PM2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应

doi:10.11958/20220086

天津医药 ›› 2022, Vol. 50 ›› Issue (11): 1139-1145.doi: 10.11958/20220086

PM_2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应

黄秋阳¹(), 王伟¹, 罗书², 郑文武¹, 冯健¹, 叶强¹, 郑舒展¹^,^△()

1 西南医科大学附属医院心血管内科（邮编646000）
2 四川省泸州市生态环境监测中心站

收稿日期:2022-01-24 修回日期:2022-06-14 出版日期:2022-11-15 发布日期:2022-11-11
通讯作者: 郑舒展 E-mail:2274152499@qq.com;toshuzhan@126.com
作者简介:黄秋阳（1993），女，硕士在读，主要从事心血管疾病研究。E-mail：2274152499@qq.com

PM_2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4

HUANG Qiuyang¹(), WANG Wei¹, LUO Shu², ZHENG Wenwu¹, FENG Jian¹, YE Qiang¹, ZHENG Shuzhan¹^,^△()

1 Department of Cardiovascular Medicine, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
2 Luzhou City Ecological Environment Monitoring Center Station, Sichuan Province

Received:2022-01-24 Revised:2022-06-14 Published:2022-11-15 Online:2022-11-11
Contact: ZHENG Shuzhan E-mail:2274152499@qq.com;toshuzhan@126.com

黄秋阳, 王伟, 罗书, 郑文武, 冯健, 叶强, 郑舒展. PM_2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应[J]. 天津医药, 2022, 50(11): 1139-1145.

HUANG Qiuyang, WANG Wei, LUO Shu, ZHENG Wenwu, FENG Jian, YE Qiang, ZHENG Shuzhan. PM_2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4[J]. Tianjin Medical Journal, 2022, 50(11): 1139-1145.

摘要/Abstract

摘要：

目的探讨PM_2.5对Raw264.7巨噬细胞自噬及炎症反应的影响及机制。方法 CCK-8检测PM_2.5对Raw264.7巨噬细胞活性的影响，透射电镜观察自噬结构；Ad-mCherry-GFP-LC3B转染细胞后荧光显微镜观察自噬通量，以探讨PM_2.5对巨噬细胞活性和自噬流的影响。再设置对照组、PM_2.5组、雷帕霉素（Rap，自噬诱导剂）组、羟氯喹（HCQ，自噬抑制剂）组、PM_2.5+HCQ组、PM_2.5+TAK-242[Toll样受体4（TLR4）抑制剂]组。Western blot法检测自噬相关蛋白，包括微管相关轻链蛋白3比值（LC3Ⅱ/Ⅰ）、p62、组织蛋白酶B（CTSB）、溶酶体膜蛋白2（LAMP2）和炎症相关蛋白，包括Toll样受体4（TLR4）、核苷酸结合域样受体蛋白3（NLRP3）的表达水平；酶联免疫吸附试验检测相关炎性因子白细胞介素（IL）-1β、IL-18、IL-10分泌水平。结果 Raw264.7巨噬细胞活性随着PM_2.5浓度的增加和作用时间的延长而降低。透射电镜观察到PM_2.5组有较多自噬空泡和双膜自噬体，自噬溶酶体少见，且荧光显微镜下PM_2.5组绿色荧光淬灭不明显，Merge后黄红色荧光比值高于对照组和Rap组（P<0.05）。相较于对照组，PM_2.5组NLRP3、LC3Ⅱ/Ⅰ、p62、CTSB、IL-1β、IL-18的表达上调，LAMP2、IL-10表达下调（P<0.05），而HCQ抑制自噬的同时，促进NLRP3、IL-1β、IL-18的表达，抑制IL-10表达（P<0.05）。相较于对照组、PM_2.5组和HCQ组，PM_2.5+HCQ组NLRP3、IL-1β、IL-18的上调及IL-10下调更为显著（P<0.05）。此外，PM_2.5组TLR4表达高于对照组，TAK-242抑制TLR4后NLRP3、LC3Ⅱ/Ⅰ、p62、CTSB、IL-1β、IL-18的表达较PM_2.5组下调，LAMP2和IL-10的表达上调（P<0.05）。结论 PM_2.5可呈时间和浓度依赖性地降低巨噬细胞活性；且PM_2.5诱导Raw264.7巨噬细胞发生自噬，但阻断自噬流，加重巨噬细胞炎症反应，且该过程可能由TLR4介导。

关键词: 颗粒物, 自噬, 炎症, Toll样受体4, NLR家族，热蛋白结构域包含蛋白3, 动脉粥样硬化, PM_2.5

Abstract:

Objective To explore the effect and mechanism of PM_2.5 on the autophagy and inflammatory response of Raw264.7 macrophages. Methods CCK-8 was used to detect the effect of PM_2.5 on the viability of Raw264.7 cells, and transmission electron microscopy was used to observe the autophagy structure. The autophagic flux was observed by fluorescence microscope after Ad-mCherry-GFP-LC3B transfection to explore the effect of PM_2.5 on macrophage activity and autophagy. Then the blank control group, the PM_2.5 group, the rapamycin (Rap, autophagy inducer) group, the hydroxychloroquine (HCQ, autophagy inhibitor) group, the PM_2.5+HCQ group and PM_2.5+TAK-242 [Toll-like receptor 4 (TLR4) inhibitor] group were set. Western blot method was used to detect levels of autophagy-related proteins, including microtubule-associated light chain protein 3 ratio (LC3Ⅱ/Ⅰ), p62, cathepsin B (CTSB), lysosomal membrane protein 2 (LAMP2) and inflammation-related proteins TLR4 and nucleotide binding Domain-like receptor protein 3 (NLRP3)]. ELISA method was used to detect the levels of inflammatory factors, including interleukin (IL)-1β, IL-18 and IL-10. Results The activity of Raw264.7 macrophages decreased with the increase of PM_2.5 concentration and the prolonged action time. Transmission electron microscopy showed that there were more autophagic vacuoles and double-membrane autophagosomes in the PM_2.5 group, while autophagic lysosomes were rare, and under the fluorescence microscope, the ratio of yellow fluorescent dots to red fluorescent dots was higher in the PM_2.5 group than that in the control group and the Rap group (P<0.05). Compared with the blank control group, the expressions levels of NLRP3, LC3Ⅱ/Ⅰ, p62, CTSB, IL-1β and IL-18 were up-regulated in the PM_2.5 group, and the expressions of LAMP2 and IL-10 were down-regulated (P<0.05). While HCQ inhibited autophagy, it promoted the expression of NLRP3, IL-1β and IL-18, and inhibited the expression of IL-10 (P<0.05). Compared with the blank control group, the PM_2.5 group and the HCQ group, NLRP3, IL-1β and IL-18 were significantly up-regulated and IL-10 was significantly down-regulated in the PM_2.5+HCQ group (P<0.05). In addition, the expression of TLR4 was high in the PM_2.5 group. Expressions of NLRP3, LC3Ⅱ/Ⅰ, p62, CTSB, IL-1β and IL-18 were down-regulated after TLR4 was inhibited by TAK-242, and expressions of LAMP2 and IL-10 were up-regulated (P<0.05). Conclusion PM_2.5 reduces macrophage activity in a time- and concentration-dependent manner. PM_2.5 induces autophagy in Raw264.7 macrophages, but blocks autophagic flow and aggravates the inflammatory response of macrophages, which may be mediated by TLR4.

Key words: particulate matter, autophagy, inflammation, Toll-like receptor 4, NLR family, pyrin domain-containing 3 protein, atherosclerosis, PM_2.5

中图分类号:

R364.5

图/表 8

表1 2组不同时间PM2.5对巨噬细胞活性影响的比较 (n=3,%, $\bar{x}\pm s$)

Tab.1 Comparison of the effect of PM2.5 on macrophage activity at different times between the two groups

组别	0 h	6 h	12 h	18 h	24 h	48 h
对照组	100.0±1.1	99.0±1.2	101.0±1.1	100.0±1.4	100.0±1.3	100.0±1.2
PM_2.5组	100.0±1.6	91.0±2.0	82.3±3.2	67.0±2.4	52.0±2.3	41.0±2.8
t	0.292	1.345^＊	3.363^＊	4.178^＊	4.765^＊	5.193^＊

图1 透射电镜下观察自噬结构（×8 000）

Fig.1 Observation of autophagy structure under transmission electron microscope (×8 000)

图2 Ad-mCherry-GFP-LC3B腺病毒转染后Raw264.7细胞内自噬情况的变化

Fig.2 Changes of autophagy in Raw264.7 cells transfected with Ad-mCherry-GFP-LC3B

图3 Western blot检测各组LC3、CTSB、p62、LAMP2蛋白表达 A：Western blot检测图；B~E分别为LAMP2、LC3、CTSB、p62蛋白表达半定量分析。

Fig.3 Western blot detection of LAMP2, LC3, CTSB, p62 protein expression in each group

表2 各组炎性因子表达水平比较 (n=3,ng/L, $\bar{x}\pm s$)

Tab.2 Comparison of expressions of inflammatory factor levels between the four groups

组别	IL-1β	IL-18	IL-10
对照组	25.47±8.70	25.32±5.79	45.59±7.55
PM_2.5组	156.79±13.77^a	129.71±9.75^a	24.31±3.82^a
HCQ组	124.09±11.46^a	106.57±9.13^a	19.32±3.40^a
PM_2.5+HCQ组	205.55±29.13^abc	218.54±11.56^abc	7.24±2.52^abc
F	55.819^＊＊	217.096^＊＊	61.207^＊＊

图4 Western blot检测自噬蛋白、NLRP3蛋白表达 A：Western blot检测图；B~D为p62、LC3、NLRP3蛋白表达半定量分析；1为对照组，2为PM2.5组，3为HCQ组，4为PM2.5+HCQ组；a与对照组比较，b与PM2.5组比较，c与HCQ组比较，P<0.05。

Fig.4 Western blot detection of expression levels of autophagy and NLRP3 protein

表3 抑制TLR4后各组炎症因子水平比较 (n=3,ng/L, $\bar{x}\pm s$)

Tab.3 Comparison of inflammatory factor levels after inhibition of TLR4 between the three groups

组别	IL-1β	IL-18	IL-10
对照组	25.54±8.70	24.99±5.81	46.11±7.56
PM_2.5组	157.19±11.84^a	130.21±9.65^a	23.10±4.12^a
PM_2.5+TAK-242组	74.54±6.47^ab	72.56±7.53^ab	86.92±10.13^ab
F	129.037^＊＊	132.806^＊＊	52.373^＊＊

图5 抑制TLR4后Western blot检测各组自噬和炎症相关蛋白表达 A：Western blot检测图；B~G分别为TLR4、NLRP3、LAMP2、LC3、CTSB、p62蛋白表达半定量分析；1为对照组，2为PM2.5组，3为PM2.5+TAK-242组；a与对照组比较，b与PM2.5组比较，P<0.05。

Fig.5 After inhibiting TLR4, the expression of autophagy and inflammation-related proteins detected by Western blot assay

参考文献 24

[1]	KOLLANUS V, PRANK M, GENS A, et al. Mortality due to Vegetation Fire-Originated PM_2.5 Exposure in Europe-Assessment for the Years 2005 and 2008[J]. Environ Health Perspect, 2017, 125(1):30-37. doi:10.1289/EHP194.
[2]	HOEK G, KRISHNAN R M, BEELEN R, et al. Long-term air pollution exposure and cardio- respiratory mortality:a review[J]. Environ Health, 2013, 12(1):43. doi:10.1186/1476-069X-12-43.
[3]	GENG J, LIU H, GE P, et al. PM2.5 promotes plaque vulnerability at different stages of atherosclerosis and the formation of foam cells via TLR4/MyD88/NFκB pathway[J]. Ecotoxicol Environ Saf, 2019, 176:76-84. doi:10.1016/j.ecoenv.2019.03.068.
[4]	ZHU X C, ZHAO P, LU Y G, et al. Potential injurious effects of the fine particulate PM2.5 on the progression of atherosclerosis in apoE-deficient mice by activating platelets and leukocytes[J]. Arch Med Sci, 2019, 15(1):250-261. doi:10.5114/aoms.2018.81039.
[5]	GOTTLIEB R A, MENTZER R M. Autophagy during cardiac stress: joys and frustrations of autophagy[J]. Annu Rev Physiol, 2010, 72:45-59. doi:10.1146/annurev-physiol-021909-135757.
[6]	EVANS T D, JEONG S J, ZHANG X Y, et al. TFEB and trehalose drive the macrophage autophagy-lysosome system to protect against atherosclerosis[J]. Autophagy, 2018, 14(4):724-726. doi:10.1080/15548627.2018.1434373.
[7]	MARTINET W, COORNAERT I, PUYLAERT P, et al. Macrophage death as a pharmacological target in atherosclerosis[J]. Front Pharmacol, 2019, 10:306. doi:10.3389/fphar.2019.00306.
[8]	DAI P Y, SHEN D, SHEN J K, et al. The roles of Nrf2 and autophagy in modulating inflammation mediated by TLR4 - NFκB in A549 cell exposed to layer house particulate matter 2.5 (PM_2.5)[J]. Chemosphere, 2019, 235:1134-1145. doi:10.1016/j.chemosphere.2019.07.002.
[9]	ZHOU Z, SHAO T, QIN M, et al. The effects of autophagy on vascular endothelial cells induced by airborne PM2.5[J]. J Environ Sci (China), 2018, 66:182-187. doi:10.1016/j.jes.2017.05.019.
[10]	LANDRIGAN P J, FULLER R, ACOSTA N J R, et al. The Lancet Commission on pollution and health[J]. Lancet, 2018, 391(10119):462-512. doi:10.1016/S0140-6736(17)32345-0.
[11]	MCGUINN L A, SCHNEIDER A, MCGARRAH R W, et al. Association of long-term PM_2.5 exposure with traditional and novel lipid measures related to cardiovascular disease risk[J]. Environ Int, 2019, 122:193-200. doi:10.1016/j.envint.2018.11.001.
[12]	GOLD D R, LITONJUA A, SCHWARTZ J, et al. Ambient pollution and heart rate variability[J]. Circulation, 2000, 101(11):1267-1273. doi:10.1161/01.cir.101.11.1267.
[13]	ZHANG J, LIANG S, NING R, et al. PM_2.5-induced inflammation and lipidome alteration associated with the development of atherosclerosis based on a targeted lipidomic analysis[J]. Environ Int, 2020, 136:105444. doi:10.1016/j.envint.2019.105444.
[14]	RAO X, ZHONG J, MAISEYEU A, et al. CD36-dependent 7-ketocholesterol accumulation in macrophages mediates progression of atherosclerosis in response to chronic air pollution exposure[J]. Circ Res, 2014, 115(9):770-780. doi:10.1161/CIRCRESAHA.115.304666.
[15]	TABAS I. Consequences and therapeutic implications of macrophage apoptosis in atherosclerosis: the importance of lesion stage and phagocytic efficiency[J]. Arterioscler Thromb Vasc Biol, 2005, 25(11):2255-2264. doi:10.1161/01.ATV.0000184783.04864.9f.
[16]	ENDO Y, FURUTA A, NISHINO I. Danon disease:a phenotypic expression of LAMP-2 deficiency[J]. Acta Neuropathol, 2015, 129(3):391-398. doi:10.1007/s00401-015-1385-4.
[17]	VINDIS C. Autophagy:an emerging therapeutic target in vascular diseases[J]. Br J Pharmacol, 2015, 172(9):2167-2178. doi:10.1111/bph.13052.
[18]	WANG Y, ZHONG Y, LIAO J, et al. PM2.5-related cell death patterns[J]. Int J Med Sci, 2021, 18(4):1024-1029. doi:10.7150/ijms.46421.
[19]	HOSEINI Z, SEPAHVAND F, RASHIDI B, et al. NLRP3 inflammasome:Its regulation and involvement in atherosclerosis[J]. J Cell Physiol, 2018, 233(3):2116-2132. doi:10.1002/jcp. 25930.
[20]	ROSHAN M H, TAMBO A, PACE N P. The Role of TLR2, TLR4, and TLR9 in the pathogenesis of atherosclerosis[J]. Int J Inflam, 2016, 2016:1532832. doi:10.1155/2016/1532832.
[21]	SAITOH T, AKIRA S. Regulation of inflammasomes by autophagy[J]. J Allergy Clin Immunol, 2016, 138(1):28-36. doi:10.1016/j.jaci.2016.05.009.
[22]	BIASIZZO M, KOPITAR-JERALA N. Interplay between NLRP3 inflammasome and autophagy[J]. Front Immunol, 2020, 11:591803. doi:10.3389/fimmu.2020.591803.
[23]	LAI M Y, YAO H, SHAH S Z A, et al. The NLRP3-Caspase 1 inflammasome negatively regulates autophagy via TLR4-TRIF in prion peptide-infected microglia[J]. Front Aging Neurosci, 2018, 10:116. doi:10.3389/fnagi.2018.00116.
[24]	LI M Z, HUA Q H, SHAO Y T, et al. Circular RNA circBbs9 promotes PM_2.5-induced lung inflammation in mice via NLRP3 inflammasome activation[J]. Environ Int, 2020, 143:105976.doi:10.1016/j.envint.2020.105976.

PM_2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应

PM_2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 8

参考文献 24

相关文章 15

编辑推荐

Metrics

本文评价

[1]	陈晶晶, 农章嵩, 谭良源, 杨培培, 梁英业, 唐宏亮, 王开龙. 小胶质细胞极化在神经病理性疼痛中作用研究进展[J]. 天津医药, 2024, 52(9): 1000-1003.
[2]	范慧慧, 任玉梅, 田新磊, 张凯, 李晓丽. 止咳平喘方对支气管哮喘小鼠气道炎症及TLR4/TRAF6/NF-κB通路的影响[J]. 天津医药, 2024, 52(9): 924-929.
[3]	贾维宁, 鲍亚玲, 雷慧, 殷晓宁. 夏枯草提取物对脓毒症小鼠炎症反应和腹腔巨噬细胞的影响[J]. 天津医药, 2024, 52(9): 930-935.
[4]	方杰, 黄芮, 郑红慧, 贾倩倩, 鲍静. miR-9-5p靶向TIMP2诱导多发性骨髓瘤细胞自噬和凋亡的机制[J]. 天津医药, 2024, 52(8): 785-790.
[5]	李大强, 李坚, 陆哲明, 曹阳. 毛蕊异黄酮对脊髓损伤大鼠神经元自噬及凋亡的影响[J]. 天津医药, 2024, 52(8): 798-803.
[6]	钟敏, 施震, 周劲松, 李晋杰. GABA信号通路对脓毒症大鼠急性肺损伤内质网应激和线粒体自噬的影响[J]. 天津医药, 2024, 52(7): 733-737.
[7]	吴波, 朱卓农, 郑丽娟, 陈俊如. 苦参碱对特应性皮炎炎症、氧化应激和伤口愈合的影响[J]. 天津医药, 2024, 52(6): 566-571.
[8]	袁满, 冯子瀚, 谢敏, 王柏军. 大黄素对骨关节炎模型小鼠痛觉行为的调节机制[J]. 天津医药, 2024, 52(6): 572-577.
[9]	蒋韬, 程红艳, 吴琼. 棕矢车菊素调节SDF-1α/CXCR4信号通路对妊娠糖尿病大鼠炎症反应的影响[J]. 天津医药, 2024, 52(6): 594-598.
[10]	霍晶辰, 王悦, 李华, 邱嵘, 苏景伟, 王卓凡, 杨洁. 系统免疫炎症指数对根治性放疗Ⅲ期肺鳞癌患者长期生存的预测价值[J]. 天津医药, 2024, 52(6): 634-638.
[11]	叶朝阳, 马建中, 李后俊, 魏鲲鹏. 急性胰腺炎患者外周血TLR4、IL-1β、NLR水平与疾病进展和预后的关系[J]. 天津医药, 2024, 52(6): 648-652.
[12]	慕静然, 骆延, 梁璇, 徐陶, 曾俊伟, 刘晓红. 补体系统激活参与阿尔茨海默病的研究进展[J]. 天津医药, 2024, 52(6): 663-668.
[13]	韩正怡, 李锐, 陈齐, 王家友, 盛奎, 宋洁, 张野. 收肌管阻滞联合全麻对老年全膝关节置换术患者术后疼痛和认知功能的影响[J]. 天津医药, 2024, 52(5): 523-527.
[14]	吕梦娜, 李建斌, 吴锐. 自身炎症性疾病患者合并COVID-19严重程度的早期预测指标探讨[J]. 天津医药, 2024, 52(5): 528-531.
[15]	贾西瑞, 刘莉洁. 小胶质细胞在脓毒症相关性脑病中的作用及研究进展[J]. 天津医药, 2024, 52(5): 557-560.

PM2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应

PM2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4

引用本文

RichHTML

PDF (PC)

可视化

摘要/Abstract

使用本文

图/表 8

参考文献 24

相关文章 15

编辑推荐

Metrics

本文评价

PM_2.5通过TLR4破坏自噬流加重Raw264.7巨噬细胞炎症反应

PM_2.5 aggravates the inflammatory response of Raw264.7 macrophages by disrupting autophagic flow through TLR4