CERKL通过激活SIRT1/E2F1轴减轻蓝光导致的人视网膜色素上皮细胞氧化应激损伤
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海南省卫生健康行业科研项目(No.20A200078)


CERKL attenuates blue light-induced oxidative stress in human retinal pigment epithelial cells by activating the SIRT1/E2F1 axis
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Hainan Health Industry Scientific Research Project(No.20A200078)

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    摘要:

    目的:探讨神经酰胺类似蛋白(CERKL)是否通过激活沉默信息调节因子1(SIRT1)/E2F转录因子1(E2F1)轴减轻蓝光导致的视网膜色素上皮(RPE)细胞氧化应激损伤。

    方法:培养人视网膜色素上皮-19(ARPE-19)细胞,蓝光照射后观察细胞形态变化,PCR与蛋白免疫印迹法测定细胞CERKL的表达情况; 分别采用siRNA-CERKL与pcDNA3.1-CERKL转染ARPE-19细胞,蓝光暴露处理后,采用MTT法测定细胞活力,TUNEL法检测细胞凋亡情况,分析氧化应激标志物的含量与SIRT1/E2F1轴的表达情况; 随后转染siRNA-SIRT1至ARPE-19细胞,再次测定蓝光照射下细胞氧化应激损伤状况。

    结果:蓝光照射后,ARPE-19细胞逐渐收缩成圆球状,且出现空泡; 蓝光照射导致CERKL表达水平升高(P<0.05),同时观察到细胞活力降低(P<0.05),凋亡率升高(P<0.05),活性氧、丙二醛与8-羟基脱氧鸟苷含量升高(P<0.05); 沉默CERKL会加剧此现象,而上调CERKL则能缓解此变化(P<0.05); 上调CERKL同时激活了SIRT1表达,促进了E2F1的脱乙酰化(P<0.05),而沉默SIRT1能逆转上调CERKL对蓝光导致ARPE-19细胞氧化应激损伤的缓解作用(P<0.05)。

    结论:CERKL通过激活SIRT1表达,促进E2F1脱乙酰化,从而减轻蓝光诱发的ARPE-19细胞氧化应激损伤。

    Abstract:

    AIM:To investigate whether ceramide kinase-like protein(CERKL)alleviates oxidative stress injury of retinal pigment epithelial(RPE)cells induced by blue light via activating the silent information regulator 1(SIRT1)/E2F transcription factor 1(E2F1)axis.

    METHODS:Cultured human retinal pigment epithelial-19(ARPE-19)cells were irradiated with blue light to observe the morphological changes, and the expression of CERKL was detected by PCR and Western blot. ARPE-19 cells were transfected with siRNA-CERKL and pcDNA3.1-CERKL respectively. After exposure to blue light, cell viability was determined by MTT assay, apoptosis was detected by TUNEL assay, content of oxidative stress markers and the expression of SIRT1/E2F1 axis was analyzed. Then siRNA-SIRT1 was transfected into ARPE-19 cells, and the oxidative stress damage of ARPE-19 cells under blue light irradiation was detected again.

    RESULTS:ARPE-19 cells gradually contracted into spheres and appeared vacuoles after exposure to blue light. Blue light irradiation led to the increase of CERKL expression level(P<0.05), meanwhile, the rate of cell viability was decreased(P<0.05), the rate of the apoptosis was increased(P<0.05), contents of reactive oxygen species, malondialdehyde and 8-hydroxydeoxyguanosine were increased(P<0.05). Silence of CERKL aggravated this phenomenon, while up-regulation of CERKL could alleviate this change(P<0.05). Up-regulation of CERKL also activated the expression of SIRT1 and promoted the deacetylation of E2F1(P<0.05). Silencing SIRT1 could reverse the alleviating effect of up-regulating CERKL on oxidative stress injury of ARPE-19 cells induced by blue light(P<0.05).

    CONCLUSION: CERKL can reduce oxidative stress damage of ARPE-19 cells induced by blue light via activating SIRT1 expression and promoting the deacetylation of E2F1.

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庄海容,吴子东,陈雪红,等. CERKL通过激活SIRT1/E2F1轴减轻蓝光导致的人视网膜色素上皮细胞氧化应激损伤.国际眼科杂志, 2022,22(8):1245-1251.

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  • 收稿日期:2021-09-16
  • 最后修改日期:2022-07-14
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  • 在线发布日期: 2022-07-27
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