中华眼底病杂志

中华眼底病杂志

叔丁基对苯二酚对高糖培养视网膜Müller细胞核因子E2相关因子2、血红素氧合酶1和磷脂酰肌醇-3激酶表达的影响

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目的观察叔丁基对苯二酚(tBHQ)对高糖环境下视网膜Müller细胞中核因子E2相关因子2(Nrf2)、血红素氧合酶1(HO-1)、磷脂酰肌醇-3激酶(PI3K)的表达影响;初步探讨tBHQ的抗氧化及抗凋亡作用。方法大鼠视网膜Müller细胞分为正常糖组(5.5 mmol/L)(N组)、高糖组(45.0 mmol/L)(HG组)、tBHQ干预组(HG+tBHQ组)。HG+tBHQ组Müller细胞培养48 h后,加入tBHQ 20 μmol/L预处理剂诱导Nrf2和HO-1的表达。免疫荧光染色法鉴定Müller细胞;蛋白免疫印迹法和实时荧光定量聚合酶链反应检测各组Müller细胞中Nrf2、HO-1、PI3K、B淋巴细胞瘤-2(Bcl-2)、Bax蛋白和基因的表达;流式细胞仪检测各组Müller细胞的凋亡。结果培养的Müller细胞细胞体大,细胞浆丰富;细胞核呈圆形或卵圆形,边界清晰。HG组Müller细胞中Nrf2(t=4.114)、HO-1(t=9.275)蛋白表达较N组升高,差异有统计学意义(P=0.006、0.000)。HG+tBHQ组Müller细胞中Nrf2(t=7.847)、HO-1(t=7.947)、PI3K(t=5.397)、Bcl-2(t=6.825)蛋白表达较HG组升高,差异有统计学意义(P=0.000、0.000、0.002、0.000);Bax蛋白表达较HG组降低,差异有统计学意义(t=14.998、P=0.000)。HG组Müller细胞中Nrf2(t=7.292)、HO-1(t=15.014)mRNA较N组升高,差异有统计学意义(P=0.000、0.000)。HG+tBHQ组Müller细胞中Nrf2(t=18.046)、HO-1(t=39.458)、PI3K(t=4.979)、Bcl-2(t=9.535)mRNA较HG组升高,差异有统计学意义(P=0.000、0.000、0.003、0.000);Bax mRNA较HG组降低,差异有统计学意义(t=16.520、P=0.000)。HG组Müller细胞凋亡率较N组增高,差异有统计学意义(t=39.905、P=0.000);HG+tBHQ组Müller细胞凋亡率较HG组降低,差异有统计学意义(t=21.083、P=0.000)。结论tBHQ通过上调视网膜Müller细胞中Nrf2、HO-1、PI3K的表达,抑制Müller细胞的凋亡。

ObjectiveTo observe the effect of tert-Butylhydroquinone (tBHQ) on the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase (HO)-1 and phosphatidylinositol 3-kinase (PI3K) in high glucose cultured retinal Müller cells; and to investigate the anti-oxidative stress and anti-apoptotic effects of tBHQ.MethodsRetinal Müller cells were divided into normal glucose group (5.5 mmol/L, N group), high glucose group (45 mmol/L, HG group) and tBHQ intervention group (HG+tBHQ group). After retinal Müller cells were cultured with high glucose for 48 hours, the pretreatment with tBHQ (20 μmol/L) induced the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. The Müller cells were identified by immunofluorescence staining. The expressions of Nrf2, HO-1, PI3K, B-cell lymphoma-2 (Bcl-2) and Bax were detected by Western blot and real-time fluorescence quantitative PCR. Flow cytometry was used to detect the apoptosis of retinal Müller cells in rats.ResultsMüller cytoplasm and nucleus GS showed strong positive, large cell body, abundant cytoplasm, uniform green fluorescence; nuclear DAPI staining round or oval, clear boundary. The expression of Nrf2 protein (t=4.114, P=0.006), HO-1 protein (t=9.275, P=0.000), Nrf2 mRNA (t=7.292, P=0.000) and HO-1 mRNA (t=15.014, P=0.000) in the HG group were higher than those in the N group. The expressions of Nrf2 protein (t=7.847, P=0.000) ,HO-1 protein (t=7.947, P=0.000), PI3K protein (t=5.397, P=0.002), Bcl-2 protein (t=6.825, P=0.000), Nrf2 mRNA (t=18.046, P=0.000), HO-1 mRNA (t=39.458, P=0.000), PI3K mRNA (t=4.979, P=0.003) and Bcl-2 mRNA (t=9.535, P=0.000) in the HG+tBHQ group were significantly higher than those in the HG group. The protein and mRNA expressions of Bax protein in the HG+tBHQ group were significantly lower than those in the HG group (t=14.998, 16.520; P=0.000, 0.000). Flow cytometry showed that the apoptosis rate of Müller cells in the HG group was significantly higher than that in the N group (t=39.905, P=0.000). The apoptosis rate of Müller cells in the HG+tBHQ group was significantly lower than that in the HG group (t=21.083, P=0.000).ConclusiontBHQ can inhibit the apoptosis of retinal Müller cells by up-regulating the expression of Nrf2, HO-1 and PI3K.

关键词: 氢醌类; NF-E2相关因子2; 血红素氧化酶(脱环); 磷酸肌醇3-激酶; Müller细胞

Key words: Hydroquinones; NF-E2-related factor 2; Heme oxygenase (decyclizing); Phosphatidylinositol 3-kinase; Müller cell

引用本文: 田敏, 吴进川, 何薇, 余曦, 吕红彬. 叔丁基对苯二酚对高糖培养视网膜Müller细胞核因子E2相关因子2、血红素氧合酶1和磷脂酰肌醇-3激酶表达的影响. 中华眼底病杂志, 2018, 34(4): 382-387. doi: 10.3760/cma.j.issn.1005-1015.2018.04.015 复制

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