N-乙酰半胱氨酸对PM2.5致大鼠肺损伤时MAPK主要通路蛋白活化及氧化炎症反应的影响

doi:10.24920/003527

摘要/Abstract

摘要： 目的探讨N-乙酰半胱氨酸（N-Acetylcysteine,NAC）对PM2.5致大鼠肺损伤时丝裂原激活蛋白激酶（Mitogen activated protein kinases,MAPK）主要通路蛋白活化及炎症反应的影响。方法 48只雄性Wistar大鼠随机分为6组：空白对照组（C1组）、滴水对照组（C2组）、PM2.5染毒组（P组）及低（L组）、中（M组）、高剂量（H组）NAC干预组。滴水对照组给予气管滴注灭菌注射用水（1 ml/kg）1次/周,共4次;PM2.5染毒组大鼠给予气管滴注PM2.5悬液1 ml/kg（7.5 mg/kg）1次/周,共4次;低、中、高剂量NAC干预组分别给予125 mg/Kg,250 mg/Kg,500 mg/Kg NAC灌胃（10 ml/Kg）6天,第7天气管滴注PM2.5悬液1 ml/kg（7.5 mg/kg）,重复此过程3次。所有大鼠于四周后处死。采用苏木素–伊红（HE）染色、阿新蓝-过碘酸希夫（AB-PAS）染色观察大鼠肺组织病理变化;ELISA法检测大鼠血清及支气管肺泡灌洗液（BALF）中IL-6、肺组织匀浆的MUC5AC水平;分光光度法测定血清及BALF中的GSH-PX活力;Western blot法检测肺组织p-ERK1/2、p-JNK1/2和p-p38蛋白表达情况。所有检测结果进行组内及组间统计学比较。结果经PM2.5染毒的各组（P,L,M及H组）大鼠肺组织破坏和支气管上皮细胞粘液分泌增加,随着NAC干预剂量增加,肺组织破坏和粘液分泌程度减轻。PM2.5染毒组（P组）及NAC干预各组（L,M及H组）的肺组织MUC5AC含量、血清和BALF中IL-6水平显著高于对照组（C1和C2组）,L,M及H组低于P组（均P<0.05）,且随着NAC干预剂量增加呈下降趋势;血清和BALF中GSH-PX活力在P,L,M及H各组均显著低于对照组,在L,M及H组显著高于P组（均P<0.05）,并随着NAC干预剂量增加呈上升趋势;肺组织中p-ERK1/2、p-JNK1/2、p-p38蛋白表达在P,L,M及H各组均高于对照组,L,M及H组低于P组（均P<0.05）,并随着NAC干预剂量的增加肺组织内p-ERK1/2、p-JNK1/2和p-p38蛋白表达水平呈下降趋势。结论 NAC可拮抗PM2.5致大鼠肺MAPK主要通路蛋白活化及肺氧化炎症损伤,对PM2.5诱导的肺损伤具有保护作用。

关键词: 细颗粒物, N-乙酰半胱氨酸, 丝裂原活化蛋白激酶, 氧化应激, 炎症, 大鼠

Abstract: Objective To evaluate the antagonistic effects of N-acetylcysteine (NAC) on mitogen-activated protein kinases (MAPK) pathway activation, oxidative stress and inflammatory responses in rats with lung injury induced by fine particulate matter (PM_2.5).Methods Forty eight male Wistar rats were randomly divided into six groups: blank control group (C1), water drip control group (C2), PM_2.5 exposed group (P), low-dose NAC treated and PM_2.5 exposed group (L), middle-dose NAC treated and PM_2.5 exposed group (M), and high-dose NAC treated and PM_2.5 exposed group (H). PM_2.5 suspension (7.5 mg/kg) was administered tracheally once a week for four times. NAC of 125 mg/kg, 250 mg/kg and 500 mg/kg was delivered intragastrically to L, M and H group respectively by gavage (10 ml/kg) for six days before PM_2.5 exposure. The histopathological changes and human mucin 5 subtype AC (MUC5AC) content in lung tissue of rats were evaluated. We investigated IL-6 in serum and bronchoalveolar lavage fluid (BALF) by Enzyme-linked immunosorbent assay (ELISA), MUC5AC in lung tissue homogenate by ELISA, glutathione peroxidase (GSH-PX) in serum and BALF by spectrophotometry, and the expression of p-ERK1/2, p-JNK1/2 and p-p38 proteins by Western blot. All the measurements were analyzed and compared statistically.Results Lung tissue of rats exposed to PM_2.5 showed histological destruction and increased mucus secretion of bronchial epithelial cells. Rats receiving NAC treatment showed less histological destruction and mucus secretion. Of P, L, M and H group, MUC5AC in lung tissue, IL-6 in serum and BALF were higher than controls (C1 and C2) (all P<0.05), with the highest levels found in the P group and a decreasing trend with increase of NAC dose. The activity of GSH-PX in serum and BALF of PM_2.5 exposed rats (P, L, M and H) was lower than that of controls (all P<0.05), with higher activities found in NAC treated rats (L, M, and H), and an increasing trend with increase of NAC dose. The expressions of p-ERK1/2, p-JNK1/2 and p-p38 proteins in PM_2.5 exposed lung tissue (P, L, M and H) was higher than controls (all P<0.05), with decreased levels and dose dependent downregulation found in NAC treated rats.Conclusion NAC can antagonize major MAPK pathway activation, lung oxidative stress and inflammatory injury induced by PM_2.5 in rats.

Key words: fine particulate matter (PM_2.5), N-acetylcysteine, mitogen-activated protein kinases, oxidative stress, inflammatory response, rats

Ping Fen, Cao Qin, Lin Hua, Han Shuzhi. Antagonistic Effects of N-acetylcysteine on Mitogen-activated Protein Kinase Pathway Activation, Oxidative Stress and Inflammatory Responses in Rats with PM_2.5 Induced Lung Injuries[J].Chinese Medical Sciences Journal, 2019, 34(4): 270-276.

图/表 5

参考文献 22.

1.	Roy A, Gong J, Thomas DC , et al. The cardiopulmonary effects of ambient air pollution and mechanistic pathways: a comparative hierarchical pathway analysis. PLoS ONE 2014; 9(12):e114913.doi: 10.1371/journal.pone.0114913.
2.	Ghio AJ, Carraway MS, Madden MC . Composition of air pollution particles and oxidative stress in cells, tissues, and living systems. J Toxicol Environ Health B Crit Rev 2012; 15(1):1-21.doi: 10.1080/10937404.2012.632359.
3.	Schulz AJ, Mentz GB, Sampson NR , et al. Effects of particulate matter and antioxidant dietary intake on blood pressure. Am J Public Health 2015; 105(6):1254-61. doi: 10.2105/AJPH.2014.302176.
4.	Du X, Jiang S, Bo L , et al. Combined effects of vitamin E and omega-3 fatty acids on protecting ambient PM2.5-induced cardiovascular injury in rats. Chemosphere 2017; 173:14-21. doi: 10.1016/j.chemosphere.2017.01.042.
5.	Thai P, Loukoianov A, Wachi S , et al. Regulation of airway mucin gene expression. Annu Rev Physiol 2008; 70:405-29. doi: 10.1146/annurev.physiol.70.113006.100441.
6.	Cao J, Qin G, Shi R , et al. Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells. J Appl Toxicol 2016; 36(4):609-17. doi: 10.1002/jat.3249.
7.	Jarvis IW, Bergvall C, Morales DA , et al. Nanomolar levels of PAHs in extracts from urban air induce MAPK signaling in HepG2 cells. Toxicol Lett 2014; 229(1):25-32. doi: 10.1016/j.toxlet.2014.06.013.
8.	Samoli E, Stafoggia M, Rodopoulou S , et al. Associations between fine and coarse particles and mortality in Mediterranean cities: results from the MED-PARTICLES project. Environ Health Perspect 2013; 121(8):932-8. doi: 10.1289/ehp.1206124.
9.	Zhou B, Shen H, Huang Y , et al. Daily variations of size-segregated ambient particulate matter in Beijing. Environ Pollut 2015; 197:36-42. doi: 10.1016/j.envpol.2014.11.029.
10.	Li R, Kou X, Xie L , et al. Effects of ambient PM2.5 on pathological injury, inflammation, oxidative stress, metabolic enzyme activity, and expression of c-fos and c-jun in lungs of rats. Environ Sci Pollut Res Int 2015; 22(24):20167-76. doi: 10.1007/s11356-015-5222-z.
11.	Chuang KJ, Chan CC, Su TC , et al. The effect of urban air pollution on inflammation, oxidative stress, coagulation, and autonomic dysfunction in young adults. Am J Respir Crit Care Med 2007; 176(4):370-6. doi: 10.1164/rccm.200611-1627oc.
12.	Wang G, Zhao J, Jiang R , et al. Rat lung response to ozone and fine particulate matter (PM2.5) exposures. Environ Toxicol 2015; 30(3):343-56. doi: 10.1002/tox.21912.
13.	Jin X, Su R, Li R , et al. Amelioration of particulate matter-induced oxidative damage by vitamin c and quercetin in human bronchial epithelial cells. Chemosphere 2016; 144:459-66. doi: 10.1016/j.chemosphere.2015.09.023.
14.	Elbini DI, Jallouli M, Annabi A , et al. A minireview on N-acetylcysteine: An old drug with new approaches. Life Sci 2016; 151:359-63. doi: 10.1016/j.lfs.2016.03.003.
15.	Nouri A, Heidarian E, Nikoukar M . Effects of N-acetyl cysteine on oxidative stress and TNF-alpha gene expression in diclofenac-induced hepatotoxicity in rats. Toxicol Mech Methods 2017; 27(8):1-7. doi: 10.1080/15376516.2017.1334732.
16.	Thai P, Loukoianov A, Wachi S , et al. Regulation of airway mucin gene expression. Annu Rev Physiol 2008; 70:405-29. doi: 10.1146/annurev.physiol.70.113006.100441.
17.	Iwanaga K, Elliott MS, Vedal S , et al. Urban particulate matter induces pro-remodeling factors by airway epithelial cells from healthy and asthmatic children. Inhal Toxicol 2013; 25(12):653-60. doi: 10.3109/08958378.2013.827283.
18.	Schuh K, Pahl A . Inhibition of the MAP kinase ERK protects from lipopolysaccharide-induced lung injury. Biochem Pharmacol 2009; 77(12):1827-34. doi: 10.1016/j.bcp.2009.03.012.
19.	Xia Z, Dickens M, Raingeaud J , et al. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995; 270(5240):1326-31. doi: 10.1126/science.270.5240.1326.
20.	Seriani R , Junqueira MS, de Toledo AC, et al. Diesel exhaust particulates affect cell signaling, mucin profiles, and apoptosis in trachea explants of Balb/C mice. Environ Toxicol 2015; 30(11):1297-308. doi: 10.1002/tox.22000.
21.	Song K S, Lee W J, Chung K C , et al. Interleukin-1 beta and tumor necrosis factor-alpha induce MUC5AC overexpression through a mechanism involving ERK/p38 mitogen-activated protein kinases-MSK1-CREB activation in human airway epithelial cells. J Biol Chem 2003; 278(26):23243-50. doi: 10.1074/jbc.M300096200.
22.	Wang J, Huang J, Wang L , et al. Urban particulate matter triggers lung inflammation via the ROS-MAPK-NF-kB signaling pathway. J Thorac Dis 2017; 9(11):4398-412. doi: 10.21037/jtd.2017.09.135.

Group	Lung tissue MUC5AC (pg/mg)	Serum		BALF
Group	Lung tissue MUC5AC (pg/mg)	IL-6 (pg/mg)	GSH-PX (U)	IL-6 (pg/ml)	GSH-PX (U)
C1	108.63±9.39	29.50±2.60	4568.56±203.53	41.27±2.24	190.53±2.71
C2	110.54±4.94	30.36±3.49	4531.75±129.02	41.47±2.31	189.46±2.46
P	192.25±13.52^ab	56.20±4.89^ab	1512.11±278.37^ab	71.85±2.44^ab	120.22±1.91^ab
L	171.21±12.58^abc	52.59±4.38^ab	1527.53±145.73^ab	69.63±1.95^ab	125.77±2.93^abc
M	153.06±4.60^abcd	46.43±3.29^abcd	2487.14±247.75^abcd	63.22±2.71^abcd	145.71±2.29^abcd
H	139.04±3.67^abcde	40.40±2.77^abcde	2659.91±197.28^abcd	56.54±1.46^abcde	181.58±1.57^abcde
F value	88.409	72.203	296.754	275.291	1333.633
P value	0.000	0.000	0.000	0.000	0.000

Group	p-ERK1/2	p-JNK1/2	p-p38
C1	0.40±0.02	0.31±0.01	0.20±0.02
C2	0.40±0.02	0.31±0.02	0.20±0.02
P	0.81±0.03^ab	0.82±0.03^ab	0.88±0.04^ab
L	0.75±0.01^abc	0.66±0.01^abc	0.63±0.01^abc
M	0.61±0.02^abcd	0.50±0.02^abcd	0.47±0.01^abcd
H	0.49±0.01^abcde	0.37±0.02^abcde	0.29±0.02^abcde
F value	201.256	333.001	524.187
P value	0.000	0.000	0.000

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