| Objective: To study the effect of Wuhu Decoction through PI3K/Akt/mTOR signaling pathway to regulate DCs autophagy on the proliferation and activation of T cells and the secretion of inflammatory factors in RSV-induced asthmatic mice,and to further clarify the mechanism of action of Wuhu Decoction in the treatment of RSV-induced asthma..Methods:(1)The effect of Wuhu Decoction on the secretion of inflammatory factors in Th17 cells of RSV-induced asthmatic mice through DCs autophagy: Establish a mouse model of RSV-induced asthma.Divide the mice into normal group,model group,Wuhu Decoction low,medium and high dose groups,dexamethasone group,rapamycin and chloroquine groups,each with 10 groups.Only,after corresponding drug intervention,ELISA detects the expression of IL-17A and IL-17F in alveolar lavage fluid,HE,Masson,PAS detects lung tissue pathological changes and calculates collagen deposition and mucus reserve,and observes lung tissue under transmission electron microscope In autophagosomes,Western Blot method was used to detect the expression of autophagy proteins in dendritic cells.(2)The effect of Wuhu Decoction Drug-containing serum on the secretion of inflammatory factors in Th17cells of RSV-induced asthmatic mice through DCs autophagy: In vitro primary isolation of mouse bone marrow-derived DCs and spleen-derived CD4+T cells were co-cultured at a ratio of 1:10 to prepare high,medium,and low doses of Wuhu Decoction medicated serum,and construct RSV-induced autophagy of DCs in vitro Cell model,observe T cell proliferation and activation after adding medicated serum,perform anti-mouse CD4-PE/CD3-APC antibody and CD69 staining,flow cytometry analysis of CD4+ T cell proliferation and CD69 on T cell surface expression.(3)The regulatory effect of Wuhu Decoction on PI3K/Akt/mTOR signaling pathway in DCs autophagy in RSV-induced asthmatic mice: Establish RSV-induced asthma mouse models in vitro,and divide the mice into normal group,model group,Wuhu Decoction low,medium and high dose groups,dexamethasone group,rapamycin and chloroquine groups.After drug intervention,Western Blot detection of DCs in isolated lung tissues detected PI3K,Akt,phosph-Akt,phosph-mTOR,ULK1,phosph-ULK1,p70s6 k,phosph-p70s6 and other protein expressions on the PI3K-Akt-mTOR signaling pathway,and analyzed The effect of Wuhu Decoction on different links of the PI3K-Akt-mTOR signaling pathway in dendritic cells.(4)Wuhu Decoction Drug-containing serum regulates the autophagy of DCs induced by RSV through PI3K/Akt/mTOR signaling pathway: After the primary isolation of DCs,the RSV-induced DCs autophagy model was constructed and transfected with mRFP-eGFP-LC3 plasmid.The occurrence of autophagy was observed under a laser confocal microscope.Western Blot was used to detect the PI3K/Akt/mTOR signaling pathway.Expression of key proteins.Results:(1)The results of in vivo experiments showed that the levels of IL-17A and IL-17F in the alveolar lavage fluid of the model group were higher than those in the normal group.The expression levels of IL-17A and IL-17F decreased after intervention with different doses of Wuhu Decoction;Pathological staining showed that the lung tissue of the model group had inflammatory exudation,luminal stenosis,and increased levels of collagen deposition index and mucus reserve index,which improved after treatment with various doses of Wuhu Decoction.Transmission electron microscopy and autophagy protein detection showed that the model group was small The autophagy level of mice increased compared with mice in the normal group,and the autophagy level further increased after treatment in each dose group of Wuhu Decoction.(2)The results of in vitro experiments show that after DCs and T cells are co-cultured and infected with RSV,CD4+ T cells proliferate and activate,whether it is through the autophagy inducer rapamycin or the autophagy inhibitor chloroquine,it can act on DCs,It affects the autophagy function of DCs,and then affects the proliferation and activation of CD4+T cells,and restores the hyperproliferation of T cells to normal.Compared with the model control group(P<0.01),low,medium,and high drug-containing serum acts on DCs can significantly inhibit the abnormal proliferation and activation of CD4+ T cells,and the inhibitory effect increases with the increase of concentration,which is significantly different from the control group(P<0.01).This has a similar effect to the autophagy regulators rapamycin and chloroquine.(3)In vivo experiments show that the PI3K/Akt/mTOR signaling pathway is activated in RSV-induced asthma mouse models.After the intervention of Wuhutang,the PI3K/Akt/mTOR signaling pathway is inhibited from the upstream and the expression of autophagy-related proteins.(4)The results of in vitro experiments show that the RSV-induced DCs autophagy model has a higher autophagy level under the laser confocal microscope,and the autophagy level of DCs further increases after the intervention of the Wuhu Decoction Drug-containing serum.Western Blot detects the Wuhu Decoction Drug-containing serum can decrease the expression of PI3K,phosph-Akt,phosph-mTOR,ULK1,phosph-ULK1,and phosph-p70s6 protein.Conclusion:(1)In RSV-induced asthma,the autophagy level of DCs is increased,the expression of IL-17A and IL-17F is increased,the T cell has excessive activation and proliferation,and the PI3K/Akt/mTOR signaling pathway is activated.(2)Wuhu Decoction up-regulates the autophagy level of DCs and reduces the expression of IL-17A and IL-17F in in vivo experiments.In in vitro experiments,it can inhibit over-activated proliferation of T cells to improve airway inflammation and airway remodeling in asthma.(3)In vivo and in vitro experiments,Wuhu Decoction can inhibit the expression of proteins on the PI3K/Akt/mTOR signaling pathway from upstream and increase the autophagy level of DCs,thereby affecting the proliferation and activation of T cells and the secretion of inflammatory factors,then exerting therapeutic effects. |
PDF Full Text Request