Protective Effect Of Exercise Training On Blomycin-induced Pulmonary Fibrosis And Obesity Associated-Lung Injury

ObjectivesLung is the only tissue in contact with the outside that is vulnerable to a variety of damage.As a nonpharmacologic treatment,exercise training has been advocated for the treatment of chronic inflammation and obesity-related metabolic syndromes.Recently,hydrogen sulfide?H₂S?,a gaseous transmitter,has been found to engage in many physiological and pathophysiological processes.Pulmonary fibrosis has been involved in a variety of lung injury and influenced the development and outcome of these diseases.Herein,the first part aimed to investigate whether exercise training could improve Bleomycin?BLM?-induced lung fibrosis by regulating endogenous H₂S.Glucocorticoids?GCs?,the widely used anti-inflammatory hormones,are synthesized mainly in adrenal.While accumulating evidence demonstrates GCs can also been synthesized or metabolized in extra-adrenal organs and affects local tissue functions.In addition,we have previously shown that endogenous H₂S generation as critical regulators of adrenocortical responsiveness might improve relative adrenal insufficiency during sepsis.Whether H₂S could regulate GCs synthesis or metabolism in the lung?This study aimed to examine whether exercise training could affect biosynthesis and metabolic regulation of GCs and improve lung fibrosis via upregulating H₂S.Obesity is a metabolic disorder characterized by the low-grade chronic inflammation.So,the second part of this article aimed to investigate whether exercise training could improve obesity-associated pulmonary inflammation by regulating local GCs synthesis or metabolism.Whether exercise training could improve pulmonary fibrosis and regulating local GCs synthesis or metabolism via H₂S will be investigate in the future.methods1^st part:We first analyzed the role of H₂S in BLM-induced lung fibrosis.The ICR mice were randomly assigned to four groups?10 mice per group?:control group?Control?,bleomycin group?BLM?,hydrogen sulfide group?H₂S?,bleomycin plus hydrogen sulfide group?BLM+H₂S?.And then discussed the effect of exercise training on pulmonary fibrosis:control group?Control?,bleomycin group?BLM?,exercise training group?Ex?,bleomycin plus exercise training group?BLM+Ex?.After running 45min per day?12m/min?and 5d per week all 4 weeks,BLM or an equal volume saline intratracheally administered.After continues two weeks' training,the lungs were harvested.For histological assessment,the lungs were harvested and using hematoxylin and eosin staining and Masson's trichrome staining.The content of hydroxyproline,type ? collagen levels and transforming growth factor?TGF-??were determined by ELISA?Enzyme-linked immuno sorbent assay?.Total protein were isolated to determine the change of E-cadherin,a-smooth muscle actin?a-SMA?,phosphorylated Smad2/Smad3/Smad4 andCBS/CSE.2^nd part:WT and ob/ob mice were randomly assigned to the following four groups:?1?Sed-WT group?n=10?,WT mice that were subjected to sedentary conditions;?2?Ex-WT group?n=10?,WT mice that were subjected to regular treadmill exercise;?3?Sed-ob?n=10?,ob/ob mice that were subjected to sedentary conditions;?4?Ex-ob group?n=10?,ob/ob mice that were subjected to regular treadmill exercise.Exercised animals had run 6 day per week,1h per day?12m/min?without any exhaustion.After six weeks,the lungs were harvested for analysis.The levels of interleukin?IL?-1?,IL-6,IL-18.monocyte chemotactic protein?MCP?-1,myeloperoxidase?MPO?,corticosterone were determined by ELISA.Detected the expression of CD68 and assessment of GCs synthetases?StAR?CYP11A1?CYP21?3?-HSD?CYP11B1?and metabolic enzymes?11?-HSD1?11?-HSD2?by histological.Total RNA and protein were isolated to determine the expression of 11?-HSD1 and 11?-HSD2.RU486,a glucocorticoid receptor antagonist,was used to investigate whether it could block the anti-inflammatory effects of GCs in lung tissues of obese mice.WT and ob/ob mice were randomly assigned to the following six groups:?1?Sed-WT group?n=7?,non-trained WT mice that received only vehicle;?2?Ex-WT group?n=7?,exercise-trained WT mice that received only vehicle;?3?Ex-WT RU486 group?n=7?,non-trained WT mice that received RU486 injections;?4?Sed-ob?n=7?,non-trained ob/ob mice received only vehicle;?5?Ex-ob group?n=7?,exercise-trained ob/ob mice that received only vehicle;?6?Ex-ob RU486 group?n=7?,non-trained WT mice that received RU486 injections.Once-daily subcutaneous injections of RU486?30 mg/kg/d?or vehicle?0.50%gelatin,50%mannitol?at treatment period.Exercise training protocol was same as that in the first set of experiments.Body weight was monitored weekly.Results1^st part:1.H₂S treatment BLM-associated pulmonary epithelial and aging:Double immunofluorescence staining demonstrated that H₂S attenuated BLM-associated lung fibrosis as measured by increased expression of E-cadherin,as well as decreased expression of p21and p53 than control.2.H₂S attenuates BLM-associated lung fibrosis:Compared with control group,hydroxyproline.I collagen and TGF-? contents rise of BLM group?P<0.01,p<0.01,p<0.01?;After exercise six weeks.the expression of hydroxyproline,I collagen and TGF-? were decreased Compared with BLM group?p<0.05,p<0.01,p<0.01?.3.Exercise training improve the BLM-induced pulmonary fibrosis in mice:HE and Masson staining results showed that compared with the Control group,the structure of lung tissue in BLM group was damaged,collagen deposition increased;Compared with BLM group,the structure of lung tissue in BLM+Ex group was relatively intact,collagen deposition decreased.ELISA results showed,compared with the Control group,the levels of hydroxyproline,collagen type I and TGF-?in BLM group were significantly higher?p<0.01,p<0.01,p<0.05?.Compared with BLM group,the levels of hydroxyproline,collagen type I and TGF-? in BLM+ Ex group were significantly lower?p<0.01,p<0.01,p<0.05?.4.Exercise training increases the epithelial and reduce mesenchyma:BLM induced increasing of a-SMA as well as the loss of E-cadherin?p<0.01,p<0.01?.While Exercise training attenuated the effects of BLM.Exercise training increased the expression of E-cadherin with the decreased expression of a-SMA?p<0.01,p<0.05?.5.Exercise training ameliorates BLM-induced TGF-? signaling in lung tissue:The phosphorylation of Smad2/3 and Smad4 expression were elevated after BLM administration compared with control group?p<0.01,p<0.05,p<0.01?.which were ameliorated after Exercise training treatment compared with BLM group?p<0.01,p<0.05,p<0.01?.6.Exercise training increased the expression of CBS and CSE:The levels of CBS and CSE were decreaseed in BLM group were decreased compared with Control group?p<0.01,p<0.01?.After Exercise training,the expression of CBS and CSE were increased compared with BLM group?p<0.01,p<0.05?.2^nd part:1.Exercise training decreases pulmonary cytokine levels in obese mice:The Sed-ob group pulmonary levels of IL-1?,IL-18 and MCP-1 were significantly increased in the ob/ob mice compared with Sed-WT?p<0.01,p<0.01,p<0.01?.Exercise training resulted in a significant decrease in the protein levels of IL-1?,IL-18 and MCP-1 in the ob/ob mice compared with Sed-ob group?p<0.05,p<0.05?P<0.01?.Neither obesity nor exercise training affected pulmonary level of IL-6.2.Exercise training attenuates pulmonary leukocyte infiltration in obese mice:CD68-positive staining macrophages were significantly increased in the lung tissue of Sed-ob mice compared with Sed-WT group mice?p<0.01?.Exercise training resulted in a significant decrease of CD68-positive staining macrophages in the lung tissue of ob/ob mice compared with Sed-ob group?p<0.05?.3.Exercise training decreases MPO activity in obese mice:We found that the MPO activity in Sed-ob group was significantly higher than Sed-WT group?p<0.01?.Exercise training significantly decreased the obesity-induced MPO activity in lung tissues compared with Sed-ob group?p<0.05?.4.Exercise training increases pulmonary corticosterone levels in obese mice,but there was no effect in plasma:it was found that pulmonary corticosterone levels were significantly decreased in the Sed-ob group compared with the Sed-WT group?p<0.05?.Exercise training not only reversed the decreased corticosterone levels in the lung tissues of ob/ob mice,but also significantly increased pulmonary corticosterone levels in the C57 mice?p<0.01?.No statistically significant change in plasma corticosterone levels was found in four experimental groups.5.No positive immunoreactivity for CYP11B1 in the mice lung obtained from C57 or ob/ob mice with or without exercise training:By using immunohistochemistry technique,we detected positive immunostaining for StAR,CYP11A1,3p-HSD,CYP21as well as11?-HSD1 and 11?-HSD2 in lung tissue sections obtained from mice.However,immunohistochemistry revealed no positive immunoreactivity for CYP11B1 in the adult mouse lung obtained from C57 or ob/ob mice with or without exercise training.6.Effect of exercise training on pulmonary expression of 11?-HSD1 and 11?-HSD2 in obese mice:Exercise training significantly increased pulmonary 11?-HSD1 expression not only in the C57 mice,but also in the ob/ob mice by using Real time PCR and Western Blot Analysis,respectively.obesity per se had no effect on both mRNA and protein levels of 11?-HSD1 in the lung tissues,as compared with the C57 mice.The expression of 11?-HSD2 was significantly increased in the lung tissues of ob/ob mice compared with the C57 mice.However,exercise training per se had no effect on pulmonary 11?-HSD2 expression in both the C57 and ob/ob mice.7.RU486 blocks the anti-inflammatory effects of exercise training:In Ex-WT/RU486 group,RU486 significantly increased pulmonary levels of IL-1??IL-18,MCP-1,and MPO activity as compared with Ex-WT group?p<0.05,p<0.01,p<0.01,p<0.05?.In Ex-ob/RU486 group,RU486 blocked exercise-mediated decreases in IL-1?,IL-18,MCP-1,and MPO activity as compared with Ex-ob group?p<0.01,p<0.01,p<0.05,P<0.01?.8.The changes of mice weight:The mice weight in In Ex-WT group were significantly decreased compared with Sed-WT group?p<0.05?,while were increased in Ex-WT+RU486 group?p<0.05?.The decreased mice weight in Ex-ob group were reversed in Ex-ob+RU486 group?p<0.01?.9.Exercise training increases the lung tissue of mice H₂S synthetase CBS and protein expression of CSE:The levels of CBS and CSE were decreaseed in Sed-ob group were decreased compared with Sed-WT group?p<0.01,p<0.01?.After Exercise training,the expression of CBS and CSE were increased compared with Sed-ob group?p<0.01,p<0.05?.ConclusionsExercise could promote the activity of GCs,improve the BLM induced pulmonary fibrosis and lung tissue inflammation associated with obesity by inhibiting activation of the TGF-? signaling pathways and its possible mechanism is that exercise training can improve the levels of the H₂S in the mice lung tissue.