The Effects And Mechanism Of Icariin On OVA And Endotoxin-induced Inflammation

partⅠEffects of icariin on ovalbumin-induced asthma inflammation.Objective:Observation of icariin on ovalbumin-induced airway inflammation.Methods:(1) 72 male SD rats were randomly divided into 6 groups of 12 rats were the control group (PBS), model group (OVA), the positive control group (OVA+ dexamethasone group) and OVA+ icariin low, medium and high dose group (5,10,20 mg/kg-1); (2) Using ovalbumin (OVA) sensitization and the establishment rat asthma model; (3) Observation effects of icariin on the OVA-induced lung inflammation and pathological score; (4) Observation 6 groups of peripheral blood cells, total peripheral blood cell counts, eosinophil and macrophage counts; (5) Using enzyme-linked immunosorbent assay detect serum IL-4, IFN-γ, IL-13 and TNF-a levels; (6) Obtained BALF of rats, using enzyme-linked immunosorbent assay detection of BALF in IL-4 and IFN-y levels.Results:(1) Effects of icariin on the lung tissue of asthmatic rats and inflammatory grading score of the results:OVA+ high dose icariin bronchus, blood vessels and around submucosal inflammatory cell infiltration in lung tissue, visible part of the bronchial epithelial mucosal injury, compared with model group, bronchial epithelium and vascular inflammation around some relief. Model of lung tissue inflammation scores were significantly higher than the control group, OVA+ icariin high dose group compared with model group, inflammation score decreased, the difference was significant (P＜0.05); (2) Asthma model of rat peripheral blood cell counts showed that:the model group peripheral blood cells, eosinophils and macrophages compared with the control group were significantly increased, OVA+ icariin high dose group on eosinophils cells and macrophages significantly inhibited, the difference was significant (P<0.05); (3) Effects of icariin on peripheral blood cytokines of asthmatic rats showed:model group, serum levels of IL-4, IL-13 and TNF-a levels compared with the control group were significantly increased, serum IFN-γlevels decreased significantly. OVA+ icariin high dose group on asthma serum IL-4, IL-13 and inhibition of TNF-a levels significantly, the difference was significant (P＜0.05), icariin high dose can increase asthma model of serum levels of IFN-γexpression, the difference was significant (P＜0.05); (4) Effects of icariin on IL-4 and IFN-γ expression in BALF of asthmatic rats:model group, IL-4 were significantly higher than the normal control group in BALF; OVA+ icariin high dose group compared with the asthma model group, IL-4 expression was lower in BALF, there was a significant difference (P＜0.05), asthma model group IFN-y expression was significantly lower than the control group in BALF and icariin treatment group compared with the asthma model group, IFN-y expression was increased in BALF, the difference was significant (P＜0.05).Conclusion:(1) Icariin improve the asthma model in rats with lung tissues inflammation; (2) Inhibit the asthma model peripheral blood eosinophils and macrophages; (3) Icariin inhibit the asthma model of rat cytokines in serum and BALF.PartⅡEffects of icariin on asthmatic airway inflammation in molecular mechanisms.Objective:Observation of icariin on ovalbumin-induced imbalance of Thl/Th2 cytokine expression and its mechanism.Methods:(1) 60 male SD rats were randomly divided into control group (PBS), model group (OVA-induced), dexamethasone group, OVA+ icariin low, medium and high dose group (5,10,20 mg/kg-1); (2) Using ovalbumin (OVA) sensitization and the establishment rat asthma model; (3) Using ELISA, observation effects of icariin on IL-4 and IFN-y in the lung tissue; (4) Using immunohistochemical staining assay detect T-bet and GATA-3 in the lung tissue; (5) Using Realtime RT-PCR, observation effects of icariin on T-bet and GATA-3 mRNA expression in the lung tissue and spleen lymphocytes; (6)Using Western blot, observation T-bet, GATA-3 and NF-κB p65 protein expression of icariin in the lung tissue of rats.Results:(1) The ELISA lung tissue showed that, OVA+ icariin, the high dose group compared with the asthma model group, rat lung tissue IL-4 expression was significantly lower, the difference was significant (P<0.05) the lung tissue expression of IFN-y tended to increase, but no statistical significance (P>0.05); (2) lung tissue of rats in each group GATA-3 and T-bet immunohistochemical staining showed that, OVA+ prostitution high dose of sheep Lophanthus rugosus glycosides staining decreased GATA-3, T-bet staining did not significantly reduced; (3) the lung tissue and spleen lymphocytes T-bet and GATA-3 mRNA expression showed that icariin treatment group compared with the asthma model group, the lung tissue of T-bet and GATA-3 mRNA expression was lower, the difference was significant (P＜0.05); asthma model in rat spleen lymphocyte GATA-3 and T-betmRNA was significantly higher than the normal control group, icariin treatment group compared with the asthma model group, the lung tissue of GATA-3 mRNA expression was lower, the difference was significant (P<0.05), T-bet mRNA expression decreased, but the difference was not statistically significant (P>0.05); (4) of the lung tissue T-bet and GATA-3 protein expression showed that the model group T-bet and GATA-3 expression compared with the PBS control group, a significant increase in icariin inhibits GATA-3 protein increased, while no significant inhibition of T-bet; (5) The lung tissue NF-κB p65 immunohistochemistry results, OVA+ high dose icariin the bronchus, blood vessels and submucosal surrounding lung tissue with inflammatory cell infiltration, NF-κB p65 cells on small, compared with the model group, NF-κB p65 expression less; (6) The lung tissue NF-κB p65 protein show, icariin treatment group decreased expression of p65 total protein, cytoplasmic p65 protein expression.Conclusion:(1) Asthma model icariin can regulate lung tissue expression of Thl/Th2 cytokines imbalance; (2) Icariin can regulate lung tissue of asthmatic rats and the spleen lymphocytes of Thl/Th2 associated transcription factors (T-bet/GATA-3) expression imbalance; (3) Icariin inhibits asthmatic lung tissue activation of NF-κB p65 protein in the role.PartⅢIcariin regulation of allergic inflammation induced by endotoxin in vivo and in vitro experimental studyObjective:This study purpose to explore the icariin in inhibiting LPS-induced inflammatory response in vivo and in vitro function and signal transduction mechanism.Methods:(1) Observation effects of icariin on LPS-induced acute inflammation by HE staining; (2) Observation effects of icariin on lung tissue myeloperoxidase; (3) Using enzyme-linked immunosorbent assay detect serum cytokines (TNF-α, PGE2 and NO); (4) Using Real-time RT-PCR observation of icariin on the lung tissue of mice in each group TNF-α, iNOS and COX-2mRNA expression; (5) Using western blot observe the activation of the PI3K/AKT; (6) Using EMSA confocal observation effect of icariin on the activation of NF-κB p65 inhibition.Results:Icariin (20 mg/kg-1) after the intervention, can inhibit the LPS-induced mouse lung tissue TNF-a, IL-6 and increased expression of COX-2mRNA; Icariin can reduce peroxidase (MPO) activity; In RAW 264.7 cells, effects of icariin on the cytotoxicity of LPS has a protective effect; Western blot and confocal microscopy analysis showed that icariin pretreatment RAW 264.7 macrophage cells, can reduce the p65 nuclear translocation; Icariin to activate PI3K/AKT signaling pathway.Conclusions:(1) Icariin can inhibit LPS-induced inflammatory response in mice the role of lung tissue; (2) Icariin can inhibit LPS-induced inflammation in mouse lung tissue factor gene expression; (3) Icariin can inhibit p65 from the cytoplasm into the nucleus; (4) Icariin can activate PI3K/AKT signaling pathway.