The Mechanism Of Hydrogen Delay The Development Of Chronic Obstructive Pulmonary Disease

Objective: Through research the effects of hydrogen in lung function and pathology to a cigarette-smoke induced chronic obstructive pulmonary disease rat model,observe the expression of inflammation factors(TNF-?,IL-6,IL-17,IL-23),the metalloproteinase / tissue inhibitor of metalloproteinase(MMP-12 / TIMP-1)and apoptosis protein(caspase-3,caspase-8)in COPD rat lung tissue,to discuss the effects of hydrogen in COPD through anti-inflammatory,adjust the balance of protease / resistant protease and antiapoptotic.Methods:1 Healthy male SD rats(160-200g)were randomized into the following seven groups(10 rats in each group): control group,COPD group,low-hydrogen group(Hl group),Middle Hydrogen group(Hm group),High Hydrogen group(Hh group),Very high Hydrogen and Oxygen groups(Hvh group),Oxygen group(O2 group).The rats with COPD were established by the smoking method.The daily condition of rats and weight changes were observed;lung function and right ventricular hypertrophy index(RVHI)were assessed;And white blood cells were assessed in bronchoalveolar lavage fluid(BALF).Pathological changes in lung tissue were analyzed using light microscopy.2 Protein of TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 was detected by immunohistochemistry and western blot of the lung tissue.Gene expression of TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 in the lung was assessed by RT-PCR.Results:1 Body weight of ratBefore smoking there is no obvious difference between the body weight of every group of rat(P > 0.05).In comparison with the control group,rat from the COPD group suffer loss of weight(P < 0.05),while body weights of rat from hydrogen groups and oxygen groups have no changes.And compared with the COPD group,weights of rat in the Hl group and Hh group increase(P < 0.05),Hm,Hvh and O2 group have no changes,and no differences are observed between every hydrogen groups and oxygen groups.2 Pulmonary functionFEV0.2 of COPD group is significantly smaller than that of Control group.Compared with COPD group,FEV0.2 of Hl,Hm,Hh and O2 groups are significantly larger(P < 0.01),while that of Hvh group is of no change.Hvh group has a significant smaller FEV0.2 than Hl,Hm,Hh and O2 groups(P < 0.01).COPD group's FVC is much larger than that of Control group(P < 0.01).And in contrast with COPD group,Hl,Hm,Hh and O2 groups have significantly smaller FVC(P < 0.01),Hvh group has no changes.FVC of Hl group is much smaller than Hh and Hvh groups(P < 0.01),and that of Hm,Hh and O2 groups are all significantly smaller than Hvh group(P < 0.01).In comparison with COPD group,FEV0.2 / FVC of Control group is much larger(P < 0.01),while Hl,Hm,Hh and O2 groups' FEV0.2 / FVC are significantly larger(P < 0.01),and Hvh group has no changes.Hvh group's FEV0.2 / FVC is significantly smaller than Hl,Hm,Hh and O2 groups(P < 0.01).3 BALF count and classificationCOPD group's BALF white blood cell count,neutrophil count and macrophage count are much larger than the Control group(P < 0.01),and the lymphocyte count of COPD is slightly smaller(P > 0.05).Compared with COPD group,the counts of white blood cell,neutrophil and macrophage of Hm,Hh,Hvh and O2 groups all decrease significantly(P < 0.01),Hl groups' white blood cell count and neutrophil count decrease significantly(P < 0.01)and macrophage count decrease slightly(P < 0.05).While the counts of Hm,Hh and Hvh groups have no differences(P > 0.05),they are all much smaller than that of Hl and O2 groups(P < 0.01).In comparison with COPD group,lymphocyte cell count of every hydrogen groups and oxygen groups are slightly larger(P > 0.05).4 Right Ventricular Hypertrophy Index(RVHI)Rat from COPD group have a significantly larger RVHI(P < 0.01).Compared with COPD group,RVHIs of every hydrogen groups and oxygen groups decrease significantly(P < 0.01).RVHIs of Hm,Hh and Hvh groups are smaller than Hl group(P < 0.01),and that of Hvh group is smaller than O2 group(P < 0.05).5 Pathologic changes of lung tissuesCompared with Control group,the lungs of COPD group's rat get increases in volume,and their general appearances are of white color,rough and bumpy,and of bad elasticity.In microscopic examination,it can be observed that the alveolar duct,sac and alveolar are significantly expanded,the structure of alveolar is in disorder,alveolar wall is thinning and even to divide,some alveolar merge into pulmonary large cell,degeneration and necrosis are in ciliated cells,the pulmonary arterioles narrows from thickening,different levels of infiltration by neutrophils,lymphocyte cells and mononuclear macrophages.In contrast with COPD group,the volumes of Hvh group's lungs are slightly smaller,the wall of pulmonary arterioles thickens,inflammatory cell infiltration can be observed,alveolar wall is slightly thinning and divided to different levels,the alveolar spaces are larger,and some begin to merge,but have slight improvement over COPD group.Rat from Hl,Hm,Hh and O2 groups have smaller lung volume than COPD group and have better appearances,the pulmonary arterioles is slightly thickened,alveolar wall is slightly thinner and division better,still have pulmonary large cells but less than COPD group,and inflammatory cell infiltration is also less than COPD.Compared with Control group,COPD group of MLI and BWT / D significantly increased(P < 0.01),while MAN of COPD is decreased significantly(P < 0.01).Hm and Hh groups' MLI are much higher than COPD group(P < 0.01)and the difference between Hm and Hh is negligible(P > 0.05).In contrast with COPD group,every hydrogen groups' MAN is significantly lowered(P < 0.01).Hh group is lower than Hm and Hvh groups(P < 0.01),while Hm and Hvh have no differences(P < 0.01).Compared with COPD group,BWT / D of Hm and Hvh groups are much lower(P < 0.01),Hvh and O2 groups are slightly lower(P < 0.05).No differences are observed within all the hydrogen groups and oxygen groups.6 Protein of TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 of the lung tissue was detected by immunohistochemistry.TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 expression was visible in the bronchi of all sizes,as well as in the cytoplasm of the alveolar infiltrated inflammatory cells.Compared with the Control group,the expression of all these proteins was increased in the COPD group.The expression levels of TNF-?,IL-6,IL-17,IL-23,MMP-12,caspase-3,and caspase-8 was reduced in each of the hydrogen groups,O2 group has no significant difference as compared with the COPD group.There was no significant difference in the expression levels of these proteins between Hm,Hh and Hvh groups,but in these groups were lower than in the Hl group.Compared with the COPD group,the protein expression level of TIMP-1 was increased in all hydrogen groups,O2 group has no significant difference;there were no significant difference in expression levels between Hm,Hh and Hvh groups,but in these groups were higher than in the Hl group.7 Protein of TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 of the lung tissue was detected by Western blot.Compared with the Control group,the expression of all these proteins was increased in the COPD group.The expression levels of TNF-?,IL-6,IL-17,IL-23,MMP-12,caspase-3,and caspase-8 was reduced in each of the hydrogen groups,O2 group has no significant difference as compared with the COPD group.There was no significant difference in the expression levels of these proteins between Hm,Hh and Hvh groups,but in these groups were lower than in the Hl group.Compared with the COPD group,the protein expression level of TIMP-1 was increased in all hydrogen groups,O2 group has no significant difference;there were no significant difference in expression levels between Hm,Hh and Hvh groups,but in these groups were higher than in the Hl group.8 Gene expression of TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 in the lung was assessed by RT-PCR.In the COPD group,the mRNA expression level of genes encoding TNF-?,IL-6,IL-17,IL-23,MMP-12,TIMP-1,caspase-3,and caspase-8 were significantly increased as compared with the Control group(P < 0.01).Compared with the COPD group,the gene expression level of TNF-? was significantly decreased in all hydrogen groups(all P < 0.01),O2 group was decreased(P < 0.05);there was no significant difference between this expression in Hm,Hh and Hvh groups(P > 0.05),were significantly lower than the Hl and O2 group(P < 0.01).Compared with the COPD group,the IL-6 mRNA expression level was significantly decreased in Hm,Hh and Hvh groups(P < 0.01),while Hl and O2 group were decreased(P < 0.05),Hvh group was decreased than Hm and Hh group(P < 0.05),there was no significant difference in the IL-6 mRNA level between the Hm group and the Hh group(P > 0.05).Compared with the COPD group,the expression level of IL-17 was significantly decreased in all the hydrogen groups(all P < 0.01);there was no significant difference in this expression level between Hm,Hh and Hvh groups(P > 0.05),were significantly decreased compared with O2 group(P < 0.01);although the expression level in the Hm and Hvh group were significantly lower than in the Hl group(P < 0.01),and in the Hh group was decreased compared with the Hl group(P < 0.05).Compared with the COPD group,the mRNA expression level of IL-23 was significantly decreased in all hydrogen groups and O2 group(all P < 0.01);no difference in the expression of this gene was discovered between Hm,Hh and Hvh groups(P > 0.05),which both showed reduced levels of this mRNA as compared to that of the Hl group(P < 0.05),Hvh group reduced compared to O2 group(P < 0.05).Compared with the COPD group,the mRNA expression level of MMP-12 was significantly decreased in all hydrogen groups(all P < 0.01),O2 group was decreased(P < 0.05);no significant differences were found among Hm,Hh and Hvh groups(P > 0.05).The mRNA expression level of TIMP-1 was significantly increased in Hm,Hh and Hvh groups(P < 0.01),and in the Hl group(P < 0.05)as compared with the COPD group;no significant difference was shown between Hm,Hh and Hvh groups(P > 0.05).Compared with the COPD group,the expression levels of caspase-3 and caspase-8 were significantly decreased in all hydrogen groups(P < 0.01),but no significant difference was shown among hydrogen groups(P > 0.05).Conclusions:1 Cigarette smoke exposure was used to prepare COPD model in rats,this model of COPD was determined to be successful.Inhaling hydrogen can reduced inflammatory cells of BALF,reduced lung injury,improve lung function and right ventricular hypertrophy index.Effect of 22%,41.6%,66.7% hydrogen showed enhanced effects over 2% hydrogen.2 Inflammation factors(TNF-?,IL-6,IL-17,IL-23),the metalloproteinase / tissue inhibitor of metalloproteinase(MMP-12 / TIMP-1)and apoptosis protein(caspase-3,caspase-8)has obvious links in the development of COPD.The effects of hydrogen in COPD through anti-inflammatory,adjust the balance of protease / resistant protease and antiapoptotic.