The Molecular Mechanism Of PPARγ Involving The Smoke-induced Chronic Obstructive Pulmonary Disease By The AMPK Pathway

Background:Chronic obstructive pulmonary disease(COPD)is a common chronic inflammatory lung disease characterized by incompletely reversible and progressive airway obstruction.Data shows that with the aging of the population and the increase in the number of youngsters smoking,the incidence of COPD is showing an upward trend year by year.It is expected that by the year 2020,COPD will become the third largest cause of death in the world.Studies have shown that smoking is the primary factor in inducing COPD.According to relevant literature reports,in the cigarette smoke-induced COPD rat model,the rat lung tissue exhibits airway epithelial cell damage,inflammatory cell infiltration,intrabronchial cell blockage and so on.With the increase of cigarette smoke induction time,the number of epithelial cells that develop emphysema,hyperemia,degeneration,and necrosis increases.In serum and/or bronchoalveolar lavage fluid(BALF)of rats with COPD,there was an increase in the levels of pro-inflammatory cytokines TNF-α,IL-6 and IL-8,and a decrease in the content of anti-inflammatory IL-10.However,due to the complicated pathogenesis of COPD,there is still no effective treatment for COPD.Therefore,it is urgent to elucidate the pathogenesis of COPD and develop effective treatment methods for COPD according to the relevant pathogenesis.Autophagy is an evolutionarily conserved cellular process that is recovered through long-lived proteins and damaged organelles to maintain energy balance.Data shows that autophagy is essential for maintaining homeostasis and development.A large number of literatures indicate that autophagy is closely related to COPD.According to related studies,the expression of autophagy-related proteins ATG4 B,ATG5-ATG12,ATG7 and the ratio of LC3 BII to LC3 BI increased significantly in the lung tissues of COPD patients.In lung tissue of rats with COPD,the expression levels of autophagy-related proteins LC3 II,Beclin-1,ATG5,and ATG7 increased significantly.Therefore,autophagy disturbance may become an important pathogenic factor of COPD.Peroxisome(PPARs)are members of the nuclear hormone receptor superfamily.There are three subtypes of PPARα,PPARβ/δ and PPARγ.Data shows that PPARγ is mainly expressed in adipose tissue,closely related to the body’s energy metabolism and immune regulation.Studies have found that PPARγ is closely related to COPD.According to relevant literature reports,mi R-27-3p was highly expressed in lung tissue and alveolar macrophages of COPD mice in a mouse COPD model induced by combination of cigarette smoke and lipopolysaccharide(LPS).Mi R-27-3p could inhibit the activation of PPARγ by targeting and activated the TLR2/4 signaling pathway,NF-κB signaling pathway,JNK/P38 signaling pathway and JAK/STAT signaling pathway of inflammation-related signaling pathways,leading to the production of lung tissue inflammation.The above literatures suggest that PPARγ may be a protective factor for COPD,and the increased expression of PPARγ may contribute to the improvement of COPD.AMP-activated protein kinase(AMPK)is a heterotrimeric complex composed ofα-catalytic subunit,β-regulatory subunit and γ-regulatory subunit.It is a key molecule which regulates the metabolism of biological energy.Data shows that disturbance of AMPK activity is closely related to many diseases such as cancer diseases,cardiovascular diseases,and lung diseases.Studies have shown that AMPK plays an important regulatory role in COPD.According to related literature,AMPK activation could significantly reduce the contents of pro-inflammatory cytokine IL-6 and IL-8 in the COPD model of human airway epithelial cells BEAS-2B cells and small airway epithelial SAEC cells induced by cigarette extracts.Subsequent in vivo experiments also confirmed that activation of AMPK in the elastase-induced mouse emphysema model can significantly reduce the levels of pro-inflammatory cytokines IL-6 and IL-8 in mouse BALF.In addition,studies have shown that in the COPD model of rats induced by combination of cigarette smoke and lipopolysaccharide,the levels of inflammatory cytokines TNF-α in serum and skeletal muscle of rats were significantly increased,and the expression levels of AMPK m RNA and protein in skeletal muscle of rats were significantly decreased.The above literatures suggest that COPD may be treated with AMPK as a target.In this study,COPD cells and animal models were established through cigarette smoke extract(CSE)or LPS combined with cigarette smoke exposure,PPARγ activators rosiglitazone(Ros)and troglitazone(Tro),PPARγ inhibitor BDAGE were used to promote or inhibit PPARγ expression,LC3 si RNA was used to suppress the occurrence of autophagy,compound C(Com C)was used to suppress activation of AMPK signaling pathway on those foundations to explore the relationship between COPD,PPARγ,autophagy,and AMPK signaling pathways and to elucidate the pathogenesis of COPD.The successful development of this study will provide new targets,new perspectives and new technologies for the clinical treatment of COPD,and provide a substantial theoretical basis for the clinical treatment of COPD with PPARγ as a target.Method:Western blot was used to detect the relative expression of autophagy-related proteins Beclin-1,ATG5,ATG7,p62 and LC3(LC3I、LC3II)in cigarette bronchial epithelial cells 16 HBE cells induced by cigarette smoking extract(CSE).Real-time PCR and Western blot were used to detect the m RNA and protein relative expression of PPARγ in 16 HBE cells induced by CSE.The COPD model of rats and cells were established by induction of cigarette smoke or CSE.The inhibition of PPARγ expression was used by PPARγ inhibitor BADGE.The increasion of PPARγexpression was used by PPARγ activators rosiglitazone(Ros)and troglitazone(Tro)and LC3 si RNA was transfected to inhibit autophagy production on this basis.The relative expression levels of autophagy-related proteins Beclin-1,ATG5,ATG7,p62 and LC3(LC3I、LC3II)were detected by Western blot.The expression levels of inflammation-related proteins i NOS and COX-2 were detected by Western blot and immunofluorescence.Elisa was used to detect the levels of pro-inflammatory cytokines IL-6 and IL-8.The expressions of AMPK signaling proteins AMPK and p-AMPK in16 HBE Cells induced by CSE were detected by Western Blot.Western blot was used to detect the relative expression of proteins in AMPK signal pathway and downstream proteins in m TOR signal pathway AMPK,p-AMPK,S6 K and p-S6 K in COPD models of rats and cells.AMPK inhibitor compound C was used to inhibit the activation of AMPK signal in COPD model of cells and Elisa was used to detect the levels of pro-inflammatory cytokines IL-6 and IL-8on this basis.Western blot was used to detect the relative expression levels of m TOR signal pathway downstream proteins S6 K and p-S6 K proteins,inflammation-related proteins i NOS and COX-2,autophagy-related proteins p62 and LC3(LC3I、LC3II).Result:Compared with cells of CSE 0 group,the relative expression levels of PPARγ m RNA and protein in 16 HBE cells were significantly decreased after 1% and 5%CSE induction,the relative expressions of autophagy-related proteins Beclin-1,ATG5,ATG7,and p62 were significantly decreased and the ratio of relative expression of LC3 II to LC3 I proteins were significantly increased.Compared with the cells of 0 h group,the relative expression levels of PPARγ m RNA and protein in 16 HBE cells were significantly decreased at 1 h,3 h,6 h,12 h,and 24 h after 1%CSE induction.The autophagy-related protein Beclin-1 and the ratio of relative expression of LC3 II to LC3 I proteins at 24 h,the relative expression levels of ATG5 and p62 proteins at 6 h,12 h and24 hand the relative expression level of ATG7 protein at 3 h,6 h,12 h and 24 h in 16 HBE cells were significantly increased after 1%CSE induction compared with the cells of 0 h group.Compared with cells of control group,the levels of pro-inflammatory cytokines IL-6 and IL-8,the relative expression of inflammation-related proteins i NOS and COX-2and the relative expression of autophagy-related proteins Beclin-1,ATG5,ATG7,p62 and the ratio of relative expression of LC3 II to LC3 I proteins in cells of CSE group were significantly increased.Compared with the cells of CSE group,the levels of pro-inflammatory cytokines IL-6 and IL-8,the relative expression of inflammation-related proteins i NOS and COX-2 and the autophagy-related protein p62 in cells of Ros and Tro group were significantly decreased and the relative expression levels of Beclin-1,ATG5,ATG7 and the ratio of relative expression of LC3 II to LC3 I proteins in cells of Ros and Tro group were significantly increased.In cells of BADGE group,the levels of pro-inflammatory cytokines IL-6 and IL-8,the relative expression levels of inflammation-related proteins i NOS and COX-2,and autophagy-related protein p62 were significantly increased and the relative expression levels of autophagy-related proteins Beclin-1,ATG5,ATG7 and the ratio of relative expression of LC3 II to LC3 I proteins were significantly decreased compared with cells of CSE group.Animal experiments were consistent with the results of cell experiments.Compared with cells of Ros or Tro group,the levels of pro-inflammatory cytokines IL-6 and IL-8,the relative expression levels of inflammation-related proteins i NOS and COX-2,and autophagy-related protein p62 in cells of LC3 si RNA group(LC3 si RNA+Ros or LC3 si RNA+Tro)were significantly increased and ratio of relative expression of LC3 II to LC3 I proteins in cells of LC3 si RNA group(LC3 si RNA+Ros or LC3 si RNA+Tro)was significantly decreased.Compared with cells of 0 h group,the ratio of relative expression of AMPK signal pathway proteins p-AMPK to AMPK in 16 HBE cells was significantly increased at 0.5 h and 1 h after CSE induction and the ratio of relative expression of AMPK signal pathway proteins p-AMPK to AMPK in 16 HBE cells was significantly decreased at 6 h,12 h,and24 h after CSE induction.Compared with cells of control group,the ratio of relative expression of AMPK signal pathway proteins p-AMPK to AMPK in cells of CSE group was significantly decreased and the ratio of relative expression of downstream proteins in m TOR signal pathway p-S6 K to total S6 K was significantly increased.Compared with cells of CSE group,the ratio of relative expression of AMPK signal pathway proteins p-AMPK to AMPK in cells of 10 μM and 30 μM Ros groups and each concentration(1,10,30 μM)of Tro groups was significantly increased and the ratio of relative expression of downstream proteins in m TOR signal pathway p-S6 K to total S6 K proteins in cells of10 μM and 30 μM Ros groups and each concentration(1,10,30 μM)of Tro groups was significantly decreased.The ratio of relative expression of downstream proteins in m TOR signal pathway p-S6 K to total S6 K proteins in cells of 1 μM Ros group was significantly decreased and the ratio of relative expression of AMPK signal pathway proteins p-AMPK to AMPK proteins in cells of 1 μM Ros group was not significantly changed compared with cells of CSE group.It was consistent with the results of animal experiments.Compared with cells of Ros or Tro group,the levels of pro-inflammatory cytokines IL-6 and IL-8,the relative expression of inflammation-related proteins i NOS and COX-2,the autophagy-related protein p62 and the ratio of relative expression of downstream proteins in m TOR signal pathway p-S6 K to total S6 K proteins in cells of Compound C(CSE+Ros+Com C or CSE+Tro+Com C)group were significantly increased and the ratio of relative expression of LC3 II to LC3 I proteins in cells of Compound C(CSE+Ros+Com C or CSE+Tro+Com C)group was significantly decreased.Conclusion:In COPD,defective autophagy is occured and the expression of PPARγis significantly reduce;PPARγ can promote autophagy and improve COPD in an autophagy-dependent manner;PPARγ promotes the occurrence of autophagy through the AMPK/S6 K signaling pathway,thereby improving COPD.