Effects Of Astragaloside Ⅳ On Immune Function In Asthmatic Model Mice

Objective: Asthma is an inflammatory disease of respiratory system.In order to study the effects of astragaloside Ⅳ on immune Th cells in OVA-induced asthma mouse model in vivo and in vitro and find new targets of asthma disease with astragaloside Ⅳ treatment,we used a mouse asthma model,in which airway inflammation and airway remodeling was produced by ovalbumin sensitization and challenge.Methods:(1)Proteomics technology was used to analyze the protein profiles from serum of OVA-induced mice by protein extraction,trypsin digestion,peptide fractionation,and high-precision mass spectrometry.The mass spectrometry data were compared for relative quantification of proteins before and after OVA-induction.Mass spectrometry used data-independent acquisition(DIA)to search for specific proteins by bioinformatics methods.(2)OVA-induced asthma model were established and divided into control,model,astragaloside Ⅳ and dexamethasone group randomly.Pulmonary pathological changes were determined by HE staining method.The changes of IFN-γ,IL-5,IL-13,IL-17 A and TGF-β from lung homogenate were examined by ELISA.m RNA expressions of transcription factors like T-bet,GATA-3,STAT-6,RORγt,and Foxp3 in spleen were studied by RT-PCR.(3)Primary spleen cells from OVA-induced mice were cultured in vitro.After treated with different concentration of astragaloside Ⅳ of cultured cells,cell viability assay was performed by CCK-8 kit.Meantime,the expression of Th2 cytokines like IL-4,IL-5 and IL-13 in the supernatant were determined by ELISA.(4)Immature dendritic cells,extracted from bone marrow of mice,were incubated with IL-4 and GM-CSF,and stimulated simultaneously by LPS.The expression of IL-12 was detected after incubation of astragaloside Ⅳ.Cell morphology was observed by microscope and CD11 c expression on the surface of BMDCs was examined by flow cytometry.Results:(1)A total 764 proteins were identified from the serum,in which 603 came from both groups.101 of them were found significant difference in which 23 proteins were up-regulated and 78 proteins were down-regulated by induction with OVA.The up-regulated proteins were mainly involved in the immune system and stress response.(2)Irregular tissue structure and severe inflammation were found in asthma group.For the astragaloside Ⅳ treatment group,inflammatory cell infiltration and airway epithelial cell damage in mice were improved significantly.The expressions of Th2cytokines(IL-5 and IL-13)in lung homogenate were down-regulated compare with asthma model group,however the expressions of IFN-γ and TGF-β were not changed.We found astragaloside Ⅳ could suppressed the expression of STAT-6 m RNA,GATA-3 m RNA,and ROR γt mRNA,however,no any effects for expression of Fox P3 and T-bet m RNA.(3)For the OVA-induced splenocytes,no any effects of the cell activity we found with astragaloside Ⅳ treatment in vitro.However,high concentration of astragaloside Ⅳ(200 μg/m L)had a significant inhibitory effect on the expression of IL-4,IL-5 and IL-13.(4)The shape of BMDCs was changed to long tentacles after culture in vitro.The expression of CD11 c was less than 60%,we guess we did not get enough mature dendritic cells this time.The expression of IL-12 increased significantly stimulated by OVA and decreased by astragaloside Ⅳ.Conclusions:(1)After OVA-induced in vivo,the immune system was activated of mice.The expression of some immune-related proteins was increase.This result provided a basis for the establishment of asthma model.(2)Astragaloside Ⅳ could reduce the lung inflammation through down regulation of Th2 transcription factors and pro-inflammatory cytokines IL-5,IL-13 and IL-17 A.Astragaloside Ⅳ may be use as a new active ingredient for the treatment of asthma.(3)Astragaloside Ⅳ,at high concentration,inhibit the Th2 cytokines like IL-4,IL-5 and IL-13,which secreted by splenocytes induced by OVA.However,astragaloside Ⅳ,at low concentration,no any significant inhibiting effects.(4)Astragaloside Ⅳ has a certain inhibitory effect on the cytokine IL-12 secreted by BMDCs,and the culture methods of the mature bone marrow-derived dendritic cells in this experiment need to be further verified.