视网膜色素上皮细胞表达IL-1β和IL-6与细胞自噬的关系
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摘要
目的:研究细胞自噬在缺氧条件下视网膜色素上皮(RPE)细胞表达炎性因子IL-1β和IL-6中的作用。方法:将体外培养的人RPE细胞系随机分为缺氧组、3-甲基腺嘌呤(3-MA)+缺氧组、氯喹(CQ)+缺氧组和对照组。培养24h后采用Western blot法检测各组细胞的自噬关键蛋白微管相关蛋白1轻链3B(LC3B)、Beclin-1及p62的相对表达水平;采用ELISA法检测各组细胞上清液中IL-1β和IL-6的浓度。结果:缺氧组IL-1β和IL-6的浓度明显高于对照组、3-MA+缺氧组和CQ+缺氧组(均P<0.01)。缺氧组细胞中LC3B-Ⅱ/Ⅰ和Beclin-1的相对蛋白表达量均明显高于对照组和3-MA+缺氧组,缺氧组中LC3B-Ⅱ/Ⅰ的相对蛋白表达量低于CQ+缺氧组(均P<0.01)。缺氧组细胞中p62相对蛋白表达量明显低于对照组、3-MA+缺氧组和CQ+缺氧组(均P<0.01)。结论:缺氧使RPE细胞LC3B-Ⅱ/Ⅰ、Beclin-1表达增强,p62表达减弱,并促进细胞分泌IL-1β和IL-6,自噬抑制剂3-MA和CQ能够减少RPE细胞表达IL-1β和IL-6。
·AIM:To investigate the role of cellular autophagy in the expression of IL-1β and IL-6 by retinal pigment epithelium(RPE) cells under hypoxia.·METHODS: A cultured human RPE cell line was randomly divided into the hypoxia group, 3-methyladenine(3-MA)+ hypoxia group, chloroquine(CQ)+ hypoxia group and the control group. Cells incubated in a hypoxic incubator were set as the hypoxia group. After culture for 24 h, western blot was used to analyze the relative expression of autophagy-associated key proteins in each group, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1 and p62. Then, ELISA was applied to detect the concentration of IL-1β and IL-6 in the cell culture supernatants.·RESULTS: The concentration of IL-1β and IL-6 in the hypoxia group was significantly higher than that in the control group 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01). The relative expression of LC3 B-II/I and Beclin-1 in the hypoxia group was significantly higher than that in the control group and 3-MA+hypoxia group. The relative expression of LC3 B-II/I in the hypoxia group was significantly lower than that in the CQ+ hypoxia group(all P<0.01). The relative expression of p62 in the hypoxia group was significantly lower than that in the control group, 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01).·CONCLUSION: Hypoxia can enhance the expression of LC3 B-II/I and Beclin-1 in RPE cells, weaken the expression of p62, and promote the secretion of IL-1β and IL-6. Autophagy inhibitors 3-MA and CQ can reduce the expression of IL-1βand IL-6 in RPE cells.
·AIM:To investigate the role of cellular autophagy in the expression of IL-1β and IL-6 by retinal pigment epithelium(RPE) cells under hypoxia.·METHODS: A cultured human RPE cell line was randomly divided into the hypoxia group, 3-methyladenine(3-MA)+ hypoxia group, chloroquine(CQ)+ hypoxia group and the control group. Cells incubated in a hypoxic incubator were set as the hypoxia group. After culture for 24 h, western blot was used to analyze the relative expression of autophagy-associated key proteins in each group, including microtubule associated protein 1 light chain 3 B(LC3 B), Beclin-1 and p62. Then, ELISA was applied to detect the concentration of IL-1β and IL-6 in the cell culture supernatants.·RESULTS: The concentration of IL-1β and IL-6 in the hypoxia group was significantly higher than that in the control group 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01). The relative expression of LC3 B-II/I and Beclin-1 in the hypoxia group was significantly higher than that in the control group and 3-MA+hypoxia group. The relative expression of LC3 B-II/I in the hypoxia group was significantly lower than that in the CQ+ hypoxia group(all P<0.01). The relative expression of p62 in the hypoxia group was significantly lower than that in the control group, 3-MA+hypoxia group and CQ+ hypoxia group(all P<0.01).·CONCLUSION: Hypoxia can enhance the expression of LC3 B-II/I and Beclin-1 in RPE cells, weaken the expression of p62, and promote the secretion of IL-1β and IL-6. Autophagy inhibitors 3-MA and CQ can reduce the expression of IL-1βand IL-6 in RPE cells.
引文
1 Parzych KR, Klionsky DJ. An overview of autophagy: morphology, mechanism, and regulation. Antioxid Redox Signal 2014; 20(3):460-473
2 Zeng X, Ju D. Hedgehog Signaling Pathway and Autophagy in Cancer. Int J Mol Sci 2018; 19(8):2279
3杜军辉, 李蓉, 马楼艳, 等. 自噬与炎症的关系及其在眼科疾病中的相关研究进展. 临床眼科杂志2017; 25(1):91-94
4 Araujo RS, Santos DF, Silva GA. The role of the retinal pigment epithelium and Müller cells secretome in neovascular retinal pathologies. Biochimie 2018; 155:104-108
5金伯泉.细胞和分子免疫学.北京:世界图书出版社1995:103-149
6 Du JH, Li X, Li R, et al. Role of autophagy in angiogenesis induced by high glucose condition in RF/6A cells. Ophthalmologica 2017; 237(2):85-95
7李蓉, 杜军辉, 姚杨, 等. 缺氧条件下自噬在视网膜血管内皮细胞血管生成中的作用. 中华实验眼科杂志 2018; 36(3):187-193
8王海青, 牛国桢, 张晓波, 等. RPE细胞的正常功能及其在眼科疾病中的作用. 生命科学2013; 25(9):878-882
9 Chen H, Wang H, An J, et al. Plumbagin induces RPE cell cycle arrest and apoptosis via p38 MARK and PI3K/AKT/mTOR signaling pathways in PVR. BMC Complement Altern Med 2018; 18(1):89
10 Fisher CR, Ferrington DA. Perspective on AMD Pathobiology: A Bioenergetic Crisis in the RPE. Invest Ophthalmol Vis Sci 2018; 59(4):41-47
11 Feng S, Yu H, Yu Y, et al. Levels of inflammatory cytokines IL-1β,IL-6, IL-8, IL-17A, and TNF-α in aqueous humour of patients with diabetic retinopathy. J Diabetes Res 2018; 2018:8546423
12 Tsai T, Kuehn S, Tsiampalis N, et al. Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients. PLoS One 2018; 13(3):e0194603
13 Leung KW, Barnstable CJ, Tombran-Tink J. Bacterial endotoxin activates retinal pigment epithelial cells and induces their degeneration through IL-6 and IL-8 autocrine signaling. Mol Immunol 2009; 46(7):1374-1386
14 Bansal M, Moharir SC, Swarup G. Autophagy receptor optineurin promotes autophagosome formation by potentiating LC3-II production and phagophorematuration. Commun Integr Biol 2018; 11(2):1-4
15 Zheng T, Li D, He Z, et al. Prognostic and clinicopathological significance of Beclin-1 in non-small-cell lung cancer:a meta-analysis. Onco Targets Ther 2018; 11:4167-4175
16 Battista RA, Resnati M, Facchi C, et al. Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation. PLoS One 2018; 13(8):e0201621
17 Li R, Tian J , Du J, et al. Manipulation of autophagy:a novelly potential therapeutic strategy for retinal neovascularization. BMC Ophthalmol 2018; 18:110
18 Mitter SK, Rao HV, Qi X, et al. Autophagy in the retina: a potential role in age-related macular degeneration. Adv Exp Med Biol 2012; 723:83-90
19 Szatmari-Toth M, Kristof E, Vereb Z, et al. Clearance of autophagy-associated dying retinal pigment epithelial cells-a possible source for inflammation in age-related macular degeneration. Cell Death Dis 2016; 7(9):e2367
20 Mitter SK, Song C, Qi X, et al. Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD. Autophagy 2014; 10(11):1989-2005
21 Chen PM, Gombart ZJ, Chen JW. Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipidaccumulation:possible implications of lysosomal dysfunction in macular degeneration. Cell Biosci 2011; 1(1):10
22 Yoon YH, Cho KS, Hwang JJ, et al. Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells. Invest Ophthalmol Vis Sci 2010; 51(11):6030-6037
23周延冲. 多肽生长因子基础与临床. 北京:中国科学技术出版社1993:253-267
24 Schett G. Physiological effects of modulating the interleukin-6 axis. Rheumatology(Oxford) 2018; 57(suppl2):43-50
25 Ghanemi A, St-Amand J. Interleukin-6 as a “metabolic hormone”. Cytokine 2018; 112:132-136
26 Ge Y, Huang M, Yao YM. Autophagy and proinflammatory cytokines:Interactions and clinical implications. Cytokine Growth Factor Rev 2018; 43:38-46
27 Mao C, Yan H. Roles of elevated intravitreal IL-1β and IL-10 levels in proliferative diabetic retinopathy. Indian J Ophthalmol 2014; 62(6):699-701
28 Symeonidis C, Papakonstantinou E, Androudi S, et al. Comparison of interleukin-6 and matrix metalloproteinase expression in the subretinal fluid and the vitreous during proliferative vitreoretinopathy:correlations with extent, duration of RRD and PVR grade. Cytokine 2014; 67(2):71-76
2 Zeng X, Ju D. Hedgehog Signaling Pathway and Autophagy in Cancer. Int J Mol Sci 2018; 19(8):2279
3杜军辉, 李蓉, 马楼艳, 等. 自噬与炎症的关系及其在眼科疾病中的相关研究进展. 临床眼科杂志2017; 25(1):91-94
4 Araujo RS, Santos DF, Silva GA. The role of the retinal pigment epithelium and Müller cells secretome in neovascular retinal pathologies. Biochimie 2018; 155:104-108
5金伯泉.细胞和分子免疫学.北京:世界图书出版社1995:103-149
6 Du JH, Li X, Li R, et al. Role of autophagy in angiogenesis induced by high glucose condition in RF/6A cells. Ophthalmologica 2017; 237(2):85-95
7李蓉, 杜军辉, 姚杨, 等. 缺氧条件下自噬在视网膜血管内皮细胞血管生成中的作用. 中华实验眼科杂志 2018; 36(3):187-193
8王海青, 牛国桢, 张晓波, 等. RPE细胞的正常功能及其在眼科疾病中的作用. 生命科学2013; 25(9):878-882
9 Chen H, Wang H, An J, et al. Plumbagin induces RPE cell cycle arrest and apoptosis via p38 MARK and PI3K/AKT/mTOR signaling pathways in PVR. BMC Complement Altern Med 2018; 18(1):89
10 Fisher CR, Ferrington DA. Perspective on AMD Pathobiology: A Bioenergetic Crisis in the RPE. Invest Ophthalmol Vis Sci 2018; 59(4):41-47
11 Feng S, Yu H, Yu Y, et al. Levels of inflammatory cytokines IL-1β,IL-6, IL-8, IL-17A, and TNF-α in aqueous humour of patients with diabetic retinopathy. J Diabetes Res 2018; 2018:8546423
12 Tsai T, Kuehn S, Tsiampalis N, et al. Anti-inflammatory cytokine and angiogenic factors levels in vitreous samples of diabetic retinopathy patients. PLoS One 2018; 13(3):e0194603
13 Leung KW, Barnstable CJ, Tombran-Tink J. Bacterial endotoxin activates retinal pigment epithelial cells and induces their degeneration through IL-6 and IL-8 autocrine signaling. Mol Immunol 2009; 46(7):1374-1386
14 Bansal M, Moharir SC, Swarup G. Autophagy receptor optineurin promotes autophagosome formation by potentiating LC3-II production and phagophorematuration. Commun Integr Biol 2018; 11(2):1-4
15 Zheng T, Li D, He Z, et al. Prognostic and clinicopathological significance of Beclin-1 in non-small-cell lung cancer:a meta-analysis. Onco Targets Ther 2018; 11:4167-4175
16 Battista RA, Resnati M, Facchi C, et al. Autophagy mediates epithelial cancer chemoresistance by reducing p62/SQSTM1 accumulation. PLoS One 2018; 13(8):e0201621
17 Li R, Tian J , Du J, et al. Manipulation of autophagy:a novelly potential therapeutic strategy for retinal neovascularization. BMC Ophthalmol 2018; 18:110
18 Mitter SK, Rao HV, Qi X, et al. Autophagy in the retina: a potential role in age-related macular degeneration. Adv Exp Med Biol 2012; 723:83-90
19 Szatmari-Toth M, Kristof E, Vereb Z, et al. Clearance of autophagy-associated dying retinal pigment epithelial cells-a possible source for inflammation in age-related macular degeneration. Cell Death Dis 2016; 7(9):e2367
20 Mitter SK, Song C, Qi X, et al. Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD. Autophagy 2014; 10(11):1989-2005
21 Chen PM, Gombart ZJ, Chen JW. Chloroquine treatment of ARPE-19 cells leads to lysosome dilation and intracellular lipidaccumulation:possible implications of lysosomal dysfunction in macular degeneration. Cell Biosci 2011; 1(1):10
22 Yoon YH, Cho KS, Hwang JJ, et al. Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells. Invest Ophthalmol Vis Sci 2010; 51(11):6030-6037
23周延冲. 多肽生长因子基础与临床. 北京:中国科学技术出版社1993:253-267
24 Schett G. Physiological effects of modulating the interleukin-6 axis. Rheumatology(Oxford) 2018; 57(suppl2):43-50
25 Ghanemi A, St-Amand J. Interleukin-6 as a “metabolic hormone”. Cytokine 2018; 112:132-136
26 Ge Y, Huang M, Yao YM. Autophagy and proinflammatory cytokines:Interactions and clinical implications. Cytokine Growth Factor Rev 2018; 43:38-46
27 Mao C, Yan H. Roles of elevated intravitreal IL-1β and IL-10 levels in proliferative diabetic retinopathy. Indian J Ophthalmol 2014; 62(6):699-701
28 Symeonidis C, Papakonstantinou E, Androudi S, et al. Comparison of interleukin-6 and matrix metalloproteinase expression in the subretinal fluid and the vitreous during proliferative vitreoretinopathy:correlations with extent, duration of RRD and PVR grade. Cytokine 2014; 67(2):71-76