基于Nrf2通道研究柚皮素磷脂复合物对氧化损伤ARPE-19细胞的保护作用

Protective effects of naringin phospholipid complex on oxidative injury in ARPE-19 cells associated with activation of Nrf2 pathway

摘要:

目的：研究柚皮素(Nar)及其磷脂复合物(NPC)对叔丁基氢过氧化物(t-BHP)诱导产生的氧化损伤人视网膜色素上皮细胞(ARPE-19细胞)的保护作用及其作用机制。

方法：根据文献最佳制作工艺制备NPC。将体外培养的ARPE-19细胞分为溶媒组(用DMSO培养)、模型组(用200μmol/L t-BHP干预)、Nrf2-siRNA组(针对Nrf2基因进行细胞转染)、柚皮素组(200μmol/L柚皮素培养基预处理后加入200μmol/L t-BHP)、NPC组(200μmol/L NPC培养基预处理后加入200μmol/L t-BHP)、Nrf2-siRNA+柚皮素组(200μmol/L柚皮素预处理后Nrf2基因干扰，再加入200μmol/L t-BHP)、Nrf2-siRNA+NPC组(200μmol/L NPC预处理后Nrf2基因干扰，再加入200μmol/L t-BHP)。检测各组细胞内活性氧、丙二醛、超氧化物歧化酶、总抗氧化能力水平，分别采用RT-PCR和Western blot法检测各组细胞内血红素加氧酶-1(HO-1)、醌氧化还原酶-1(NQO-1)、谷氨酸半胱氨酸连接酶(GCL)和核因子E2相关因子2(Nrf2)的表达水平。

结果：NPC较柚皮素能明显降低ARPE-19细胞内丙二醛、活性氧的含量，提高细胞内超氧化物歧化酶、总抗氧化能力水平。柚皮素和NPC预保护ARPE-19 细胞后，Nrf2、HO-1、NQO-1和GCL mRNA的相对表达量和蛋白表达水平均较模型组和Nrf2-siRNA组升高。柚皮素组与NPC组，Nrf2-siRNA+柚皮素组与Nrf2-siRNA+NPC组细胞内4种基因和蛋白相对表达水平均有差异。Nrf2-siRNA+柚皮素组与Nrf2-siRNA组Nrf2、HO-1、NQO-1蛋白表达无显著差异。Nrf2-siRNA+NPC组与Nrf2-siRNA组4种蛋白表达均有差异，NPC作用明显强于柚皮素。

结论：柚皮素磷脂复合物能明显提高细胞内抗氧化能力，降低氧化水平，其通过激活Nrf2/ARE抗氧化应激通路上调Nrf2及其下游抗氧化酶和Ⅱ相解毒酶的表达对氧化损伤的ARPE-19细胞起到更好的保护作用。

Abstract:

AIM:To investigate the protective effects of naringin(Nar)phospholipid complex(NPC)on oxidative injury in retinal pigment epithelium cells(ARPE-19 cells)induced by tert-butyl hydroperoxide(t-BHP)and elucidate the underlying mechanism.

METHODS:The NPC was prepared by solvent method. Experimental cells are divided into seven groups: control group \〖cultured with dimethylsulfoxide(DMSO)\〗, model group(intervention with 200μmol/L t-BHP), nuclear factor erythroid 2-related factor 2(Nrf2)-siRNA group(cell transfection for Nrf2 gene), naringin group(add 200μmol/L t-BHP after pretreatment with 200μmol/L naringin medium), NPC group(add 200μmol/L t-BHP after pretreatment with 200μmol/L NPC medium), Nrf2-siRNA+ naringin group(after 200μmol/L naringin pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP)and Nrf2-siRNA+ NPC group(after 200μmol/L NPC pretreatment, Nrf2 gene interference, then add 200μmol/L t-BHP). The intracellular levels of superoxide dismutase(SOD), malondialdehyde(MDA)and total antioxidant capacity(T-AOC)were detected, intracellular level of reactive oxygen species(ROS)was detected by DCFH-DA staining method. The mRNA and protein expressions of HO-1, NQO-1, GCL and Nrf2 were detected by real-time PCR and western blot, respectively.

RESULTS:NPC more significantly increased the levels of SOD and T-AOC, reduced the contents of ROS and MDA than naringin in t-BHP-treated ARPE-19 cells. After naringin and NPC pre-protected ARPE-19 cells, the relative expression and protein expression of Nrf2, HO-1, NQO-1 and GCL mRNA were higher than those of the model group and Nrf2-siRNA group. There were statistically significant differences in the relative expression of 4 genes and the expression levels of 4 proteins in the naringin group and the NPC group, the Nrf2-siRNA+naringin group and the Nrf2-siRNA+NPC group. The expression of Nrf2, HO-1 and NQO-1 protein in the Nrf2-siRNA+naringin group was not significantly different than that in the Nrf2-siRNA group. Compared with the Nrf2-siRNA group, the expression of 4 proteins in the Nrf2-siRNA+NPC group was statistically significant, and the effect of NPC was significantly stronger than that of naringin.

CONCLUSION: After naringin forms a phospholipid complex, it can significantly increase the antioxidant capacity in cells and reduce the oxidation level. It up-regulates the expression of Nrf2 and its downstream antioxidant enzymes and phase Ⅱ detoxification enzymes by activating the Nrf2/ARE antioxidative stress pathway to better protect ARPE-19 cells from oxidative damage.