Effect And Mechanism Of Chromofungin Regulating Autophagy Of Alveolar Macrophages On Sepsis Induced Acute Lung Injury

Objective:The purpose of this study was to evaluate the effects of Chromofungin(CGA_47-66,CHR),a peptide derived from Chromogranin A（CGA）,regulating alveolar macrophage autophagy on lung tissue and function of alveolar macrophage during lipopolysaccharide（LPS）induced acute lung injury（ALI）,and to explore the possible mechanism.Methods:In vivo,C57BL/6 male mice were intraperitoneally injected with LPS（10 mg/kg）to establish the model of sepsis acute lung injury.After 30 minutes of pretreatment with CHR and autophagy inducer Rapa,the 5-day survival rate and general situation of the mice were observed.After treated with LPS for 6 h,the mice were sacrificed to extract lung tissue,and the pathological damage of lung tissue was evaluated by HE staining.The levels of pulmonary inflammatory factors in bronchoalveolar lavage fluid were determined by ELISA.In order to verify the protective effect of CHR on ALI after LPS induction and the effect of CHR on autophagy in lung tissues,the fluorescence intensity expression of autophagy related proteins in lung tissues were further observed by immunofluorescence.In the mouse alveolar macrophage cell line（MH-S cells）,the levels of autophagy related proteins and TLR4-related signaling pathway related proteins were detected by Western blot,and the changes of autophagy flux in MH-S were observed by transmission electron microscopy（TEM）,so as to verify the effect of CHR on promoting autophagy in alveolar macrophages after LPS stimulation through TLR4-related signaling pathway.Results:Compared with mice in control group,after 6 hours of LPS stimulation,the mice ate less food,were motionless,suffered diarrhea and the purulent secretion in eyes were increased,the 5-day survival rate of mice was only 12.5%（P<0.05）.Compared with the LPS group,the general conditions were relatively improved after pretreatment with CHR and Rapa,and the survival rate increased,among which the 5-day survival rate in the CHR group was 68.75%（P<0.05）,while the 5-day survival rate of mice pretreated by Rapa was 81.25%（P<0.05）,showing significant differences.Histopathological analysis was performed on the lung tissue,Hematoxylin-Eosin staining（HE staining）section were observed by inverted microscope,compared with the control group,acute inflammatory reactions were observed in the lung tissue after LPS stimulation,such as interstitial edema and thickening,alveolar integrity destruction,and a large number of inflammatory cell infiltration.Compared with LPS group,the pathological damage of CHR and Rapa group was significantly reduced.The fluorescence expression of autophagy markers in the lung tissues of mice were detected by immunofluorescence assay,compared with the control group,the fluorescence expression intensity of autophagy marker proteins LC3B,Atg7 and P62 in the LPS group were increased.After pretreatment with CHR and Rapa,the fluorescence intensity of LC3B and Atg7 was significantly increased compared with that of LPS group,while the fluorescence intensity of P62 was decreased.The levels of inflammation-related cytokines in the alveolar lavage fluid of mice were determined by ELISA,compared with the control group,the concentration of TNF-α（34.12±3.35 vs 4.19±0.13,P<0.05）in the LPS group was significantly increased,while the expression of IL-10 was decreased（359.80±50.87 vs 1014±34.88,P<0.05）.Compared with LPS group,the relative level of TNF-αwas decreased（20.37±2.17 vs 34.12±3.35,P<0.05）,and the level of IL-10 was increased（582.90±8.26 vs359.80±50.87,P<0.05）after CHR pretreatment.In Rapa group,the levels of inflammatory cytokine TNF-α（15.51±0.21 vs 34.12±3.35,P<0.05）were decreased,and the expressions of anti-inflammatory cytokine IL-10（923.20±46.29 vs 359.80±50.87,P<0.05）were increased,the differences were statistically significant.Western Blot analysis of mouse alveolar macrophage strain MH-S showed that compared with the Control group,LC3B protein level was significantly decreased in the LPS group,and the expression level of autophagy substrate protein P62 was increased.Compared with LPS group,LCB protein expression was increased in CHR group and Rapa group,while P62 protein expression was significantly decreased.The results of transmission electron microscopy showed that the cell morphology of the Control group was intact and the mitochondria structure was normal.There were autophagosomes in CHR,and the number of autophagosomes was significantly increased in Rapa group,but there was no obvious autophagosomes in CGA_4-16group.The cells in the LPS group were swollen and contained lamellar bodies,and autophagosomes were forming.After pretreatment with CHR,autophagy was observed in all groups,and a large number of autophagy lysosomes were observed,and the occurrence of autophagy lysosomes was more obvious than that in the LPS group.After pretreatment with Rapa as autophagy inducer,autophagy lysosomes appeared,and the number of autophagy lysosomes was also significantly increased.Autophagosome formation was not observed in the CGA_4-16group.Conclusions:CHR can promote the protective autophagy of alveolar macrophages by regulating TLR4-related signaling pathway,and has a protective effect on ALI after LPS stimulation.