| 唐广明,莫银欢,何苑琳,等.硇洲马尾藻褐藻糖胶通过清除ROS和抑制PERK-ATF4-CHOP通路抑制UVB诱导HaCaT细胞凋亡[J].中国海洋药物,2026,(1):-. |
| 硇洲马尾藻褐藻糖胶通过清除ROS和抑制PERK-ATF4-CHOP通路抑制UVB诱导HaCaT细胞凋亡 |
| Sargassum naozhouense fucoidan inhibits UVB-induced apoptosis in HaCaT cells by scavenging ROS and inhibiting the PERK-ATF4-CHOP pathway |
| 投稿时间：2024-09-27 修订日期：2024-11-25 |
| DOI： |
| 中文关键词: 马尾藻 褐藻糖胶 HaCaT细胞 UVB PERK-ATF4-CHOP通路 |
| English Keywords:sargassum fucoidan HaCaT cells UVB PERK-ATF4-CHOP pathway |
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| 中文摘要: |
| 目的 本研究将探讨紫外线B(UVB)照射人永生化角质形成细胞(HaCaT)内质网(ER)应激后诱发的信号通路影响情况,以及从硇洲马尾藻提取的褐藻糖胶(Sargassum naozhouense fucoidan, SNF)是否通过ER应激诱导的信号通路发挥抗凋亡作用,从而保护细胞免受UVB照射的损害。方法 CCK-8法测定细胞活力,膜联蛋白Ⅴ(AV)/碘化丙啶(PI)双染法检测各组细胞的凋亡情况；免疫荧光染色测定CAAT增强子结合蛋白同源蛋白(CHOP)和活化转录因子4(ATF4)蛋白的荧光水平变化情况；蛋白质免疫印迹法检测凋亡相关蛋白胱天蛋白酶-3(Caspase-3)和胱天蛋白酶-9(Caspase-9)蛋白的激活程度以及p53、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)、细胞色素C(Cytochrome C)表达水平,还有对内质网应激相关蛋白蛋白激酶样内质网激酶(PERK)、ATF4、CHOP的影响；用DCFH-DA探针检测HaCaT细胞内活性氧(ROS)水平变化。结果 50 μg/mL SNF 、100 μg/mL SNF、100 μmol/L 维生素c(Vc)对HaCaT细胞均无毒害作用,且可提高被UVB辐射所损伤的细胞活力；AV/PI双染荧光结果显示50 μg/mL SNF 和100 μg/mL SNF组可以抑制细胞凋亡；蛋白质免疫印迹结果显示SNF剂量依赖性地降低了Caspase-9、Caspase-3和p53以及Cytochrome C表达水平,特别在100 μg/mL的浓度下,SNF展现出的效果与100 μmol/L Vc的抑制效果相媲美。同时SNF显著提高了UVB照射细胞中Bcl-2/Bax的比率。SNF处理可显著抑制PERK、ATF4和 CHOP表达水平,且100 μg/mL的SNF抑制效果最强,表现出与100 μmol/L Vc相当的效力；免疫荧光结果显示50 μg/mL SNF、100 μg/mL SNF和100 μmol/L Vc均可减弱ATF4、CHOP荧光强度；SNF可以有效地清除UVB照射的HaCaT细胞中的ROS。结论 SNF减轻了UVB照射下HaCaT细胞的细胞活力丧失,并抑制caspase-9、caspase-3和p53以及Cytochrome C表达,显著提高了Bcl-2/Bax的比率,有效缓解了细胞凋亡过程。SNF通过降低ROS的积累和阻断PERK-ATF4-CHOP促凋亡途径来保护细胞免受UVB照射的损害。 |
| English Summary: |
| Objective This study aims to investigate the effect of signaling pathways induced by endoplasmic reticulum (ER) stress after ultraviolet B (UVB) irradiation of human immortalized keratinocytes (HaCaT), and whether fucoidan extracted from Sargassum naozhouense (SNF) exerts anti-apoptotic effects through ER stress-induced signaling pathways, thereby protecting cells from damage caused by UVB irradiation. Methods Cell viability was determined by CCK-8 assay, and apoptosis was detected by Annexin V (AV)/ Propyl iodide (PI) double staining in each group of cells. Immunofluorescence staining determined changes in the fluorescence levels of CAAT enhancer binding protein homologous protein (CHOP) and Activating transcription factor 4 (ATF4) proteins. Western blotting was employed to detect the degree of activation of the apoptosis-related proteins Caspase-3 and Caspase-9 proteins, as well as the expression levels of p53, Bcl-2-associated X-protein (Bax), B-cell lymphoma-2 (Bcl-2), and Cytochrome C, and the effect on endoplasmic reticulum stress-related proteins protein kinase-like endoplasmic reticulum kinase (PERK), ATF4, CHOP. Changes in the level of reactive oxygen species (ROS) in HaCaT cells were detected by the DCFH-DA probe. Results 50 μg/mL SNF, 100 μg/mL SNF, and 100 μmol/L Vitamin C (Vc) had no toxic effects on HaCaT cells and increased the viability of cells damaged by UVB radiation. AV/PI double-staining fluorescence results showed that apoptosis could be inhibited in the 50 μg/mL SNF and 100 μg/mL SNF groups. Protein immunoblotting result indicated that the SNF dose-dependently decreased the level of PERK, ATF4, and CHOP. The DCFH-DA probe was used to detect the changes in the level of reactive oxygen species in HaCaT cells. The protein immunoblotting results showed that SNF dose-dependently reduced the expression levels of caspase-9, caspase-3 and p53, and Cytochrome C. Especially at the concentration of 100 μg/mL, SNF exhibited an effect comparable to that of 100 μmol/L Vc. Meanwhile, SNF significantly increased the ratio of Bcl-2/Bax in UVB-irradiated cells. SNF treatment significantly inhibited the expression levels of PERK, ATF4, and CHOP, and the strongest inhibitory effect was shown by SNF at 100 μg/mL, which demonstrated a potency comparable to that of 100 μmol/L Vc. Immunofluorescence results showed that the inhibitory effects of 50 μg/mL SNF, 100 μg/mL SNF, and 100 μmol/L Vc could all attenuate the fluorescence intensity of ATF4 and CHOP. SNF could effectively remove ROS from UVB-irradiated HaCaT cells. Conclusion SNF reduced the cell damage caused by UVB radiation in HaCaT cells. It also decreased the expression of caspase-9, caspase-3, p53, and Cytochrome C. Additionally, SNF increased the ratio of Bcl-2/Bax and effectively prevented the process of apoptosis. SNF protected the cells from UVB radiation-induced damage by decreasing ROS accumulation and blocking the PERK-ATF4-CHOP pro-apoptotic pathway. |
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