The Study On The Protective Mechanism Of Heme Oxygenase-1/ Bilirubin On The Development Of Chronic Obstructive Pulmonary Disease And Comparison Of Their Differences

Chronic obstructive pulmonary disease(COPD), which is one of the most common chronic inflammational diseases in respiratory system, is characterized by irreversible airflow limitation and can be prevented and treated. The restricted airflow resulted in progressive decline of lung function, serious harm to the health of the people and the huge economic burden on society. COPD not only mainly involved the lung that emphysema is the basic pathological change, but also caused systemic(or extrapulmonary) adverse effects. The pathogenesis of COPD may be associated with inflammation, oxidant/antioxidant imbalance, and protease/anti-protease imbalance. Now it is recognized widely that smoking is an important factor in the pathogenesis of COPD. Cigarette smoke contains a lot of harmful particles and gases that can cause inflammation and oxidative stress.Neutrophils, alveolar macrophage and T lymphocytes, which are important inflammatory cells in the development of COPD, are activated by stimuli in vivo and release a variety of inflammatory mediators such as LTB4, TNF-α, IL-17, chemokines, adhesion molecules and so on. In addition, anti-inflammatory cytokine IL-10 secreted by regulatory T cells and the enzymes or drugs which exert an anti-inflammatory or antioxidant effect, play an important role in the development of COPD. Therefore, in recent years, people got more and more attention on the protective effect of heme oxygenase and bilirubin which have anti-inflammatory and anti-oxidant.Heme oxygenase(HO) is a rate-limiting enzyme for heme metabolism and catalyzes heme into equivalent amount of biliverdin, carbon monoxide(CO) and free iron. Subsequently,biliverdin was reducted by biliverdin reductase to bilirubin(BR).Three known HO isoenzymes were reported, including inducible HO-1, constitutive HO-2 and isomer HO-3newly found. HO-1 belongs to stress protein or heat shock protein(HSP)-32, widely presents in mammalian body tissues especially microsomes of monocyte-macrophage cell.HO-1 can be induced not only by the substrate heme, but also by a variety of stimuli associated with oxidative stress and inflammation. Numerous studies have demonstrated that HO-1, as a protective protein, has many significant functions of anti-inflammatory,anti-oxidative stress, anti- apoptotic, anti-proliferation and cytoprotective effects in many diseases. Bilirubin, which is a downstream product of heme metabolism and a strong endogenous antioxidant, has the effects of anti-oxidative, anti-inflammatory, promoting the immune response capability and enhancing the antioxidative capacity of vitamin C and E.Objectives:1. To explore the relation between bilirubin levels and smoking, quit-smoking and COPD disease state, in order to provide a theoretical basis for quit-smoking, the prevention and treatment of emphysema and COPD.2. To explore the mechanism of HO-1/Bilirubin playing the protective role of anti-inflammatory and anti-oxidative on emphysema by establishing animal emphysema model and giving different interventions.Methods:1. Clinical study: checked out the medical histories of qualified patients diagnosed with and without COPD, compared their bilirubin levels and explored the relations between bilirubin levels and smoking, quit-smoking and COPD disease state.2. Animal study: the Wistar rats were either exposed or sham-exposed to cigarete smoke for 12 weeks. The rats were intraperitoneal injected with Hemin or Sn PP to induce HO-1expression or inhibit its activity, or gavaged with bilirubin to raise the level of bilirubin in the body before smoke-exposed. Then the rats were executed and tested by biopsy,evaluation of emphysema, counting cells in bronchoalveolar lavage fluid. ELISA was used to detect the levels of the associated inflammatory and anti-inflammatory cytokine such as IL-17, IL-8, TNF- α and IL-10, chemokine MCP-1, MIP-2α, and so on. The associated genes and proteins expression such as IL-17, HO-1, ICAM-1, TGF-β1, NF-κB p65, p38,p-p38 in lung tissue were tested by Realtime-PCR, immunohistochemistry and Westernblot.To explore that whether HO-1 and bilirubin exert anti-inflammatory effects in emphysema model by inhibiting the secretion of pro-inflammatory cytokine, chemokines and adhesion molecules to inhibit inflammatory cell infiltration, whether play an antioxidative role by inhibiting generation of oxidative stress products and the depletion of antioxidant enzymes,and whether influence the activation and expression of NF-κB and p38 MAPK signaling pathways, and to compare the differences of their effects.Results:1. Indirect bilirubin, the main bilirubin model playing protective role in vivo, is influenced by smoking and COPD, and quit-smoking is helpful to restore the levels of bilirubin.2. The evaluation of rat emphysema model exposured to smoke: the results of pathological,emphysema evaluation, bronchoalveolar lavage fluid(BALF) cell counting support that the model is successful. The numbers of total inflammatory cells, neutrophils and macrophages in BALF, the levels of inflammatory cytokines, chemokines, and adhesion molecules in BALF, serum and lung tissue supernatant, oxidative stress products malondialdehyde increased in smoking group increased compared with the control group,but the levels of anti-inflammatory cytokine IL-10 reduced, antioxidant enzymes SOD actvity and GSH level decreased.3. HO-1 played protective roles of anti-inflammatory and anti-oxidant in the model by reducing the numbers of inflammatory cells in BALF, promoting the secretion of IL-10,reducing the levels of inflammatory cytokines, chemokines, and adhesion molecules, and decreasing the generation of oxidative products and depletion of antioxidant enzymes4. The intervention of bilirubin also achieved the effects of an anti-inflammatory and antioxidative and its effects were better than Hemin’s because of its less toxicity.5. The activation of NF-κB and p38 MAPK signaling pathways maybe related to inflammation and oxidative stress in emphysema model. HO-1 and bilirubin may play protective roles by influencing their activation and expression.Conclusions:HO-1 and bilirubin exerted anti-inflammatory effects by promoting the secretion of IL-10,inhibiting the synthesis and secretion of inflammatory cytokines, chemokines and adhesion molecules, reducing the infiltration of inflammatory cells, and played antioxidant roles by reducing the generation of oxidative stress products and consumption of antioxidant enzymes, and inhibited airway remodeling and reduced emphysema lesions. HO-1 and bilirubin may play protective roles by influencing the activation and expression of NF-κB and p38 MAPK signaling pathways in rat emphysema models exposured to smoke. Indirect bilirubin, the main bilirubin model playing anti-oxidative role in vivo, is influenced by smoking and COPD, and quit-smoking is helpful to restore the levels of bilirubin.