Role Of A_2B Adenosine Receptor-Dependent Adenosine Signaling In Multi-Walled Carbon Nanotube-Triggered Lung Fibrosis In Mice

Carbon nanotubes（CNTs）have been used in a variety of applications because of their unique properties and functions,one of the most common forms is multi-walled carbon nanotube（MWCNT）.MWCNT have been shown to induce lung fibrosis in experimental animals with deposition of collagen fibers and formation of tissue scars,raising concern over possible toxic effects of MWCNT exposure in humans.The adenosine（ADO）is produced in response to injury and serves a detrimental role in lung fibrosis.However,the mechanisms underlying fibrosis pathogenesis remains unclear.The current study focuses on investigating the ADO signaling in the progression of lung fibrosis induced by MWCNT,in order to facilitate the understanding,monitoring,and treatment of MWCNT-induced lung lesions that might occur in exposed populations.Male C57BL/6 mice（6-8 weeks old）were exposed to MWCNT in a single dose of 40 μg by pharyngeal aspiration to examine the fibrotic responses of MWCNT exposure.（1）Mice were randomly divided into control（dispersion medium）group and MWCNT group（n=6）.At 1,3,7,and 14 days post-exposure,mice were randomly sacrificed and then bronchoalveolar lavage fluid（BALF）and lung tissue were collected.The lung tissue was stained with hematoxylin and eosin（HE）for routine morphologic assessment and with Masson’s Trichrome for evaluating fibrotic changes.The m RNA expressions of ecto-5’-nucleotidase（CD73）and A_2B adenosine receptor(A_2BAR)were analyzed by q RT-PCR.The ADO level in BALF was analyzed by high-performance liquid chromatography（HPLC）.Myeloperoxidase（MPO）in lung tissue were detected by commercial kits（2）Mice were randomly divided into control,MWCNT,MWCNT+CVT-6883,and CVT-6883,6 of each group.The CVT-6883（1 mg/kg）was used in MWCNT studies,where twice daily intraperitoneal injections were given on days 3-7.At 7 days post-exposure,mice were sacrificed and then blood samples,bronchoalveolar lavage fluid（BALF）,and lung tissue were collected.Percentage of neutrophils（Neu（%））in peripheral blood of mice were obtained using complete blood cell count.Lactate dehydrogenase（LDH）in serum were detected by kits.The concentration of Interleukin-6（IL-6）in the BALF was measured by enzyme-linked immunosorbent assay（ELISA）.The protein levels of IL-6 and transforming growth factor-β1（TGF-β1）signaling pathway related proteins in lung tissues were detected by western blot.The m RNA expressions of follistatin-like 1（Fstl1）and TGF-β1 in lung tissues were analyzed by q RT-PCR.The results showed:（1）Histopathological studies determined that MWCNT exposure caused rapid development of pulmonary fibrosis by 7 days post-exposure,that granulomatous inflammation persisted throughout the 14-day post-exposure period.MWCNT exposure led to a significant increase in disease pathology compared to control group.The fibrotic lesions significantly increased in MWCNT treatment lungs compared with control at all the time points examined.（2）Treatment of mice with MWCNT significantly increased CD73 gene expression on whole lung homogenate at 7 days post-exposure.However,CD73 gene expression appeared to be more variable at day 14 post exposure.MWCNT exposed mice exhibited significantly increased in the m RNA expression of A_2BAR at 7 days post-exposure,and this elevation remained throughout 14 days after MWCNT exposure.MWCNT treatment significantly elicited ADO level in BALF from day 3 to 14.（3）MWCNT treatment enhanced MPO activity from day 7 to 14.From these results,we concluded that MWCNT induced significant pulmonary inflammation and fibrosis after 7 days exposure and ADO signaling involved in MWCNT-induced lung fibrosis.（4）CVT-6883 treatment significantly reduced MWCNT-induced Neu（%）elevation and cytotoxicity.（5）The levels of IL-6 significantly increased in BALF of the MWCNT-induced group as compared with control mice.In contrast,this augmentation was significantly inhibited by the treatment of CVT-6883.MWCNT treatment resulted in a significant rise in the protein level of IL-6,TGF-β1,p-Smad3,collagen I,fibronectin 1（FN1）,follistatin-like 1（Fstl1）,α-smooth muscle actin（α-SMA）,platelet-derived growth factor receptor-β（PDGFR-β）,heat shock protein 47（HSP47）,and fibroblast-specific protein 1（FSP1）.CVT-6883 treatment inhibited the increased in these protein levels induced by MWCNT.CVT-6883 treatment reduced the elevated m RNA expressions of TGF-beta 1 and Fstl1 induced by MWCNT.Our results reveal that accumulation of extracellular ADO promotes the process of the fibroblast-to-myofibroblast transition via A_2BAR/TGF-β1/Fstl1 signaling in MWCNT-induced lung fibrosis.CVT-6883 attenuates collagen deposition and progressive pulmonary fibrogenesis induced by MWCNT.To the best of our knowledge,modulation of ADO levels and antagonism of A_2BAR-mediated responses may be a novel therapeutic approach for MWCNT-induced lung fibrosis.