The Molecular Mechanism Of Exercise Intervention In Improving Acute And Chronic Lung Injury Based On GPR116 Regulation

Objectives:Pulmonary Fibrosis is a chronic progressive disease,which is characterized by the differentiation of pulmonary fibroblasts into myofibroblasts,collagen deposition,and extracellular matrix accumulation,leading to the destruction of lung tissue structure and function.Even in the later stage,the quality of life decreased and the prognosis was poor.Acute lung injury is characterized by diffuse pulmonary infiltrates with high mortality,as indicated by excessive secretion of proinflammatory cytokines,cell death,disruption of the alveolar endothelial barrier,and neutrophil infiltration in lung tissue.Previous studies have shown that exercise has effects on the pathological function of lung tissue and immune cells,cytokines and oxidative stress markers in acute lung injury.The 2020 COVID-19 pandemic has caused millions of deaths globally,and this high mortality is known to be associated with acute lung injury and pulmonary fibrosis;However,there is no effective drug therapy for these pathological conditions.Exercise training can effectively inhibit the progression of pulmonary fibrosis and inflammation in mice with pulmonary fibrosis and acute lung injury.In clinical practice,exercise rehabilitation has gradually become an auxiliary method for the prevention and treatment of lung diseases.However,the principle and mechanism of its action need to be further studied and demonstrated.At the same time,many previous studies have shown that GPR116 is involved in the protective effect of lung injury,especially as a key regulator of alveolar surfactant.Therefore,the aim of exercise is to change the level of GPR116 in the body to improve the process of pulmonary fibrosis and inflammation.Therefore,in this study,bleomycin was used to establish a mouse model of pulmonary fibrosis and LPS-induced acute lung injury,and to explore whether exercise can improve acute and chronic lung injury by increasing the level of GPR116 in the body,so as to provide more theoretical basis for exercise to improve the symptoms of lung diseases Methods:In the first part,GPR116 was used to explore the molecular mechanism of exercise intervention in improving pulmonary fibrosis.First,to verify whether exercise can improve BLM-induced pulmonary fibrosis in mice,28 eight-week-old male C57BL/6 mice were randomly divided into 4 groups: Control group(CON),bleomycin group(BLM),exercise group(EX)and exercise bleomycin group(EX+BLM),7 rats in each group.After 6 weeks of treadmill exercise at a dose of 5days/week,45min/ day,and 12m/min,the pulmonary fibrosis model was established.Bleomycin was instilled in the BLM group and BLM+EX group,and saline was instilled in the CON and EX groups.Then,to explore the mechanism of exercise intervention in reducing bleomycin induced pulmonary fibrosis in mice,twenty-one eight-week-old male C57BL/6 mice were randomly divided into three groups: control group(CON),bleomycin group(BLM)and adenovirus overexpressing GPR116 bleomycin group(AD-GPR116+BLM),7 mice in each group.Lung tissue samples were collected 2 weeks later.Dynamic lung compliance and forced expiratory volume were measured by pulmonary function tester.HE and Masson staining were used to detect the pathological changes and collagen deposition in lung tissue.In the second part,GPR116 was used to explore the molecular mechanism of exercise intervention in improving endotoxemia-induced ALI.Firstly,we tested whether exercise could improve LPS-induced ALI in mice.Twenty-eight eight-week-old male C57BL/6 mice were randomly divided into 4 groups: control group(CON),lipopolysaccharide group(LPS),exercise group(EX)and lipopolysaccharide exercise group(EX +LPS),with 7mice in each group.All the subjects underwent treadmill exercise for 6 weeks with an exercise program of 5 days/week,45min/d,and 12m/min.Lung tissue samples were collected after the exercise intervention.To further verify that GPR116 may be involved in exercise intervention to alleviate endotoxemic acute lung injury,we randomly divided 28 eight-week-old male C57BL/6 mice into 4 groups: Control group(Con),lipopolysaccharide group(LPS),GPR116 lyz-cre group(Gpr116-c KO group)and GPR116 lyz-cre LPS group(Gpr116-c KO +LPS),with 7 rats in each group.The exercise protocol was the same as above.Lung tissues were collected at the end of the training.The levels of tumor necrosis factor(TNF),interleukin-1β(IL-1β),interleukin-6(IL-6),INOS and MCP-1 in lung tissue were detected by RT-PCR.The infiltration of macrophages and neutrophils in lung tissue was detected by immunohistochemical fluorescence staining with F4/80 and Ly6 G.The expression of GPR116 was quantitatively analyzed by RT-PCR and Western blot.Results:1.Exercise intervention improved lung function in mice with bleomycin-induced pulmonary fibrosis.Compared with the CON group,dynamic lung compliance(Cdyn)and forced vital capacity(FVC)in BLM group were significantly decreased;EX+BLM group compared with BLM group(##P <0.01): EX+BLM group mice dynamic lung compliance(Cdyn)and forced vital capacity(FVC)increased significantly(* P<0.05);2.Exercise intervention improved lung tissue structural damage and fibrosis degree in mice with bleomycin-induced pulmonary fibrosis.3.Adenovirus overexpressing GPR116 improved lung function in mice with bleomycin-induced pulmonary fibrosis.There were significant decreases in dynamic lung compliance(Cdyn)and forced vital capacity(FVC)in BLM group(#P <0.05);Compared with BLM group,Ad-ADGRF5+BLM group: Ad-ADGRF5+BLM group mice dynamic lung compliance(Cdyn)and forced vital capacity(FVC)were significantly increased(* P<0.05).4.GPR116 overexpression ameliorated lung tissue structural damage and fibrosis in mice with bleomycin-induced pulmonary fibrosis.Exercise intervention improved5.lung tissue structural damage in LPS-induced endotoxemia-induced ALI mice.6.Exercise intervention reduced the expression of inflammatory cytokines in lung tissue of LPS-induced endotoxemic ALI mice.Compared with CON group,the m RNA expression of IL-1β,IL-6,INOS and MCP-1 in lung tissue of LPS group was significantly increased(* P<0.05).Compared with LPS group,the expressions of IL-1β,IL-6,INOS and MCP-1 in EX+LPS group were significantly decreased(*P<0.05).But the difference of TNF-α was not significant.7.Myeloid GPR116 knockout aggravated lung tissue injury in LPS-induced LPS-induced endotoxemic ALI mice.8.Myeloid GPR116 knockout increased the m RNA expression of inflammatory cytokines in lung tissues of LPS-induced LPS-induced endotoxemic ALI mice.Compared with CON group,the m RNA expression of TNF-α,IL-1β,IL-6,INOS and MCP-1 in lung tissue of LPS group was significantly increased.However,compared with LPS group,the expression of TNF-α,IL-1β,IL-6,INOS and MCP-1 in ADGRF5-c KO+LPS group increased significantly(**P<0.01).9.Myeloid GPR116 knockout aggravated the degree of inflammatory cell infiltration in lung tissue of LPS-induced LPS-induced endotoxemic ALI mice.Conclusion: Exercise can reduce the degree of pulmonary fibrosis induced by bleomycin and the inflammation level of lung tissue in mice with acute lung injury induced by lipopolysaccharide and endotoxemia.The possible mechanism is that exercise promotes the increase of GPR116 level in the lung tissue of mice,thus ameliorating acute and chronic lung injury.