摘要
目的研究脐带间充质干细胞上清液对脂多糖(lipopolysaccharide,LPS)诱导的急性肺损伤(acute lung injury,ALI)的保护作用及机制。方法选择6周龄C57BL/6雄性小鼠共40只,随机数字法分为假手术(sham)组,LPS模型组,LPS+人脐带间充质干细胞上清液(HucMSC-cm)(LPS+cm)治疗组,LPS+HucMSC-cm+Compound C(LPS+cm+cc)干预组,每组10只。通过气管内注射LPS 5 mg/kg构建ALI模型,4 h后予以气管内注射HucMSC-cm 50μL/只构建治疗组;干预组在LPS及HucMSC-cm前30 min予腹腔注射Compound C 15 mg/kg;72 h后留取小鼠外周血检测中性粒细胞比例,并处死小鼠留取肺组织。通过苏木精-伊红染色检测小鼠肺组织病理学改变,Western Blot及免疫组化法分析肺组织中IL-6、ICAM-1、VCAM-1和磷酸化腺苷酸活化蛋白激酶(phosphate AMP-activated protein kinase,p-AMPK)的表达。体外培养人肺微血管内皮细胞(HuLEC-5a),将细胞分为三组:control组、LPS组(10μg/mL)、LPS+HucMSC-cm组。处理24 h后,Western Blot检测p-AMPK及AMPK蛋白表达水平,RT-PCR检测IL-6及IL-8的mRNA表达。多组间比较采用单因素方差分析,两组比较采用Tukey法检验。结果与sham组相比,LPS组小鼠肺组织充血水肿加重,肺间隔增厚和炎性细胞浸润增多,肺组织IL-6(P=0.003)、ICAM-1(P<0.001)及VCAM-1(P=0.001)蛋白水平明显上升,p-AMPK蛋白水平表达下降(P=0.013),外周血中性粒细胞比例上升(P<0.001),LPS+HucMSC-cm组肺组织充血水肿及病理学损伤较LPS组得到改善,肺组织中IL-6(P=0.003)、ICAM-1(P=0.001)及VCAM-1(P=0.006)蛋白水平下降,p-AMPK蛋白水平表达上升(P=0.002),外周血中性粒细胞比例下降(P<0.001),而LPS+cm+cc组肺组织病理学损伤较LPS+HucMSC-cm组再次加重,肺组织IL-6、ICAM-1及VCAM-1蛋白水平明显上升,p-AMPK蛋白水平表达下降,免疫组化法得到与蛋白相一致的结果。体外实验中,LPS处理后,IL-6(P<0.001)及IL-8(P=0.027)的mRNA表达较control组上升,且p-AMPK蛋白水平下降(P=0.005);LPS+HucMSC-cm组较LPS组IL-6(P=0.003)及IL-8(P=0.002)的mRNA表达下降,p-AMPK蛋白水平上升(P=0.003)。结论人脐带间充质干细胞上清液通过激活AMPK活性改善LPS诱导的ALI。
Objective To investigate the protective effect of human umbilical cord mesenchymal stem cell conditioned medium(HucMSC-cm)against lipopolysaccharide(LPS)-induced acute lung injury(ALI)and relevant mechanism of action.Methods Forty 6-week-old male C57BL/6 mice were selected and randomized(random number)into the sham group,LPS group,LPS+HucMSC-cm(LPS+cm)group,and LPS+HucMSC-cm+Compound C(LPS+cm+cc)group,with 10 mice in each group.Mice were intratracheally injected with LPS(5 mg/kg)to establish ALI model,and intratracheally injected with hucMSC-CM(50μL)4 h after LPS treatment.Mice in the LPS+cm+cc group were intraperitoneally treated with Compound C(15 mg/kg)prior to LPS treatment.Neutrophils in peripheral blood were counted with the automated hematology analyzer 72 h after LPS administration.After that,mice were sacrificed,and the lung tissue pathology was observed using hematoxylin eosin(HE)staining.Besides,the expressions of IL-6,ICAM-1,VCAM-1 and P-AMP-activated protein kinase(P-AMPK)in the lung tissues were analyzed by Western blot and immunohistochemical assay.In vitro,human lung microvascular endothelial cells(HuLEC-5a)were cultured and divided into three groups:control group,LPS group(10μg/mL),and LPS+HucMSC-cm group.After 24 h of treatment,the expressions of p-AMPK and AMPK were detected by Western blot,and the expressions of IL-6 and IL-8 were detected by real-time fluorescence quantitative PCR.Oneway analysis of variance was used to compare the mean values of normally distributed measurement data between groups.Comparisons between two groups were performed using the Tukey’s multiple comparison test.Results Compared with the sham group,the LPS group showed lungs with congestion and swelling,thickened pulmonary septum,and inflammatory cell infiltration.Moreover,in the LPS group,the protein expressions of IL-6(P=0.003),ICAM-1(P<0.001)and VCAM-1(P=0.001)were increased significantly,while the expression of p-AMPK was decreased(P=0.013),accompanied by an increase in the proportion of neutrophils in peripheral blood(P<0.001).Compared with the LPS group,the LPS+HucMSC-cm group demonstrated eased congestion,edema and pathological injury of lung tissue,reversed protein expressions of IL-6(P=0.003),ICAM-1(P=0.002),VCAM-1(P=0.006)and P-AMPK(P=0.002),as well as decreased proportion of neutrophils in peripheral blood(P<0.005).Compared with the LPS+HucMSC-cm group,the LPS+cm+cc group exhibited more severe lung histopathological injury,significantly increased protein expressions of IL-6,ICAM-1 and VCAM-1 in lung tissues,as well as decreased expression of P-AMPK protein.The results of immunohistochemistry were consistent with those of protein.In vitro experiment,after LPS treatment,the mRNA expressions of IL-6(P<0.001)and IL-8(P=0.027)were increased and p-AMPK protein expression(P=0.005)was decreased as compared with the control group.In comparison with the LPS group,the LPS+HucMSC-cm group showed decreased mRNA expression levels of IL-6(P=0.003)and IL-8(P=0.002),but increased protein level of p-AMPK(P=0.003).Conclusions HucMSC-cm has a protective effect against LPS-induced acute lung injury,which is mainly attributed to the inhibited expression of adhesion molecules and inflammatory factors under the activation of AMPK.
作者
陈海洋
杨宏锋
沈嘉琪
朱伟
虞志新
Chen Haiyang;Yang Hongfeng;Shen Jiaqi;Zhu Wei;Yu Zhixin(Department of Intensive Care Medicine,the Affiliated People's Hospital,Jiangsu University,Zhenjiang 212002,China;School of Medicine,Jiangsu University,Zhenjiang 212013,China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2022年第5期636-643,共8页
Chinese Journal of Emergency Medicine
基金
江苏省自然科学基金项目(BK20160547)。
关键词
人脐带间充质干细胞上清液
腺苷酸活化蛋白激酶
急性肺损伤
粘附分子
肺微血管内皮细胞
Human umbilical cord mesenchymal stem cell conditioned medium
AMP-activated protein kinase
Acute lung injury
Adhesion molecules
Lung microvascular endothelial cell
