CGA-derived Polypeptide CHR Alleviates Lipopolysaccharide-induced Lung Injury In Mice By Regulating Autophagic Flux

Objective:To investigate the effect of chromogranin?CGA?-derived peptide CGA_47-66?Chromofungin,CHR?on lung injury in mice with sepsis induced by lipopolysaccharide?LPS?,and to explore the regulating of CHR on autophagic flux in lung tissue.Methods:Sepsis-induced lung injury animal models were constructed by intraperitoneal injection of LPS?10 mg/kg?.The groups were divided into the normal saline control group?Control group?,LPS group?LPS group,LPS=10mg/kg?and low dose CHR pretreatment group?CHR_L group,CHR_L=15.5?g/kg?,high dose CHR pretreatment group?CHR_H group,CHR_H=77.5?g/kg?.After LPS administration,the general condition and 5day survival rate of sepsis mice were observed.Lung tissue samples were collected after 6 hours intraperitoneal injection of LPS to detect the water content and EB leakage of lung tissue.The pathological changes of lung tissue were observed by HE staining.The changes of autophagy were observed by transmission electron microscopy.The expression levels of autophagy associated marker protein LC3B II,substrate protein P62,autolysosome-related protein Rab7 and LAMP2 were detected by Western blot.The expressions of LC3B and P62 were further observed by immunofluorescence.Results:The activities of animals were decreased,the reaction were slower,the hair were erect,the spirits were drooping,the purulent secretion in eyes were increased,and suffered diarrhea after LPS treatment.The general condition of CHR_L group and CHR_H group were better than LPS group,but slightly worse than Control group.The 5 day survival rate of LPS group was 33.3%and significantly lower than Control group?P<0.05?,the 5 day survival rate of CHR_L group and CHR_H group were all66.7%and improved compared with LPS group,but the differences were not statistically significant?P>0.05?.Compared with the Control group,the W/D ratio?5.093±0.275 vs 4.456±0.252,P=0.000?and the EB content?1.839±0.212 vs 1.163±0.199,P=0.000?of lung tissues in LPS group were significantly increased,while the W/D ratio?4.598±0.163 vs 5.093±0.275,P=0.003?and the EB content?1.317±0.193 vs 1.839±0.212,P=0.001?of lung tissue in CHR_H group were significantly lower compared with LPS group.HE staining of lung tissue showed that the structure of lung tissue were intact and the alveolar cavity were clear in Control group.After 6hours of LPS stimulation,excessive red blood cell exudation and inflammatory cell infiltration in the alveolar cavity were observed in LPS group,but the degree of pathological change was significantly improved in CHR_H group compared with LPS group.The results of Western blot showed that the protein expressions of LC3B II?0.553±0.028 vs0.472±0.025,P=0.008?and P62?0.343±0.031 vs 0.165±0.038,P=0.000?in LPS group were significantly increased,the protein expressions of Rab7?0.149±0.025 vs 0.984±0.068,P=0.000?and LAMP2?0.543±0.019 vs0.622±0.014,P=0.000?in LPS group were significantly decreased compared with Control group.Compare with LPS group,the protein expression levels of LC3B II?0.873±0.034 vs 0.553±0.028,P=0.000?in CHR_H group was further increased,while the protein expression of P62?0.240±0.020 vs 0.343±0.031,P=0.002?in CHR_Hgroup was significantly decreased,and the protein expression levels of Rab7?0.576±0.022 vs0.149±0.025,P=0.000?and LAMP2?0.970±0.019 vs 0.543±0.019,P=0.000?were significantly increased.Immunofluorescence results again confirmed this expression trend of LC3B and P62 protein.Conclusion:TheCGA-derivedpolypeptideCHRalleviates lipopolysaccharide-induced acute lung injury by restoring the damaged autophagic flow of lung tissue in septic mice,consequently exerting organ protection.