Bone Marrow Derived Mesenchymal Stem Cells Alleviate Acute Asthma Through Negatively Regulating The Function Of Dendritic Cells

Objective Study the mechanism of BMSCs alleviating asthma model mice,and further explore the relationship between BMSCs,the autophagy of DCs and asthma.Method 1.BMSCs relieve the severity of asthma model mice through inhibiting the autophagy of lung dendritic cells.BABL/b mice were divided into 3 groups,named control group,asthma group and BMSCs-treated group.After model made respectively,we detected the proportion of dendritic cells and co-stimulatory molecules expressed on the dendritic cells in the lung and mediastinal lymph nodes(MLN)through FACS,tested the level of Ig E in serum and TH2 type cytokines(IL-4,IL-5,IL-13)in bronchoalveolar lavage fluid(BALF)by ELISA.What's more,we observed the inflammatory cells infiltration in lung through H&E straining and mucus secretion in lung through PAS straining.In addition,we detected the expression of autophagy related protein in Lung dendritic cells.2.Observe whether BMSCs regulator the function of dendritic cell by inhibiting autophagy in vitro We collected BMSCs and BMDCs cultured in vitro respectively.After pre-treating BMDCs with BMSCs,we used LPS to stimulate BMDCs,ctr-BMDCs and BMSCs conditioned BMDCs,for several hours.Then we detected the level of cytokines(IL-6,TNF-?)by ELISA,co-stimulatory molecules expressed on BMDCs by FACS,and the ability to stimulate OT?T cell to proliferate and take in OVA antigen by FACS.What's important,we added autophagy stimulator rapamycin(Rapa)to further inspect whether these changes were affected or not.3.Investigate autophagy and function of BMDCs affected by rm IL-10 in vitro(1)BMDCs were divided into 3 groups as follows,BMDCs group,LPS stimulated BMDCs group and LPS stimulated IL-10-treated group.We utilize the FACS to test the co-stimulator on the surface of BMDCs in different group and the ability of these BMDCs to active T cells to proliferate.ELISA was used to detect the ability of BMDCs to secret cytokines.(2)Pre-treated by rm IL-10 for several hours,im DCs were divided into five groups: Ctr-BMDCs group,LPS stimulated group,LPS stimulated IL-10-DCs group,Rapa + LPSstimulated group,and Rapa + LPS stimulated IL-10-DCs group.Then we observed the functions of dendritic cells as mentioned above,including using FACS to detect the activation condition of BMDCs and the ability to stimulate T cell to proliferation and to take in antigen,utilizing ELISA to examine the ability to produce cytokines.(3)We collected BMDCs cultured in vitro stimulated by LPS at different time,and utilized western blot to detect the expression of autophagy related protein LC3 B to disclosure the best time.Based on this,we further observed if IL-10 had an effect on the autophagy of BMDCs induced by LPS.4.Statistical analysis.All the experimental data referred in this paper were analyzed by Prism Graph Pad6 software to complete the statistical analysis.All the metrological data were expressed with x±s.After normality test and variance analysis of all the groups' data,we use One-Way ANOVA to analyze the data followed the normal distribution and homogeneity of variance,otherwise we utilized Kruskal-Wallis test instead.Results with P < 0.05 for the difference is statistically significant.Results 1.BMSCs can moderate asthma model mice through inhibiting autophagy and function of lung DCs.(1)Compared to asthma model group,the number of inflammatory cells and the level of mucus secretion in lung,the total cells,eosinophils and the level of Th2 type cytokines(IL-4,IL-5)in BALF,and the level of Ig E in serum were much less in BMSCs treated group.What's important,the cytokine IL-10 in BMSCs treated group is higher than that in asthma model.(2)The proportion of dendritic cells and the co-stimulatory molecules expressed on dendritic cells in lung and mediastinal lymph nodes in BMSCs treated group was lower than in asthma model group,which had a statistically significant.(3)The level of autophagy of lung dendritic cells in asthma model group was significantly higher than that in control group,because the ratio of LC3?/LC3?is higher in asthma model group.What's important,the level of autophagy was decreased in BMSCs treated group,which had a statistically significant.2.BMSCs probably regulate function of BMSCs through inhibiting autophagy(1)Compared to ctr-DCs,after LPS stimulating,the surficial co-stimulator molecules(MHC ?,CD86)on BMSCs treated-DCs were significantly lower.And theability to secret IL-6 and TNF-? and to stimulate T cell to proliferate were impaired after BMSCs treatment,which had a statistically significant.(2)The inhibitory effect of BMSCs on BMDCs could be partly or completely reversed when we added autophagy stimulator Rapa.We found that the co-stimulator molecules(CD86,MHC ?,CD40,CD80)expressed on BMDCs were significantly higher in Rapa + BMSCs treated group than that in only BMSCs treated group.The level of cytokine secretion was in the same way.3.IL-10 regulate function of BMSCs through inhibiting autophagy(1)After LPS stimulating,the level of co-stimulatory molecules expressed on IL-10 treated-BMDC were obviously lower than that on ctr-BMDCs,as well as the ability to stimulate T cell to proliferate and to secret inflammatory-cytokines,all of which had a statistically significant.What's important,this inhibitory impact could be inverted if autophagy stimulator Rapa added in,which also had a statistically significant.(2)LPS could induce BMDCs autophagy,and we could observe distinct autotomy phenomena at 12 hour.Based on this,we detected that the autophagy of IL-10 treated-BMDCs was lower than that of ctr-BMDCs after LPS stimulating,because the ratio of LC3?/LC3? was lower.Conclusion BMSCs may inhibit autophagy of BMDCs to regulate the function through secreting IL-10,so as to alleviate the inflammation of acute asthma model in mice.