Mechanism Of Conditioned Medium Of Umbilical Cord Mesenchymal Stem Cells In Repairing Acute Lung Injury

Objective: The pathogenesis of acute lung Injury(ALI)is complex,in which the essence of early onset is uncontrolled inflammation,and a large number of inflammatory factors enter the Lung tissue.It has been proved that from conditioned medium of human umbilical cord mesenchymal stem cells(HucMSC-cm)have effects of anti-inflammatory and immunomodulatory in previous studies,but the mechanism in lipopolysaccharides(LPS)-induced ALI is still unclear.This study aimed to investigate the protective effect of HucMSC-cm against LPS-induced ALI and relevant mechanism of action.Methods:(1)HucMSCs were isolated and obtained by adherent method.Then flow cytometry was used to detect the surface markers CD105,CD90 and CD29 of HucMSCs.Next,the HucMSC-cm was collected.(2)To investigate the effect of HucMSC-cm on LPS-induced ALI,6-week-old male C57BL/6 mice were selected and randomized into sham group,LPS group,LPS + HucMSC-cm(LPS+cm)group,5mice each group.Mice were intratracheally injected with LPS(5 mg/kg)to establish ALI model,and intratracheally injected with HucMSC-cm(50 μL)4 h after 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 inflammatory factor(IL-6)and adhesion molecules(ICAM-1 and VCAM-1)in the lung tissues were analyzed by Western Blot and immunohistochemical assay.(3)To determine the role of AMPK in the protection of HucMSC-cm against LPS-induced ALI,6-week-old male C57BL/6 mice were selected and randomized into sham group,LPS group,LPS+cm group,LPS+HucMSC-cm+Compound C(LPS+cm+cc)group,5 mice each group.The intervention group was constructed by intraperitoneal injection of AMPK inhibitor Compound C(15 mg/kg)30 minutes before LPS and HucMSC-cm.The pathological changes of lung tissues were detected by HE staining as well as the expressions of inflammatory factor(IL-6),adhesion molecules(ICAM-1 and VCAM-1)and key molecule(p-AMPK)in lung tissues were analyzed by western blot and immunohistochemistry.(4)Human lung microvascular endothelial cells(Hu LEC-5a)were cultured in vitro and divided into three groups: control group;LPS group(10 μg/m L);LPS+HucMSC-cm group.After 24 h of treatment with LPS and HucMSC-cm,western blot was used to detect the protein levels of p-AMPK and AMPK.Besides,the m RNA expression levels of IL-6 and IL-8 were detected by real-time fluorescence quantitative PCR.Results:(1)HucMSCs were isolated and cultured from umbilical cord tissue.It was showed that they expressed CD105,CD90 and CD29 but not CD45,CD34 and CD19.(2)Compared with the sham group,the LPS group showed lungs with congestion and swelling,thickened pulmonary septum,and inflammatory cells infiltration.Moreover,in the LPS group,the protein expressions of IL-6,ICAM-1 and VCAM-1and the proportion of neutrophils in peripheral blood increased significantly,while the expression of p-AMPK decreased.(3)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,ICAM-1,VCAM-1 and p-AMPK,as well as decreased proportion of neutrophils in peripheral blood.(4)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.The results of immunohistochemistry were consistent with those of protein.(5)In vitro experiment,after LPS treatment,the m RNA expressions of IL-6 and IL-8increased and p-AMPK protein expression decreased as compared with the control group.In comparison with the LPS group,the LPS+HucMSC-cm group showed decreased m RNA expression levels of IL-6 and IL-8 but increased protein level of p-AMPK.Conclusion:HucMSC-cm has a protective effect against LPS-induced ALI,which is mainly attributed to the inhibited expression of adhesion molecules and inflammatory factors under the activation of AMPK.