Abstract: Objective: To observe the effects of A_2aR on pulmonary fibrosis induced by bleomycin (BLM) in wild type and A_2aR gene knockout mice.Methods: Thirty male BALB/c wild type (WT) mice and twenty male A_2aR gene knockout (A_2aR KO) mice, were randomly divided into 5 groups (n=10 each):WT control group (A), WT fibrosis group (B), A_2aR KO control group(C), A_2aR KO fibrosis group(D), WT fibrosis+A_2aR agonist (CGS21680) group (E). For the induction of pulmonary fibrosis, mice were intratracheally injected with a single dose of bleomycin (5.0 mg/kg body weight), while the control groups with an equal volume of NS. After injection, the mice were vertically rotated immediately for 3 or 5 minutes in order to make the liquor evenly distributed in lung. The mice in groups A to D received intraperitoneal injection of 0.5 ml NS, those in E group received 0.5 ml CGS-21680 (0.25 mg/kg body weight) daily for a period of 4 weeks. On the 29th day, the blood and tissue samples were taken. The chloramine T method was used to detect the content of hydroxyproline (Hyp) in lung. ELISA was used to detect transforming growth factor-β1 (TGF-β1) in sera. The immunohistochemical technique and Western blot were used to detect the protein expression of TGF-β1 and A_2aR. And the mRNA expression of TGF-β1 and A_2aR were measured by in situ hybridization. The microstructure, ultrastructure and fiber staining of lung tissue in mice were observed in each group.Results: ①Thickened and destroyed alveolar walls, disordered, stenosed or partial collapsed alveolar spaces, fibrous proliferation and infiltration of inflammation cells were observed in lung of mice in group B under microscope. The described performance was even more serious in group D, and that the intervention in group E, could significantly ameliorate the pathological changes. The vacuolation in epithelial cells of type I and Ⅱ as well as the lamellar body, were observed on the visualizing of ultrastructure of lung tissue in group B. More severe pathological lesion in lung described above were presented in Group D than those in group B, obviously lessen pathological damage were presented in group E. The lung positive region in mice of group B and D was increased significantly with a fusion of patchy distribution by masson staining of fibrous tissue, the lung positive region in group D was higher than that in group B. However, the lung positive area in group E was significantly decresded compared with that in group B and D. The results above indicated A_2aR gene knockout exacerbated the pulmonary fibrosis. ②The content of TGF-β1 in sera and the content of Hyp in lung, the expression of A_2aR protein and TGF-β1 protein, the A_2aR mRNA and TGF-β1 mRNA were significantly increased in group B compared with group A(P <0.01,P<0.05). The content of TGF-β1 in sera, the content of Hyp, the expression of TGF-β1 protein and the TGF-β1 mRNA in lung were significantly increased in group D compared with group C(P <0.01,P<0.05). Those results indicated that content of Hyp and expression of TGF-β1 in mice with lung fibrosis were increased. ③Compared to group B, the content of TGF-β1 in sera and the content of Hyp in lung, the expression of TGF-β1 protein and TGF-β1 mRNA were significantly increased in group D(P<0.01). The expression of A_2aR protein and A_2aR mRNA in group E was higher than those in group B (P<0.01), the content of TGF-β1 in sera, Hyp in lung, the expression of TGF-β1 protein and TGF-β1 mRNA were much less than those in group E (P<0.01). The results above indicated that the content of Hyp and expression of TGF-β1 were increased in lung of A_2aR gene knockout mice, A_2aR agonist might upregulate the expression of A_2aR and deregulate the expression of TGF-β1.Conclusion: Pulmonary fibrosis is significantly increased in A_2aR gene knockout mice. A_2aR response to the pulmonary fibrosis is increased in wild type mice, the effect of A_2aR on anti-inflammatory in BLM-induced pulmonary fibrosis is acted by inhibiting TGF-β1 expression.

Key words: pulmonary fibrosis, bleomycin, TGF-β1, A_2aR, mouse