Celery, Raising Cd4 ~ + Cd25 ~ + Regulatory T Cells And Suppress Asthmatic Airway Inflammation And Airway Remodeling In Mice

Part1: The Effects of Apigenin on the Airway Inflammation,Airway Hyperresponsiveness and CD4~+CD25~+Treg Cellsin Asthmatic MiceObjective：To investigate the effects of apigenin on the airway inflammation，airwayhyperresponsiveness and CD4~+CD25~+Treg cells in asthmatic mice. Methods:30BALB/c mice were randomly divided into5groups. They were control group,ovalbumin (OVA) group, CD25-depletion apigenin group, apigenin group andbudesonide group. Mice were sensitized and challenged by OVA. Airwayresponsiveness to acetylcholine chloride was measured. Hematoxylin&eosinstaining was used to assess the inflammatory cell infiltrates. Periodic acid schiff (PAS)was used to quantify airway global cells in the lungs. Levels of IL-4, IFN-γ and IL-13in bronchoalveolar lavage fluid (BALF), and total IgE and OVA-specific IgE inserum were detected by ELISA. The levels of CD4~+CD25~+Treg cells in the spleenwere measured with flow cytometry analysis. Results：The airway resistance in theOVA group was obviously increased in a dose-dependent manner by administrationof ACh, whereas only a slight increase could be detected in the control group. Therewere no significant differences in baseline airway resistance among five groups(P>0.05). Treatment with apigenin and budesonide led to a sharp decrease in airwayresistance compared with the OVA group (P<0.05). The number of eosinophils andtotal inflammatory cells in BALF in the OVA group increased significantly comparedwith the control group (P<0.05), which was significantly decreased by treatment withapigenin and budesonide (P<0.05). The levels of the Th2cytokines IL-4and IL-13inBALF, total serum IgE and OVA-specific IgE were significantly increased and thelevels of the Th1cytokines IFN-γ decreased in OVA-sensitized/challenged mice comapred with the control group (P<0.05). Administration of apigenin andbudesonide reduced the levels of these Th2cytokine in BALF, total serum IgE andOVA-specific IgE compared with the OVA group (P<0.05). Treatment with apigeninor budesonide increased the ratio of CD4~+CD25~+Treg cells in the spleen comparedwith that in the OVA group (P<0.05). Conclusion：Apigenin could inhibit the airwayinflammation and hyperresponsivenes by upregulating the expression ofCD4~+CD25~+Treg cells. Part2: The Effects of Apigenin on the Airway Inflammationand Remodelling in a Murine Model of Chronic AsthmaObjective： To observe the influence of apigenin on the airway inflammation andremodelling in a murine model of chronic asthma. Methods: BALB/c mice wererandomly divided into four groups, namely, control group, OVA group, apigeningroup and budesonide group. Mice were sensitized and challenged by OVA. Mice intreatment groups were i.p. with apigenin (20mg/kg/day) or exposed to an aerosol ofbudesonide before each challenge. Airway responsiveness to acetylcholine chloridewas measured24hours after the last OVA challenge. The sections were stained witheither hematoxylin&eosin to assess the inflammatory cell infiltrates, periodic acidschiff (PAS) to quantify airway global cells and Masson’s trichrome to determinecollagen deposition in the lungs. Total collagen content of the lung was determinedby hydroxyproline assay. Interleukin (IL)-4、 IL-13、 TGF-β1and VEGF levels inbronchoalveolar lavage fluid (BALF), and total immunoglobulin E (IgE)、OVA-sIgElevels in serum were measured by ELISA. The expression of α-SMA and PCNA in lungs was evaluated by immunohistochemistry. The levels of CD4~+CD25~+Treg cellsand DC in th spleen were measured with flow cytometry analysis. The level oflymphocyte proliferation with CFSE labeled was detected by flow cytometry analysis.The protein expressions of OX40、IκBα、NF-κB and MAPK were determined bywestern blot. Results: There were no significant differences in baseline airwayresistance among the four groups (P>0.05). The airway resistance generated byadministration of ACh at doses from30to270mg/kg increased significantly in theOVA group compared with the control group (P<0.05). Treatment with apigenin orbudesonide led to a sharp decrease in airway resistance compared with the OVAgroup (P<0.05). There were no significant differences in airway resistance betweenthe apigenin group and budesonide group (P>0.05). The number of eosinophils andtotal inflammatory cells in BALF in the OVA group increased significantly comparedwith the control group (P<0.05), and was significantly decreased by treatment withapigenin or budesonide (P<0.05). There were no significant differences between theapigenin group and budesonide group (P>0.05). The levels of the Th2cytokines IL-4and IL-13in BALF and total serum IgE、OVA-sIgE were significantly increased inOVA-sensitized/challenged mice compared with the control group (P<0.05).Administration of apigenin or budesonide reduced the levels of these Th2cytokine inBALF and total serum IgE、 OVA-sIgE compared with the OVA group (P<0.05).There were no significant differences in the levels of BALF IL-4、 IL-13and totalserum IgE,OVA-sIgE between apigenin group and budesonide group (P>0.05). Thelevels of TGF-β1and VEGF in BALF in the OVA group was significantly enhancedcompared with the negative control group (P<0.05), Administration of apigenin orbudesonide decreased the levels of TGF-β1and vascular endothelial growth factor(VEGF) compared with the OVA group (P<0.05). A significant increase in thenumber of PAS-positive epithelial cells was found in the OVA group compared with the negative control group (P<0.05). Treatment with apigenin or budesonide reducedthe number of PAS-positive cells (P<0.05). The mean area of Masson’s trichromestaining (collagen deposition) in the OVA group was significantly enhancedcompared with the negative control group (P<0.05). Administration of apigenin orbudesonide caused a marked reduction in collagen deposition compared with theOVA group (P<0.05). Total lung hydroxyproline content in the OVA group wassignificantly greater than that in the control group (P<0.05). In contrast, treatmentwith apigenin or budesonide significantly reduced total lung hydroxyproline contentcompared with the OVA group (P<0.05). The area of the α smooth muscle actin(α-SMA) and proliferating cell nuclear antigen (PCNA)-stained smooth muscle layerin OVA-sensitized and challenged mice was significantly greater than that in thesaline-treated control mice (P<0.05). Administration of apigenin or budesonidedecreased the α-SMA and PCNA-immunostained area compared with the OVA group(P<0.05). Treatment with apigenin or budesonide increased the ratio ofCD4~+CD25~+Treg cells in the spleen and decreased the ratio of DC in the spleencompared with that in the OVA group (P<0.05). The level of lymphocyteproliferation was decreased in the apigenin group compared with the OVA group(P<0.05). Treatment with apigenin or budesonide decreased the protein levels ofOX40, NF-κ B and MAPK compared with that in the OVA group. Conclusion:Apigenin alleviates the airway inflammation, AHR and airway remodelling in amurine model of chronic asthma by up-regulating the expression of CD4~+CD25~+Tregcells and inhibition of OX40/OX40L pathway and MAPK pathway.