CGA衍生多肽CHR通过调控自噬流减轻脂多糖诱导的小鼠肺损伤

Chromogranin A-derived polypeptide CHR alleviates lipopolysaccharide-induced lung injury in mice by regulating autophagy flux

目的:研究嗜铬粒蛋白A(chromogranin A,CGA)衍生多肽CGA47-66(chromofungin,CHR)对脂多糖(lipopolysaccharide,LPS)诱导脓毒症小鼠肺损伤的影响,并探讨CHR对肺组织自噬流的调节作用。方法:通过LPS(10 mg/kg)腹腔注射构建脓毒症肺损伤小鼠模型,分组依次为正常对照组(Control组)、LPS处理组(LPS组)、低剂量CHR预处理组(CHRL组,CHRL=15.5μg/kg)、高剂量CHR预处理组(CHRH组,CHRH=77.5μg/kg)。LPS腹腔注射6 h后留取肺组织标本,检测肺组织含水量及EB渗漏情况;HE染色切片观察肺组织病理变化;Western blot分别检测各组肺组织中自噬标志性蛋白LC3BⅡ和底物蛋白P62的表达;免疫荧光进一步观察各组肺组织中LC3B与P62的荧光强度表达情况。结果:与正常对照组相比,LPS组肺组织湿/干比值(5.093±0.275 vs.4.456±0.252,P=0.000)和EB含量(1.839±0.212 vs.1.163±0.199,P=0.000)明显增高,而CHRH组肺组织湿/干比值(4.598±0.163 vs.5.093±0.275,P=0.003)和EB含量(1.317±0.193 vs.1.839±0.212,P=0.001)较LPS组相比明显降低;肺组织HE染色切片光镜下可见Control组肺组织结构完整,肺泡腔清晰,LPS刺激6 h后,HE染色切片中可观察到肺泡腔内大量红细胞渗出及炎症细胞浸润,而CHRH组肺组织病理改变程度较LPS组有明显好转;Western blot结果显示,与Control组相比,LPS组LC3BⅡ蛋白表达量(0.553±0.028 vs.0.472±0.025,P=0.008)和P62蛋白表达量(0.343±0.031 vs.0.165±0.038,P=0.000)明显增高,CHRH组LC3BⅡ蛋白表达量(0.873±0.034 vs.0.553±0.028,P=0.000)较LPS组进一步升高,而P62蛋白表达量(0.240±0.020 vs.0.343±0.031,P=0.002)较LPS组明显降低;免疫荧光结果再次验证了LC3B和P62蛋白的这种表达变化趋势。结论:CGA衍生多肽CHR可改善脂多糖诱导的急性肺损伤,其机制可能与促进自噬流通畅有关。

Objective:To investigate the effect of the chromogranin A(CGA)-derived peptide CGA47-66,i.e.,chromofungin(CHR),on lung injury induced by lipopolysaccharide(LPS)in mice with sepsis,as well as the regulatory effect of CHR on autophagy flux in lung tissue.Methods:A mouse model of sepsis and lung injury was established by intraperitoneal injection of LPS(10 mg/kg).The mice were divided into normal control group(control group),LPS stimulation group(LPS group),low-dose CHR(15.5μg/kg)pretreatment group(CHRLgroup),and high-dose CHR(77.5μg/kg)pretreatment group(CHRHgroup).Lung tissue samples were collected at 6 hours after the intraperitoneal injection of LPS to measure water content and EB leakage of lung tissue.HE staining was used to observe the pathological changes of lung tissue,Western blot was used to measure the protein expression of the autophagy marker LC3 B II and the substrate protein P62,and immunofluorescence assay was used to observe the fluorescence intensity of LC3 B and P62 in lung tissue.Results:Compared with the control group,the LPS group had significantly higher wet/dry weight ratio(5.093±0.275 vs.4.456±0.252,P=0.000)and EB content of lung tissue(1.839±0.212 vs.1.163±0.199,P=0.000),while compared with the LPS group,the CHRHgroup had significantly lower wet/dry weight ratio(4.598±0.163 vs.5.093±0.275,P=0.003)and EB content(1.317±0.193 vs.1.839±0.212,P=0.001).HE staining of lung tissue showed that under a light microscope,the control group had complete structure of lung tissue and clear alveolar space;after 6 hours of LPS stimulation,excessive erythrocyte diapedesis and inflammatory cell infiltration were observed in the alveolar space in the LPS group,and the CHRHgroup had significant improvements in these pathological changes compared with the LPS group.The results of Western blot showed that compared with the control group,the LPS group had significant increases in the protein expression of LC3 BⅡ(0.553±0.028 vs.0.472±0.025,P=0.008)and P62(0.343±0.031 vs.0.165±0.038,P=0.000);compared with the LPS group,the CHRHgroup had a significant increase in the protein expression of LC3 BⅡ(0.873±0.034 vs.0.553±0.028,P=0.000)and a significant reduction in the protein expression of P62(0.240±0.020 vs.0.343±0.031,P=0.002).The results of immunofluorescence assay confirmed the changing trend of the protein expression of LC3 B and P62.Conclusion:The CGA-derived polypeptide CHR can improve LPS-induced acute lung injury,possibly by promoting autophagy flux.