6细胞总抗氧化能力和抗氧化酶(超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶)活性,并且降低了活性氧和丙二醛含量,且缓解效果的抗氧化酶活性显著高于预防效果(P<0.05)。免疫印迹结果显示,DHA和SZO处理降低了H_(2)O_(2) 诱导的IEC-6细胞NF-κB、肿瘤坏死因子-α(TNF-α)和白细胞介素-1β(IL-1β)蛋白表达量,提高了Nrf2蛋白表达量。此外,SZO处理的缓解效果使IEC-6细胞TNF-α蛋白表达量显著低于预防效果(P<0.05)。综上所述,DHA和SZO通过激活Nrf2和抑制NF-κB信号通路增强细胞抗氧化和抗炎能力,预防和缓解了H_(2)O_(2) 引起的IEC-6细胞的氧化应激,且SZO对氧化损伤的缓解效果优于预防效果。

In the present study,the rat intestinal epithelial cells(IEC-6 cells)were investigated to evaluate the effects of docosahexaenoic acid(DHA)and DHA-enriched Schizochytrium oil(SZO)on the prevention and alleviation of oxidative damage induced by hydrogen peroxide(H_(2)O_(2) )in IEC-6 cells.At first,the IEC-6 cells were incubated with 0,5,10,20,40 and 80μg/mL of DHA and SZO for 24 and 48 h to screen the optimal additive concentration and culture time by measuring the cell proliferation index with methyl thiazolyl tetrazolium(MTT)cell viability test kit.After that,in the H_(2)O_(2) induction before and after,the DHA and SZO were added to the cell culture medium with optimal additive concentration.At the end of the culture,the cells and medium supernatant were collected to test the antioxidant index and inflammatory factors expression level,as well as intracellular nuclear factor E2-related factor 2(Nrf2)and nuclear factor-κB(NF-κB)signaling pathways expression.The results showed that the optimal additive concentration and culture time of DHA were 40μg/mL and 24 h,respectively.The DHA and SZO treatment increased the total antioxidant capacity and activities of antioxidant enzymes(superoxide dismutase,catalase and glutathione peroxidase)of H_(2)O_(2) -induced IEC-6 cells,decreased the contents of reactive oxygen species and malondialdehyde,and the antioxidant enzyme activities of alleviation effect were significantly higher than those of prevention effect(P<0.05).The Western blot results indicated that the DHA and SZO treatment decreased the protein expression levels of NF-κB,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)of H_(2)O_(2) -induced IEC-6 cells,and increased the Nrf2 protein expression level.Otherwise,the TNF-αprotein expression level of alleviation effect of SZO treatment was significantly lower than that of prevention effect(P<0.05).In summary,DHA and SZO can enhance antioxidant and anti-inflammatory ability by activating Nrf2 and inhibiting NF-κB signaling pathways,to prevent and alleviate H_(2)O_(2) -induced oxidative stress in IEC-6 cells,and the alleviation effect against oxidative damage of SZO is higher than the preventive effect.