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峡皑秘
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1 94 1/2 * /2 (extracellular signal-regulated kinase, ERK1/2) (bronchial smooth muscle cells, BSMCs) BSMCs Western blot RT-PCR ERK1/2 BSMCs ERK1/2 Western blot BSMCs ERK1/2 (9.13±0.87) (4.68±0.59) ERK1/2 (p-erk1/2) ERK1/2 (0.55±0.05) (0.48±0.04) (n=10, P<0.01) ERK1 ERK2 mrna (1.83± ±0.11) (0.58± ±0.12) (n=10, P<0.01) BSMCs (2.9±0.1) ERK1/2 (epidermal growth factor EGF) (5.0±0.2) 30 mol/l PD98059 (1.7±0.2) BSMCs PD mol/l PD98059 (0.8±0.1) BSMCs (1.9±0.1) EGF (3.1±0.2) 30 mol/l PD98059 (1.45±0.2) BSMCs ERK1/2 R329 Role of extracellular signal-regulated kinase 1/2 signaling pathway in migration of bronchial smooth muscle cells of chronic asthmatic rats XIE Min, LIU Xian-Sheng *, XU Yong-Jian, ZHANG Zhen-Xiang, BAI Jing, NI Wang, CHEN Shi-Xin Department of Respiratory Medicine, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China Abstract: This work was designed to explore the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway in migration of bronchial smooth muscle cells (BSMCs) of chronic asthmatic rats. To make chronic asthma model, Wistar rats underwent ovabumin (OVA) injection and eight-week inhalation. BSMCs were cultured in vitro. The expression of ERK1/2 in BSMCs was analyzed by immunocytochemistry, Western blot and RT-PCR. Migration of BSMCs was detected by both plate test and Boyden cell test. Results showed: (1) With Western blot technique, the ratio of p-erk1/2 to total ERK1/2 in chronic asthmatic group was obviously higher than that in the control group (0.55±0.05 vs 0.48±0.04, n=10, P<0.01). (2) With RT-PCR, the relative A values of ERK1 and ERK2 mrna in airways of chronic asthmatic rats were 1.83±0.24 and 1.07±0.11, respectively, which were significantly increased compared with that in the control group (0.58±0.14 and 0.51±0.12, n=10, P<0.01). (3) In plate test, the migration of BSMCs of chronic asthmatic rats was 2.9 times of that in the control group and reached 5.0 times by epidermal growth factor (EGF) stimulation, but decreased to 1.7 times Received Accepted This work was supported by the National Natural Science Foundation of China (No ) and the Dawn Program for Youth Pioneering in Technology of Wuhan Municipality (No ). * Corresponding author. Tel: ; Liuxiansheng@tjh.tjmu.edu.cn

2 ERK1/2 BSMCs 95 by 30 µmol/l PD (4) In Boyden cell test, the migration of BSMCs of chronic asthmatic rats was 1.9 times of that in the control group, and reached 3.1 times by EGF stimulation, but decreased to 1.45 times by 30 µmol/l PD Our results indicate that the migration ability of BSMCs of chronic asthmatic rats increases, and ERK1/2 signaling pathway may play an important role in this process. Key words: extracellular signal-regulated kinase; asthma; bronchial smooth muscle cell; migration (bronchial smooth muscle cells, BSMCs) [1] BSMCs BSMCs [2] BSMCs 1/2 (extracellular signalregulated kinase, ERK1/2) [3,4] ERK BSMCs BSMCs ERK1/ (ovabumin, OVA) Sigma PD98059 Biosource (epidermal growth factor, EGF) Accurate Chemical DMEM Hyclone ERK1/2 ERK1/2 (phosphorylated-erk1/2, p-erk1/2) R&D α-actin β-actin Santa Cruz BCA Pierce (RT-PCR) Promega PCR TaKaRa TRIzol Gibcol Transwell Corning Wistar ~200 g (10 ) (10 ) [4] 1 10 mg OVA 200 mg 1 ml 1 ml ( ) % OVA 50 ml 30 min h [5] 20% 6 ml/ kg 3 ml (3 ) 800 r/ min 10 min /ml 4% 400 (eosinophil, Eos) 4% HE ( 100~200 ) [6] 2000 (Pbm) (WAt) (WAm) Pbm WAt/Pbm WAm/Pbm /Pbm BSMCs [7] BSMCs 1 mm 3 (NaCl 135 mmol/l KCl 15 mmol/l MgCl 2 22 mmol/l 10 mmol/l HEPES 10 mmol/l 2 g/l 5 g/l BSA

3 96 2 g/l) 37 20~30 min 20% (fetal bovine serum, FBS) DMEM /L (37, 5% CO 2 ) 5~7 d - α-actin 0.25% 1:2 24 h 1.5 ERK1/2 ERK1/2 ERK1/2 (oligodeoxynucleotide, ODN) 5 -GCCGCCGCCGC- CGCCAT ATGGCGGCGGCGGCGGC-3 ODN TaKaRa 2 µg/ml 5 min 1 mol/l ODN 20 min 6 h 0. 5 mol/l ODN 18 h 42 h 1.6 ERK1/2 BSMCs min PBS 5 min (p-erk 1:100 α-actin 1:100) 4 1:100 2 h PBS PBS Nikon TE HMIAS Western blot BCA 50 g 8% SDS p-erk1/2 (1:1 000) ERK1/2 (1:1 000) β-actin (1:400) 4 (1:1 000) 1 h DAB HMIAS RT-PCR TRIzol RNA RNA 2 µg cdna ERK1 5 -GCTGAATCACATCCTGGGTAT-3 5 -AGATCTGTATCCTGGCTGGAA bp ERK2 5 -GCAGGTGTTC- GACGTGGGAAT-3 5 -GTGCAGAACATTAG- GTGAATA bp β-actin 5 -GTCACCAACTGGGACGATA AGGTCTTTACGGATGTCAACG bp ERK1 ERK2 PCR 94 3 min s 58 1 min 72 1 min min MUVB-20 ( UltraLum ) ERK1 ERK2 1.7 BSMCs 4 A BSMCs B BSMCs C BSMCs 10 ng/ml EGF D BSMCs ERK1/ 2 ODN A B C µmol/l PD98059 EGF PD min Sarkar [8] 24 BSMCs FBS DMEM 24 h BSMCs 48 h BSMC 3 12 EGF Boyden (Transwell 8 µm ) FBS 10% FBS-DMEM 37 5% CO 2 24 h Hanks EGF 1.8 SPSS11.5 mean±sd t q P<0.05

4 ERK1/2 BSMCs 97 Pearson OVA Eos ( 1) [(71.42±6.77) µm 2 /µm] [(19.01±0.41) µm 2 /µm] [(0.16±0.02) /µm] Eos [(3. 82±0.29)%] [ (30.23± 4.11) µm 2 /µm (8.97±0.31) µm 2 /µm (0.09±0.01) /µm (1. 11±0.17)%] (n=10 t P <0.01) 2.2 BSMCs - actin 1. (HE ) Fig. 1. Pathological change of bronchioles in lung of chronic asthmatic rats (HE staining). Compared with normal control (A), the thickness of both airway wall and smooth muscle layer increased markedly in chronic asthmatic rats (B), companied by infiltration of inflammation cells into airway wall and lung parenchyma. Scale bar, 80.6 µm. BSMCs ( 2) 2.3 ERK1/2 BSMCs p-erk1/2 BSMCs BSMCs (80.5± 3.6) (30.1±2.4) (n=10, P<0.01) BSMCs p-erk1/2 (r=0.721, P<0.01) p-erk1/2 BSMCs Eos (r=0.652, P<0.01) Western blot β-actin A ERK1/2 p-erk1/2 BSMCs ( 3) p-erk1/2 ERK1/2 (ERK ) (0.55±0.05 vs 0.48± 2. α-actin ( ) Fig. 2. Identification of cultured bronchial smooth muscle cells by immunofluorescence cytochemical staining of anti-smooth muscle α-actin antibody. Scale bar, 20 µm. 0.04, n=10, P<0.01) RT-PCR PCR β-actin A ERK1 ERK2 mrna

5 98 3. Western blot ERK1/2 Fig. 3. Western blot analysis of ERK1/2 in bronchial smooth muscle cells of chronic asthmatic rats. The blotting straps (A) and histogram of relative A value (B) show that both total ERK1/2 and p-erk1/2 were highly expressed in BSMCs of chronic asthmatic rats after normalized by β-actin. * P<0.05 compared with control. ( 4) 1.83± ±0.11 ERK1 ERK2 A 0.58± ±0.12 (P <0.01) 2.4 BSMCs ERK1/ BSMCs BSMCs (P< 0.05) ERK1/2 EGF BSMCs EGF ERK1/2 PD98059 BSMCs BSMCs EGF BSMCs ( 5) PD98059 BSMCs 50 µmol/l 100 µmol/l 20% BSMCs 10 µmol/l PD µmol/l 60% BSMCs EGF BSMCs 10 µmol/l PD RT-PCR ERK1 ERK2 mrna Fig.4. RT-PCR analysis of ERK1/2 in bronchial smooth muscle cells of chronic asthmatic rats.the photos show the mrna expressions of ERK1 (A) and ERK2 (B) in BSMCs of chronic asthmatic rats were higher than that in the control group.

6 ERK1/2 BSMCs Fig.5. Plate migration test for bronchial smooth muscle cells (BSMCs) of chronic asthmatic rats. The photos show the migration of BSMCs 24 h after scratch. A: Control. A5 was treated with 100 mol/l PD B: Chronic asthma group. B3 was treated with 30 mol/l PD C: BSMCs of chronic asthmatic rats irritated by 10 ng/ml EGF. C3 was pretreated with 30 mol/l PD98059 before EGF stimulation. D: BSMCs of chronic asthmatic rats treated with sense ODN. D A was transfected with antisense ODN. Scale bar, 80.6 µm. E: The dot-line graph shows the concentration-dependent effect of PD98059 on BSMCs in the control, chronic asthmatic rats and EGFirritated BSMCs of chronic asthmatic rats. * P<0.05 compared with the control group without PD98059, Σ P<0.05 compared with asthma group without PD98059, # P<0.05 compared with asthma + EGF group without PD98059.

7 Fig. 6. Transmembrane migration test for bronchial smooth muscle cells (BSMCs) of chronic asthmatic rats. The photos show the migration of BSMCs 24 h after scratch. A: Control group without any medication. A5 was treated with 100 mol/l PD B: Chronic asthma group B3 was treated with 30 µmol/l PD C: BSMCs of chronic asthmatic rats irritated by 10 ng/ml EGF. C3 was pretreated with 30 µmol/l PD98059 before EGF stimulation. D: BSMCs of chronic asthmatic rats treated with sense ODN. D A was transfected with antisense ODN. Scale bar, 42.2 µm. E: The dot-line graph shows the concentration-dependent effect of PD98059 on BSMCs in the control, chronic asthmatic rats and EGF-irritated BSMCs of chronic asthmatic rats. * P<0.05 compared with the control groups without PD98059, Σ P<0.05 compared with asthma group without PD98059, # P<0.05 compared with asthma + EGF group without PD98059.

8 ERK1/2 BSMCs µmol/l 80% ODN BSMCs (P<0.01) ODN BSMCs BSMCs EGF BSMCs EGF ERK1/2 PD98059 BSMCs BSMCs EGF BSMCs ( 6) PD98059 BSMCs 50 µmol/l 100 µmol/l 20% BSMCs 10 µmol/l PD µmol/l 50% BSMCs EGF BSMCs 10 µmol/l PD µmol/l 50% ODN BSMCs (P<0.01) ODN 3 BSMCs (vascular smooth muscle cell, VSMCs) BSMCs BSMCs BSMCs [2,9] BSMCs BSMCs ERK VSMCs [2,9-11] BSMCs ERK1/2 ERK1/2 p-erk1/2 p-erk1/2 ERK1/2 ERK1/2 BSMCs mrna ERK1 ERK2 ERK1/2 BSMCs p-erk1/2 Eos ERK1/2 BSMCs BSMCs ERK ERK1/2 EGF PD98059 ERK1/2 ODN ERK BSMCs ERK1/2 EGF BSMCs ERK1/2 PD98059 BSMCs ERK1/2 ODN BSMCs ERK1/2 BSMCs BSMCs BSMCs EGF BSMCs PD98059 BSMCs PD % BSMCs PD98059 ERK1/2

9 102 BSMCs BSMCs PD98059 BSMCs BSMCs IL-13 TNF VEGF ERK1/2 ERK1/2 BSMCs ERK1/2 ERK1/2 BSMCs BSMCs ERK1/2 1 Johnson PR, Burgess JK. Airway smooth muscle and fibroblasts in the pathogenesis of asthma. Curr Allergy Asthma Rep 2004; 4: Madison JM. Migration of airway smooth muscle cells. Am J Respir Cell Mol Biol 2003; 29: Zhou L, Hershenson MB. Mitogenic signaling pathway in airway smooth muscle. Respir Physiol Neurobiol 2003; 137: Jiang Y ( ), Gong XW. Regulation of inflammatory responses by MAPK signal transduction pathways. Acta Physiol Sin ( ) 2000; 52: (Chinese, English abstract). 5 Li CQ ( ), Xu YJ, Yang DL, Shi HZ, Liu XS, Xiong WN, Chen SX, Ni W, Zhang ZX. A study of helper T cell (Th)1/Th2 immune response pattern of gammadeltat cells in asthma. Chin J Intern Med ( ) 2004; 43(5): (Chinese, English abstract). 6 Bai A, Eidelman DH, Hogg JC, James AL, Lambert RK, Ludwig MS, Martin J, McDonald DM, Mitzner WA, Okazawa M. Proposed nomenclature for quantifying subdivisions of the bronchial wall. J Appl Physiol 1994; 77: Liu XS ( ), XU YJ, Zhang ZX, Ni W, Chen SX. Effect of protein kinase C on K V channel in rat bronchial smooth muscle. Acta Physiol Sin ( ) 2003; 55: (Chinese, English abstract). 8 Sarkar R, Meinberg EG, Stanley JC, Gondon D, Webb RC. Nitric oxide reversibly inhibits the migration of cultured vascular smooth muscle cells. Circ Res 1996; 78: Gizycki, MJ, Adelroth E, Rogers AV, O Byrne PM, Jeffery PK. Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am J Respir Cell Mol Biol 1997; 16: Wang TH ( ), Tan Z, Fu XD, Yang D, Hu FX, Li YY. Effect of ERK on 17 β-estradiol-induced inhibition of VSMC proliferation in rats after vascular injury. Acta Physiol Sin ( ) 2003; 55(4): (Chinese, English abstract). 11 Zhu JH ( ), Liu Z, Huang ZY, Li S. Effects of angiotensin II on extracellular signal-regulated protein kinases signaling pathway in cultured vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats. Acta Physiol Sin ( ) 2005; 57(5): (Chinese, English abstract).

标题

环球中医药 2016 年 5 月第 9 卷第 5 期摇 Global Traditional Chinese Medicine, May 2016,Vol 郾 9, No 郾 5 摇 557 两种电针对脊髓损伤 14 天后大鼠运动功能神经元及 MEK2 p 鄄 ERK1 表达的影响论著宋良玉摇吕威摇景泉凯摇莫雨