陈诚, 陈雄, 张爱华. 金松双黄酮对砷致大鼠海马神经细胞衰老的干预效应[J]. 环境与职业医学, 2022, 39(5): 550-555. DOI: 10.11836/JEOM21587
引用本文: 陈诚, 陈雄, 张爱华. 金松双黄酮对砷致大鼠海马神经细胞衰老的干预效应[J]. 环境与职业医学, 2022, 39(5): 550-555. DOI: 10.11836/JEOM21587
CHEN Cheng, CHEN Xiong, ZHANG Aihua. Effect of sciadopitysin on sodium arsenite-induced senescence of rat hippocampal neurons[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 550-555. DOI: 10.11836/JEOM21587
Citation: CHEN Cheng, CHEN Xiong, ZHANG Aihua. Effect of sciadopitysin on sodium arsenite-induced senescence of rat hippocampal neurons[J]. Journal of Environmental and Occupational Medicine, 2022, 39(5): 550-555. DOI: 10.11836/JEOM21587

金松双黄酮对砷致大鼠海马神经细胞衰老的干预效应

Effect of sciadopitysin on sodium arsenite-induced senescence of rat hippocampal neurons

  • 摘要: 背景 长期砷暴露人群除表现出典型的皮肤损害体征外,往往还伴随神经行为异常症状和体征。

    目的 探讨金松双黄酮对亚砷酸钠致大鼠神经细胞衰老的干预效应以及细胞周期相关转录因子E2F1在其中可能的介导作用。

    方法 采用4 μmol·L−1亚砷酸钠作用于SH-SY5Y细胞24 h,并分别使用50 μg·mL−1银杏叶提取物EGb761及四种主要银杏叶双黄酮(异银杏双黄酮、7-去甲基银杏双黄酮、金松双黄酮和银杏双黄酮)干预24 h,采用CCK-8法测定细胞活性。将32只180~200 g SPF级大鼠随机分为对照组、染砷组(10 mg·L−1)、银杏叶提取物干预组(10 mg·kg−1)和金松双黄酮干预组(10 mg·kg−1),每组8只,雌雄各半。采用自由饮水方式进行染毒,连续染毒3个月;干预在染毒2个月后进行,采用灌胃的方式进行给药,干预1个月。采用HE染色法检测大鼠海马结构的改变,采用尼氏染色法检测海马形态及神经细胞数量的改变。此外,分别采用β-半乳糖苷酶(SA-β-gal)染色法和Western blotting法检测海马神经细胞的衰老情况及E2F1的表达水平。

    结果 与染砷组相比,银杏叶提取物和四种主要的银杏叶双黄酮均有不同程度的细胞活性恢复作用(均P<0.05),其中金松双黄酮相对于银杏叶提取物在拮抗亚砷酸钠神经细胞毒性方面具有更好的活性恢复作用(P<0.05)。HE染色和尼氏染色结果表明:与对照组相比,染砷组大鼠海马神经细胞明显减少且突触结构异常,细胞发生肿胀、核皱缩,出现空泡现象;与染砷组大鼠比较,银杏叶提取物干预组和金松双黄酮干预组大鼠海马神经细胞明显增多且突触结构较正常。SA-β-gal染色结果表明:与对照组(2.88±0.84)相比,染砷组(15.75±3.01)的衰老细胞数量明显增加(P<0.05);与染砷组相比,银杏叶提取物干预组(9.38±1.92)和金松双黄酮干预组(7.75±2.38)衰老细胞数量明显下降(均P<0.05)。Western blotting结果表明:与对照组(1.00±0.17)相比,染砷组(0.65±0.19)海马中E2F1蛋白表达明显降低(P<0.05);与染砷组相比,金松双黄酮干预组(0.89±0.18)海马中E2F1蛋白表达水平明显恢复(P<0.05);与银杏叶提取物干预组(0.68±0.19)相比,金松双黄酮(0.89±0.18)对E2F1表达水平的恢复效果较好(P<0.05)。相关性分析结果表明,E2F1蛋白表达水平与海马神经细胞SA-β-gal染色阳性率呈负相关(r=−0.518,P<0.05)。

    结论 金松双黄酮是银杏叶提取物的有效活性成分,其可有效抑制亚砷酸钠诱导的海马神经细胞衰老,E2F1可能在其中发挥重要的介导作用。

     

    Abstract: Background In addition to the typical signs of skin damage, long-term arsenic exposure is often accompanied by signs and symptoms of neurobehavioral abnormalities.

    Objective To investigate potential intervention effect of sciadopitysin on senescence of neurons induced by sodium arsenite in rats and possible underlying mediating effect of cell cycle-related transcription factor E2F1.

    Methods SH-SY5Y cells were treated with 4 μmol·L−1 sodium arsenite for 24 h and intervened with 50 μg·mL−1 Ginkgo biloba extract (EGb761) or four major biflavonoids in Ginkgo biloba leaves (isoginkgetin, bilobetin, sciadopitysin, and ginkgetin) for 24 h respectively. Then, cell viability was measured by CCK-8 assay. Thirty-two 180-200 g SPF rats were randomly divided into a control group, an arsenic treatment group (10 mg·L−1), a Ginkgo biloba extract intervention group (10 mg·kg−1), and a sciadopitysin intervention group (10 mg·kg−1), 8 rats in each group, half male and half female. The rats were treated with sodium arsenite by free drinking water for 3 consecutive months, and the intervention treatment was conducted after 2 months of poisoning with drug intake by gavage for 1 month. HE staining was used to detect structural changes in the hippocampus, while Nissl's staining was used to detect changes in hippocampal morphology and neuron numbers. Moreover, senescence-associated β galactosidase (SA-β-gal) staining and Western blotting were used to detect senescence of hippocampal neurons and the expression level of E2F1, respectively.

    Results Compared to the arsenic treatment group, EGb761 and the four biflavonoids in Ginkgo biloba leaves effectively antagonized the inhibitory effect of sodium arsenite on cell viability (all Ps<0.05), and sciadopitysin showed better restoration of cellular viability than Ginkgo biloba extract (P<0.05). The results of HE staining and Nissl's staining showed that the hippocampal neurons in the arsenic treatment group were reduced in cell count and the synaptic structure was abnormal, with swelling, nuclear shrinkage, and vacuole, compared with the control group. The results of SA-β-gal staining showed that the number of senescent cells in the arsenic treatment group (15.75±3.01) was significantly increased compared with the control group (2.88±0.84) (P<0.05); the numbers of senescent cells in the Ginkgo biloba extract group (9.38±1.92) and the sciadopitysin treatment group (7.75±2.38) were significantly decreased compared with the arsenic treatment group (all Ps<0.05). The results of Western blotting showed that compared with the control group, the expression of E2F1 protein in hippocampus of the arsenic treatment group was significantly decreased (1.00±0.17 vs. 0.65±0.19, P<0.05); compared with the arsenic treatment group, the protein expression level of E2F1 in hippocampus of the sciadopitysin treatment group (0.89±0.18) was significantly recovered (P<0.05); compared withGinkgo biloba extract (0.68±0.19), sciadopitysin had a better recovery effect on E2F1 expression level (0.89±0.18) (P<0.05). The results of correlation analysis showed that the E2F1 protein expression level was negatively correlated with the positive rate of SA-β-gal staining in hippocampal neurons (r=−0.518,P<0.05).

    Conclusion Sciadopitysin is an effective component of Ginkgo biloba extract. It can effectively inhibit the senescence of hippocampal neurons induced by sodium arsenite, and E2F1 may play an important mediating role.

     

/

返回文章
返回