| Background and purpose: Acute lung injury(ALI)is an inflammatory syndrome of the lungs with increased vascular permeability as the main clinical manifestation caused by a variety of pathogenic factors such as severe infection,trauma,and shock.If the disease progresses,it quickly develops into Acute Respiratory Distress Syndrome(ARDS),which pose a serious threat on the life and health of patients.The pathogenesis of ALI/ARDS involves pulmonary vascular endothelium damage,destruction of the alveolar capillary barrier,accumulation of protein-rich exudate and cell debris in the alveolar space,and increasing expression of cytokines and multiple inflammatory mediators in the lungs.The key factors leading to ALI are dysregulation of anti-inflammatory and proinflammatory balances.In the early stage of inflammation,traumatic shock and gram-negative bacterial endotoxin(LPS)can activate alveolar macrophages(PAM)to release a large amount of inflammatory cytokines,mainly tumor necrosis factor-?(TNF-?),leading to the occurrence of ALI/ARDS.Therefore,controlling the release of inflammatory factors is one of the treatment strategies for preventing lung injury.Stem cells with self-renewal and multi-differentiation capabilities have attracted worldwide attention of scientists and clinicians due to their potential to treat a variety of intractable and serious diseases.As a kind of adult stem cells,mesenchymal stem cells(MSCs)have the same characteristics as general stem cells,with an ability to differentiate into fat,muscle and even bone tissue and to secrete a variety of cytokines,participating in immune regulation,damage repair and regulation of hematopoiesis.Therefore,growing evidence has recently indicated the beneficial effect of MSCs in various disease treatment,including acute lung injury.Exosomes are vesicle-like bodies that secreted from cells by active means whatever the cells are in rest or activating condition.Exosomes containing some cytokines,growth factors,lipids,encoded or non-coding RNA,and other biologically active substances released from stem cells may account for immune regulation,damage repair,metabolism,and cell signal transduction.At the same time,owing to the properties of stability,easy preservation,selective assemble and targeted delivery,safety and effectiveness for treatment of damaged tissue,exosomes from MSCs provide a new strategy for the treatment of acute lung injury.Based on the imbalances between anti-inflammatory and pro-inflammatory in acute lung injury,in this study,dental pulp mesenchymal stem cell-derived exosomes were used to intervene in acute lung injury in a rat cell model,in order to find out new breakthroughs in the treatment of acute lung injury.Methods: DPSCs in the teeth were isolated by tissue adherent method combing with enzymatic digestion.Adipogenic and osteogenic induction was performed to identify the multidifferentiation ability of DPSCs,and the gene related to adipogenic and osteogenic were examed.The expression of surface markers in DPSCs was detected by flow cytometry.Then DPSCs-derived exosomes from supernatants of DPSCs were collected by ultracentrifugation.After the centrifugation,the concentration of exosomes was gauged by the BCA method,the morphology of exosomes was photoed by transmission electron microscopy,and the markers on the surface of exosomes were identified.NR8383 rat alveolar macrophage cell line was treated with lipopolysaccharide(LPS)to establish a cell model of acute lung injury.The experimental cells were divided into 4 groups:(1)control group;(2)LPS group: stimulation with LPS at a final concentration of 1?g/m L.(3)LPS+low-dose exosome group: the final concentration of LPS and exosomes in the culture was 1?g/m L and 40?g/m L separately;(4)LPS+high-dose exosome group: the final concentration of LPS and exosomes in the culture was 1?g/m L and 80?g/m L separately.After treatment,cells were placed in an incubator,and then cell suspension supernatants were extracted at 0,6,12,and 24 hours respectively.Inflammatory factors including TNF-?,IL-1?,and IL-6 in the supernatants were measured with ELISA kit.Cell lysate from each group was prepared by treatement with RIPA for 24 hours,and then total protein was extracted by centrifugation.The relative expression levels and phosphorylated levels of extracellular regulatory protein kinase(p44/42),transcription factor NF-?B(p65)and I?B? protein were measured by Western Blot.Results: The results showed that after treatment of rat alveolar macrophages with LPS,the levels of inflammatory cytokines including TNF-?,IL-1? and IL-6 in the culture increased significantly compared with the control group,which indicated that the cell model was developed successfully.The concentration of TNF-? in the cell culture reached a peak at 6 hours after LPS treatment and then slightly decreased.However,the TNF-? in the group treatment with high-dose exosomes significantly decreased.The peak concentration of IL-1? in the cell culture presented at 24 hours after LPS treatment,and the IL-1? values in the groups treated with low or high doses of exosomes were statistically lower than those of the cells treated with LPS.The level of IL-6 reached a peak at 12 hours after LPS treatment and significantly decreased in the group treated with high-dose exosomes,compared with LPS group.After treated with LPS for 24 hours,the phosphorylation levels of NF-?B protein and I?B? protein were significantly increased when compared with the control group,while decreased dose-dependently after the low and high concentrations of exosome treatment.In addition,compared with the control group,the phosphorylation level of p44/42 protein increased significantly after 24 hours of LPS treatment,but did not apparently change in the group treated with exosomes.Conclusions: 1.DPSCs can be isolated from teeth using enzyme digestion and can be stably propagated in vitro.2.The DPSCs-derived exosomes,extracted by ultracentrifugation,have an intact membrane structure and show characteristic cup-shaped morphology,as flattened spheres with diameters ranging from 40 to 100 nm when examined under an electron microscope,and express the markers of CD9 and CD81.3.High concentration of exosomes derived from DPSCs can inhibit the production of inflammatory factors from the rat alveolar macrophages treated with LPS.4.The role of DPSCs-derived exosomes on acute lung injury may be involved in inhibition of NF-?B and MAPK pathway. |
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