Nano-Designed CO Donor SMA/CORM2 Ameliorates Bleomycin-Induced Pulmonary Fibrosis Via Manipulating Macrophage Reprogramming

Background Idiopathic pulmonary fibrosis（IPF）is a progressive and irreversible interstitial pulmonary disease due to chronic inflammatory response.The prognosis of IPF is very poor with a median survival of 2–4 years after diagnosis,however,the therapeutic options are very limited.At present,the pathogenesis of IPF has not been fully elucidated,but ample evidences suggest that IPF is associated with immune inflammatory injury.Carbon monoxide（CO）is an important gaseous signal molecule in the body,which exerts cytoprotective effect and maintains homeostasis under physiological and pathological conditions.Previously,we developed a polymeric micellar drug delivery system of CO,i.e.,SMA/CORM2,which exhibited therapeutic potentials against dextran sulphate sodium（DSS）induced mouse colitis and acetaminophen（APAP）induced liver injury.These findings suggested the potential of SMA/CORM2 for the treatment of inflammatory diseases,which motivated us to further examine the usefulness of SMA/CORM2 on IPF.Objective : In this study,we investigated the applicability of SMA/CORM2 on interstitial pulmonary fibrosis using a bleomycin（BLM）induced pulmonary fibrosis model,and the related mechanism of action was also tackled.Methods:（1）BLM（0.035 U/g）was injected intraperitoneally（i.p.）twice a week for 4weeks to establish the pulmonary fibrosis model.In the healthy control group,saline was used instead of BLM.For SMA/CORM2 treatment groups,24 h before each BLM injection,SMA/CORM2 of different concentrations（0.1,1.0,10mg/kg）was administered intravenously（i.v.）,and saline was injected i.v.to some mice as untreated control.On the 33 rd day after the first BLM injection,the pulmonary function tests were carried out,then mice were killed and lung tissues were collected for histological examination,proteins and total RNA were extracted for the tissues for western blotting,real-time RT-PCR to examine the effect and related mechanism of SAM/CORM2 in alleviating BLM-induced interstitial pulmonary fibrosis.（2）In order to determine the effect of CO as the effector molecule of SAM/CORM2 in alleviating BLM-induced interstitial pulmonary fibrosis and the related mechanism,0.1mg/kg hydrolyzed SMA（i.v.）,0.1mg/kg inactivated SMA/CORM2（i SMA/CORM2）（i.v.）,and 0.01mg/kg native CORM2（equivalent to 0.1 mg/kg SAM/CORM2 that has the CO loading of 10%）that was dissolved in DMSO for i.p.injection,were used.（3）The safety of SAM/CORM2 was evaluated by injecting 0.1mg/kg SAM/CORM2 to healthy mice,meanwhile,liver and kidney functions were evaluated by blood biochemistry examinations from mice in the experiment（1）.Result:（1）The results of lung histomorphology,histopathology and Masson examination revealed that severe inflammation and the consequent pulmonary fibrosis were triggered by BLM,whereas SMA/CORM2 treatment remarkably suppressed the inflammation progression and ameliorated the formation of fibrosis.Western blotting and RT-PCR showed that SMA/CORM2 inhibited IPF by regulating the TGF-β/Smad2/3 signaling pathway as well as the subsequent events,epithelial-mesenchymal transition（EMT）（2）Further results showed that SAM/CORM2 significantly inhibited BLM-induced upregulation of CD206 and Arg-1 that are the makers of of anti-inflammatory M2 macrophages.These results indicate that SMA/CORM2 influences the TGF-β/Smad2/3 signaling pathway and EMT,through the regulation of anti-inflammatory macrophage polarization.Meanwhile,we found that the suppression of macrophage polarization accompanied with the downregulated hypoxia-inducible factor-1α（HIF-1α）.（3）Furthermore,we verified that CO is the effector molecule of SMA/CORM2 playing the inhibitory role on IPF,namely i SAM/CORM2 and SMA did not show any effects on the progress of inflammation and lung fibrosis.Moreover,compared to native CORM2 of equivalent CO dose,SMA/CROM2 exhibited a much better protective effect indicating its superior bioavailability.In addition,we evaluated the safety of SMA/CROM2 by examining liver and kidney function and patologivcal examination of the lung,in which no any adverse effects were found after SMA/CORM2 treatment.Conclusion: SMA/CROM2 through releasing CO remarkably alleviated BLM-induced interstitial pulmonary fibrosis by regulating macrophage reprogramming,without any apparent adverse side effects.SMA/CORM2 may thus become a candidate drug for IPF as well as other inflammatory diseases,and we anticipate its application in the future.