| Backgroud and objectives Burn are tissue damage caused by heat,electricity,chemicals,radiation,etc.,mainly skin and mucosa damage.Burns are often associated with severe inflammation and organ damage,especially in the lungs.The most common complication of patients with burns is pulmonary infection.After severe burns,the body’s immune function declines,which leads to the aggravation of lung infection and the increased mortality.Acute lung injury(ALI)is one of the most common complications in patients with extensive deep burn,which can cause hypoxia in body,and patients are prone to acute respiratory distress syndrome(ARDS).It is also one of the reasons for the high mortality rate of burn patients.Previous studies have found that inflammatory response,oxidative stress and apoptosis of lung tissue are the main pathological mechanisms for the occurrence and development of lung injury complications after severe burns.Mesenchymal stem cells(MSCs),as a promising cell therapy for human diseases through differentiation,self-renewal and immune regulation,have attracted wide attention and have great potential in developing new therapies for various diseases.MSCs can be separated from bone marrow,adipose tissue,umbilical cord and blood.However,due to the tumorigenicity of these cells,their clinical application is limited.Studies have shown that the therapeutic effect of bone marrow mesenchymal stem cells depends largely on paracrine factors,exosomes.Exosome is a nano-bilayer membrane structure containing lipids,small RNA and proteins,which plays an important role in intercellular communication.Because of its low immunogenicity,tumorigenicity andeasy management,exosome has become a promising alternative to whole-cell therapy.In addition,studies have shown that exosome can play a role in the treatment of human diseases through anti-oxidation,anti-inflammation and inhibition of apoptosis.Therefore,this study aimed to investigate the effect of exosome derived from human umbilical cord mesenchymal stem cells(hUCMSCs)on improving ALI induced by burn and its molecular mechanism.Methods After cultured for 3-5 generations,the culture supernatant of hUCMSCs was collected,and the exosome was isolated by ultracentrifugation.The morphology and size of obtained hUCMSCs-Exo were analyzed by transmission electron microscopy and nanoparticle tracking analysis,respectively.The specific markers CD63 and CD9 of exosome were detected by Western blotting.The expression of miRNA-451 in hUCMSCs and hUCMSCs-Exo were detected by qRT-PCR.In the hUCMSCs,the liposome transfection reagent was used to transfect the miR-451 inhibitor and its control sequence(inhibitor NC),and the exosomes of the two cell,named NCI-Exo and miR-451I-Exo,were collected.In vivo experiments were performed in rats,and rats were divided into 6 groups: sham,burn,burn+PBS,burn+Exo,burn+NCI-Exo,burn+miR-451I-Exo.In burn group,the rat was prepared the 30% III degree scald model by hot water scalding.In scald model,the Exo injection group was tail vein injected with hUCMSCs-Exo.The lung function index of each group was detected 6 h after scald.Peripheral blood of each group was collected at 6h,12 h,24h and 48 h after scald.The serum inflammatory factors IL-6,IL-1β and TNF-α were detected by ELISA.At 24 h and 48 h after scald,the rats were sacrificed and lung tissues were collected.ELISA was used to detect the levels of inflammatory factors(IL-6,IL-1β and TNF-α),and oxidative stress related factor(malonaldehyde(Malonaldehyde).,MDA),myeloperoxidase(MPO)and superoxide diamutase(SOD)activity)in lunghomogenate.Morphological changes of lung tissue were observed by HE staining.The proportion of apoptosis in lung tissue was detected by TUNEL.Western blotting was used to detect the activation of the TLR4/NF-κB signaling pathway.Results1.hUCMSCs-Exo was successfully isolated from the supernatant of cultured hUCMSCs.Transmission electron microscopy showed that hUCMSCs-Exo was a spherical vesicle with a diameter of 40-160 nm.Western blot results showed that hUCMSCs-Exo highly expressed exosome-specific markers CD63 and CD9.2.The qRT-PCR data demonstrated that the expression of miR-451 was enriched in hUCMSCs-Exo,and the expression of miR-451 in exosome secreted by hUCMSCs after the deletion of miR-451 was also significantly reduced(p<0.05).3.Pulmonary function index analysis: Compared with sham group,burn group had less maximal inspiratory capacity,higher central airway resistance and lower lung compliance(p<0.05).After Exo injection,the maximal inspiratory capacity was significantly increased,central airway resistance was significantly decreased and pulmonary compliance was significantly increased(p<0.05).In addition,the improvement of pulmonary function in rats was significantly weakened by exosome from hUCMSCs knockout of miR-451.4.The expression of inflammatory factors in serum: At different time points,the expression of IL-6,IL-1β and TNF-α in the serum of burn rats were all increased significantly(p<0.05).After Exo injection,the expression of inflammatory factors were significant decreased(p<0.05),and the inhibition role of exosome from hUCMSCs knockout of miR-451 on serum inflammatory factors was weakened.In addition,the levels of IL-6 and IL-1β reached their maximum at 6 hours after scald,and graduallydecreased with the prolongation of time,while the level of TNF-α reached its maximum at 24 hours after scald.5.The expression of inflammatory factors in lung tissue: compared with sham group,the expression level of inflammatory factors in lung homogenate of burn rats was increased significantly(p<0.05),while be decreased in Exo rats(p<0.05).In addition,the exosome from hUCMSCs knockout of miR-451 have weakened role in inhibiting inflammatory factors.6.The activation of TLR4/NF-κB signaling pathway: TLR4/NF-κB signaling pathway was activated in burn rat,that is,the expression of TLR4 and p-p65 was significantly up-regulated(p<0.05).Exo injection can significantly inhibit TLR4/NF-κB signaling pathway(p<0.05),while the exosome from hUCMSCs knockout of miR-451 have weakened role in inhibiting TLR4/NF-κB signaling pathway.7.The oxidative stress related molecule expression/activity: MDA level and MPO activity were significantly increased in burn rats,while SOD activity was significantly decreased(p<0.05).Exo significantly inhibited MDA level and MPO activity and increased SOD activity(p<0.05),while the antioxidant effect of exosome from hUCMSCs knockout of miR-451 was significantly reduced.8.The pathological damage of lung tissue.The lung tissue of sham rat was pale pink,the lung tissue structure was clear completed,the alveolar wall was thin without edema,and the interstitial blood vessels were not dilated.In Burn group,the alveolar structure was obviously disordered and incomplete,the lung tissue was edematous,and the interstitial lung was thickened.The lung tissue and interstitial structure of the Exo injection group were relatively intact,and the edema of alveoli and interstitium was alleviated.The results of lung injury score showed that burn group had significantly greater lung injury than sham group(p<0.05),Exo injection had significantly decreasedlung injury score(p<0.05),while exosome from hUCMSCs knockout of miR-451 had significantly increased lung injury score.9.Proportion of apoptotic cells in lung tissue: TUNEL results showed that apoptotic cells in burn group were significantly higher than that in sham group(p<0.05).Apoptotic cells in lung tissue of rats injected with Exo were significantly reduced(p<0.05),while the anti-apoptotic effect of exosome from hUCMSCs knockout of miR-451 was significantly weakened.Conclusion1.The exosome isolated from hUCMSCs is a group of small vesicles with membrane structure and low electron density,whose surface expresses the specific proteins CD63 and CD9.2.hUCMSCs inhibit the activation of TLR4/NF-κB signaling pathway and the expression of inflammatory factors in burn rats through exosomal miR-451.3.hUCMSCs reduce oxidative stress in burn rats through exosomal miR-451.4.hUCMSCs reduce the apoptosis of lung tissue cells in burn rats and effectively improve the lung function and lung injury in burn rats through exosomal miR-451. |
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