The Study On Effect Of Astragalus Polysaccharide To CD₄⁺T Cell In Asthmatic Mice

Objective:To observe the influence of astragalus polysaccharide on the balance of Th1/Th2/Th17/Treg cells and related factors of CD4+T cell in asthmatic mice.Methods:⑴Female BALB/c mice were randomly divided into asthmatic model group, normalcontrol group. Asthmatic model group in0,7,14days was intraperitoneally injected with0.1ml the allergens liquid (including OVA10μg and Aluminium Hydroxide2mg), began21days, inspired by inhalation of2%OVA, once a day, every45minutes, atomization7days,normal control group was sensitized and excited with saline instead of OVA, the two groupsof mice were sacrificed after the last excitation48h. Using Cytometric Bead Array（CBA）method to detect the level of IL-4, IFN-γ in serum and broncho-alveolar lavage fluid (BALF)of mice, HE staining of lung tissue pathological change, counted the total number of BALFcell and cell sorting, to verify the success of asthmatic model group.⑵Mice were randomlydivided into normal control group, asthmatic model group, treatment group of astragaluspolysaccharide (APS). The treatment of normal control group, asthmatic model group wereconsisitent with asthma model identification, the sensitization phase of APS in the low,medium and high molecular weight group were consisitent with the model group, theninspired after30minutes of the intraperitoneal injection of0.1ml APS solution withconcentration for4g/ml, the three groups of mice were sacrificed after the last excitation48h,to observe the therapeutic effect of APS on asthmatic mice.①Detected the level of IL-4,IFN-γ in serum and BALF of mice, HE staining of lung tissue pathological change, countedthe total number of BALF cell and cell sorting, to observe the therapeutic effect of APS at theanimal level.②Separated the single cell of spleen, after positive selection of CD4+T cellwith anti-mouse CD4Particles and detection for purity, the CD4+T cell of each group weredivided into normal group, asthma group, treatment group, then cultured in the1640culturesolution containing PMA, Ionomycin, Golgistop at37℃,5%carbon dioxide environment for5hours, collected the cell to detect the ratio of Th1, Th2, Th17, Treg cells by flow cytometry.CD4+T cell from asthmatic mice were divided into asthma group, APS group, normal groupcells still from normal control group of mice, after APS group was added APS solution for2 hours, added PMA, Ionomycin to each group, cultured for12h,24h,36h, then collected thesupernatant and-20℃cryopreservation, to measure the level of IL-4, IFN-γ, IL-17, IL-10byCBA method, to observe the therapeutic effect of APS at the cellular level.Results:⑴To verify the success of asthmatic model. Through behavioral observation in mice, wecould see visible asthma-like symptoms in asthma group, such as grabbing nose and mouth,restlessness, deep and fast respiration, gatism, no distinct changes in normal control. Lunghistopathological observation results, asthmatic group structural disordered, airway epithelialinjuryed, airway wall thickened, airway secreted amount of mucus, bronchial and peripheralvascular had amount of infiltration of inflammatory cells, lung airway structure of controlgroup was clear, airway epithelial neatly arranged, no thickening of airway smooth muscleand obvious change of inflammation. The level of IL-4, IFN-γ in serum and BALF, the levelof IL-4in asthma group elevated than normal group (P<0.05), the level of IFN-γ in asthmagroup was lower than normal group(P<0.05). Comparison of BALF leukocyte count andproportion of Eos, total white blood cell count and proportion of Eos in asthma group werehigher than normal group(P<0.05). The results showed the success of asthmatic model.⑵Toobserve the therapeutic effect of APS on asthmatic mice.①To observe the therapeutic effectof APS at the animal level. Behavioral observation in mice, spirit of the APS group wasobviously improve, basic normal breathing, high activity, no significant weight loss and othersymptoms, especially the low molecular weight group. Lung histopathological observationresults, lung tissue structure of APS group was relatively regular, airway epithelial relativelyintegrated, no secretion of mucus, infiltration of inflammatory cells in peribronchovasularinterstitum and alveolar lumen were significantly reduced, especially the low molecularweight group. The level of IL-4, IFN-γ in serum and BALF. The level of IL-4in APS groupwas lower than asthmatic group(P<0.05), the low molecular weight group was significantlyreduced, the level of IFN-γ in APS group elevated than asthma group(P<0.05), the lowmolecular weight group was significantly elevated. Comparison of BALF leukocyte count andproportion of Eos, total white blood cell count and proportion of Eos in APS group was lowerthan asthma group (P<0.05), the low molecular weight group was significantly reduced.②To observe the therapeutic effect of APS at the cellular level.The selecting purity of CD4+Tcell suggested the purity was24.8%before selection，then was75.2%after selection, theresults had significant difference(P<0.05), could be used in subsequent cell culture. Detectedeach cell proportion of Th1, Th2, Th17, Treg, the proportion of Th1, Treg cell in asthma group was lower than normal group(P<0.05), APS group was higher than asthma group(P<0.05), thelow molecular weight group was significantly elevated. The proportion of Th2, Th17cell inasthma group was higher than normal group(P<0.05), APS group was lower than asthmagroup(P<0.05), the low molecular weight group was significantly reduced. Cytokine level inthe CD4+T cell culture supernatant, the level of IFN-γ, IL-10in asthma group was lower thannormal group(P<0.05), APS group was higher than asthma group(P<0.05), the level of IL-4,IL-17in asthma group was higher than normal group(P<0.05), APS group was lower thanasthma group(P<0.05). On relationship between cytokine level and concentration of APS，with increasing concentration of APS，the level of cytokine did not change obviously(P＞0.05),which indicated that low dose of APS could affect secretion of cytokines significantly.Conclusion:1. APS could inhibit the airway inflammation in asthmatic mice.2. APS could impact theasthmatic mice by acting on the CD4+T cell to secrete cytokines and the balance ofTh1/Th2/Th17/Treg cells.3. The effect of low molecular weight group of APS on the treatmentof asthma was more significant.4. Low dose of APS might have a good treatment effect onairway inflammation in asthma.