Dynamic changes of found in inflammatory zone 1 protein and mrna expression in the lung with experimental pulmonary fibrosis of the rat

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1 Acta Physiologica Sinica, August 25, 2005, 57 (4): FIZZ1 mrna 1 2, * FIZZ1 (found in inflammatory zone 1) (5 mg/kg ) HE Masson FIZZ1 mrna (1) (7 d) (14~21 d)(28 d)(2) FIZZ1 7 d 14 d 7 d d (3) FIZZ1 mrna 7 d 14 d d FIZZ1 mrna FIZZ1 mrna Q593; R362; R563.1 Dynamic changes of found in inflammatory zone 1 protein and mrna expression in the lung with experimental pulmonary fibrosis of the rat MA Wan-Li 1, YE Hong 2,*, TAO Xiao-Nan 1, XIN Jian-Bao 1 1 Pulmonary Laboratory of Ministry of Health of China, Department of Respiratory Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China; 2 Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan , China Abstract To investigate the role of found in inflammatory zone 1 (FIZZ1) protein in the pathogenesis of experimental pulmonary fibrosis, 48 male Sprague-Dawley rats were randomly divided into two groups, the pulmonary fibrosis group and the control group. Rat pulmonary fibrosis was reproduced by an intratracheal injection of bleomycin (5 mg/kg body weight). Normal saline (1 ml/kg body weight) was given intratracheally injection in the control group. There were 24 rats in each group, and 6 animals were separately killed on the 7 th, 14 th, 21 th and 28 th day after treated with bleomycin or normal saline. Then the following tests were undertaken: (1) HE and Masson staining of lung section; (2) Determination of lung tissue hydroxyproline (HYP); (3) Immunohistochemical staining of protein of FIZZ1 in the lung; (4) In situ hybridization of FIZZ1 mrna in the lung. The results showed: (1) There were full of inflammatory cells in the lung, the interval of alveoli enlarged and many alveolar spaces disappeared on the 7 th day after treated with bleomycin in the fibrosis group. Collagen began to proliferate after 14 d. The pulmonary fibrosis was stably established on the 28 th day, full of green fibers in the Masson staining of lung section. (2) The expression of FIZZ1 protein in the lung increased after 7 d in bleomycin-treated animals (3.013±0.326 vs 0.473±0.056, P<0.01 vs control), but was slightly decreased on the 14 th day (2.124±0.197) and expensively decreased on the 21 st day (1.760±0.105) and the 28 th day (0.691±0.081). (3) The expression of FIZZ1 mrna in the lung also increased after 7 d by treated with bleomycin (3.795±0.338 vs 0.678±0.087, P<0.01 vs control), but decreased on the 14 th day (1.276±0.104) and further Received Accepted * Corresponding author. Tel: ; yehongmwl@hotmail. com.

2 494 Acta Physiologica Sinica, August 25, 2005, 57(4): decreased on the 28 th day (0.896±0.084). The expression of FIZZ1 protein and mrna in fibrosis group was higher than that in the control group (P<0.05 or P<0.01). The results suggest that FIZZ1 protein and FIZZ1 mrna are dynamically changed in the lung with experimental pulmonary fibrosis, which may contribute to the pathogenesis of pulmonary fibrosis. Key words: pulmonary; fibrosis; found in inflammatory zone 1 FIZZ (found in inflammatory zone) 2000 [1] FIZZ1 FIZZ [2-5] FIZZ (serious acute respiratory syndrome, SARS) 28 FIZZ1 mrna FIZZ Sprague-Dawley ~230 g A5 FIZZ1 Santa Cruz (USA) FIZZ1 ( ) % 3 ml/kg 5 mg/kg A5 (1 ml/kg ) % HE Masson HE Masson 60 2 h HE Masson mol/l HCl h; 6 mol/l NaOH HCl; 1 mol/l NaOH 1 mol/l HCl ph r/min 10 min 1 ml 0.5 ml 0.05 mol/l T 1ml 20 min 3.15 mol/l ml 5 min 10% 1 ml min 560 nm (SABC ) FIZZ h PBS 3 min 3 0.3% Triton X min 3% H 2 O 2 15 min 10% 30 min 1:100 FIZZ1 37 1~2 h mol/l PBS 5 min 3 IgG min 0.01 mol/l PBS 5 min 3 SAB min 0.01 mol/l PBS 5 min 4 DAB FIZZ1 mrna FIZZ1 [6] FIZZ1 FIZZ1 mrna : 5'-GAAGT CCAGA ATGAA AATAT TTGCA ACTGC CTGTG C-3' 0.5% H 2 O 2 / h 20 µl

3 : FIZZ1 mrna SSC 0.2 SSC min 0.5 mol/l PBS 3 IgG min 0.5 mol/l PBS 3 BronchioliBC min 0.5 mol/l PBS 4 DAB Image Pro-Plus (A ) mean ± SD t HE Masson 7 d 14 d 7 d 21 d 28 d Masson HE Masson ( 1 1) 1. Table 1. Content of hydroxyproline (HYP) in the lung tissue (mean ± SD, n=24) (mg/g) Hydroxyproline (mg/g lung tissue) 7 d 14 d 21 d 28 d Control 2.31± ± ± ±0.04 Fibrosis 2.29± ±0.05 * 3.11±0.05 ** 3.85±0.05 ** 2.2 FIZZ1 FIZZ1 7 d 14 d 7 d 21 d 28 d FIZZ1 mrna FIZZ1 FIZZ1 mrna 7 d 14 d 21 d 28 d ( 3 2) 2. FIZZ1 Table 2. Results of immunohistochemical staining of FIZZ1 protein in the lung (A value, mean ± SD, n=24) FIZZ1 protein (A) 7 d 14 d 21 d 28 d Control 0.473± ± ± ±0.32 Fibrosis 3.013±0.326 ** ± ** 1.760±0.105 ** 0.691±0.081 * 3. FIZZ1 mrna Table 3. Results of in situ hybridization of FIZZ1 mrna in the lung (A value, mean ± SD, n=24) FIZZ1 mrna (A) 7 d 14 d 21 d 28 d Control 0.678± ± ± ±0.091 Fibrosis 3.795±0.338 ** 1.276±0.104 ** 1.003±0.110 * 0.896±0.084 *

4 496 Acta Physiologica Sinica, August 25, 2005, 57(4): HE Masson Fig. 1. HE and Masson staining in the lung of rat from different phases with experimental pulmonary fibrosis. A: HE, control. B: HE, d 7, alveolitis phase. C: Masson, d14, fibers proliferating phase, the green staining is fiber. D: Masson, d 28, pulmonary fibrosis, full of green staining fibers. Scale bar, 25 µm. 2. FIZZ1 mrna Fig. 2. In situ hybridization of FIZZ1 mrna in the lung of rat from different phases with experimental pulmonary fibrosis. A: Control, the brown staining is the positive of FIZZ1 mrna. B: d 7, alveolitis phase, expressions of FIZZ1 mrna obviously enhanced. C: d 14, fibers proliferating phase. D: d 28, pulmonary fibrosis. Scale bar, 25 µm.

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