The Effects Of Visfatin On Apoptosis,Autophagy And PI3K/AKT Signaling Pathway In Lung During Acute Lung Injury

Visfatin,a novel adipokine found in recent years,that is involved in the body’s inflammation and immune response.Its discovery promoted the relationship between adipose tissue and inflammation.Inflammation could cause apoptosis and autophagy,so visfatin may directly or indirectly affect apoptosis and autophagy.Acute lung injury(ALI)is a respiratory system disease in which the systemic inflammatory response syndrome is manifested in the lungs and it is often used as an inflammation model.The study found that in animal models of acute lung injury,bronchoalveolar lavage fluid levels of visfatin increased,suggesting that visfatin as an inflammatory mediator may be involved in the occurrence and development of pulmonary inflammation,thus affecting apoptosis and autophagy.Therefore,in this study,Kunming mice were used as experimental animals,and replicated acute lung injury model by LPS.HE staining,TUNEL,transmission electron microscopy,immunohistochemical staining,real-time fluorescence quantitative PCR and Western blot were used to study the effects of visfatin on apoptosis,autophagy and PI3K/AKT signaling pathway in pulmonary in acute lung injury.In order to discuss the role of visfatin in inducing apoptosis and autophagy in inflammatory responses through the PI3K/AKT signaling pathway,and study the relationship between the inflammatory response and apoptosis and autophagy in the lung and its mechanism of action.The results are as follows:1.Effects of visfatin on lung tissue structure in acute lung injuryThe model of acute lung injury induced by LPS was established.The results of HE staining showed that the mice in the saline control group had intact and clear lung structure.The alveolar wall was thin and the infiltration of inflammatory cells and body fluid were not found in the alveolar cavity.In the acute lung injury model group(LPS group),the lung tissue was significantly damaged,the alveolar wall was thickened,and a large number of inflammatory cell especially neutrophil infiltration,pulmonary interstitial and alveolar hemorrhage were observed.In the LPS+Visfatin group,the degree of lung injury was reduced,the alveolar septum was thinner than that of the LPS group,the alveolus was slightly congested,and the lung interstitial and alveolar appeared slight exudation,and a small number of inflammatory cell infiltration was found.The immunohistochemical results of vascular endothelial growth factor(VEGF)showed that there was little of VEGF in alveolar epithelial cells in saline control mice.The expression of VEGF in the lung tissue of the acute lung injury model group(LPS group)was significantly increased and was mainly expressed in alveolar epithelial cells,respiratory bronchiolar epithelial cells,pulmonary interstitial neutrophils,and monocytes.The expression of VEGF in lung tissue of LPS+Visfatin group was significantly lower than that of LPS group.These results suggest that visfatin may play a role in alleviating the pathological changes of LPS-induced acute lung injury in lung inflammation.2.Effect of visfatin on apoptosis of lung tissue in acute lung injuryTUNEL technology and IPP software were used to research and statistical analysis.The results showed that apoptotic cells were mainly distributed in mouse alveolar epithelial cells and bronchial epithelial cells.The saline control group had fewer apoptotic cells in the lung tissue.The quantity of apoptotic cells in LPS group was high significantly increased(P<0.01).The number of apoptotic cells in LPS + Visfatin group was significantly lower than that in LPS group(P<0.05).The results suggest that LPS-induced acute lung injury can promote the apoptosis in lung tissue of mice,while visfatin can inhibit the apoptosis induced by acute lung injury.3.Effect of visfatin on the apoptosis factor in lung tissue of acute lung injuryThe expressions of Bax,Bik and Bcl-2,Bcl-xl mRNA in the Bcl-2 gene family were detected by q-PCR.The results showed that the mRNA expression of the pro-apoptotic factors Bax and Bik in the LPS group showed highly significant increase compared with the saline control group(P<0.01),and the anti-apoptotic factors Bcl-2 and Bcl-xl mRNA expression was significantly increased(P<0.05).The expression of pro-apoptotic factors in LPS+Visfatin group was significantly lower than that in LPS group(P<0.05),while the expression of anti-apoptosis factors in LPS+Visfatin group was significantly higher than that in LPS group(P<0.01).Western blot was used to detect the expression of apoptosis-related factors p53 and MLKL in lung tissue.The results showed that the expression of P53 and MLKL protein in the LPS group was significantly increased compared with the control group.Compared with LPS group,the protein expression of P53 and MLKL in LPS+Visfatin group showed a decreasing trend.These results suggest that visfatin can inhibit apoptosis by inhibiting pro-apoptotic genes and promote anti-apoptotic genes.4.Effect of visfatin on autophagy of lung cells in acute lung injuryTransmission electron microscopy revealed that there were few autophagosomes in the saline control group,but compared with the saline control group,there was a significant increase in autophagic corpuscles in the LPS group;the autophagic corpuscles in the LPS+Visfatin group were significantly decreased than those in the LPS group,but slightly more than the saline control group.Immunohistochemical method was used to detect the expression of autophagy markers LC3 and Beclin1,and the data were analysised with IPP software.The results showed that positive products of brown-yellow particles were found in lung.LC3 is mainly expressed in the cytoplasm and predominantly in epithelial cells of alveolar septa.Beclin1 is mainly expressed in alveolar epithelial cells and alveolar nodular swelling cells.The expression of LC3 and Beclin1 in the LPS group was significantly higher compared with the saline control group(P<0.01).The expression of LC3 and Beclin1 in the LPS+Visfatin group was significantly lower than that in the LPS group(P<0.01).The results suggest that LPS-induced acute lung injury can promote autophagy in mice’s lung tissue,while visfatin can reduce autophagy induced by acute lung injury.5.Effect of visfatin on autophagy factor of lung tissue in acute lung injuryQ-PCR was used to detect the expression of autophagic factor LC3 and Beclin1 mRNA.The results showed that the autophagic mRNA expression was lower in the saline control group,indicating that the autophagy expression was less when the body worked in correct order,and the mRNA expression of LC3 and Beclin1 was increased in the LPS group compared with the control group.Compared with LPS group,the mRNA expression of LC3 and Beclin1 was decreased in LPS+Visfatin group.The results of Western Blot showed that the protein level of LC3 and Beclin1 in the LPS group was significantly increased compared with the control group.Compared with LPS group,the protein expression of LC3 and Beclin1 in LPS+Visfatin group showed a decreasing trend.These results suggest that,in acute lung injury,the expression of autophagy is enhanced to assist the apoptosis of cells,while visfatin can inhibit apoptosis and reduce the expression of autophagy to reduce the degree of lung injury.6.Effect of visfatin on PI3K-AKT signaling pathwayWestern Blot and ImageJ software were used to detect the protein levels of PI3 K,AKT,and p-AKT,and to verify if visfatin was involved in the apoptosis and autophagy by PI3K/AKT signaling pathway in acute lung injury.The results showed that,compared with the control group,the expression of PI3 K,AKT and p-AKT were up-regulated in the LPS group.Compared with the LPS group,the expression of PI3 K and p-AKT was up-regulated and the expression of AKT was down-regulated in the LPS+Visfatin group.These results suggest that visfatin can activate PI3K/AKT signaling pathway,inhibit lung cell apoptosis,and reduce autophagy to protect the lung.