淫羊藿苷对卵蛋白和内毒素诱导炎症反应的影响及机制研究
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摘要
第一部分
淫羊藿苷对卵蛋白诱导大鼠哮喘性气道炎症的影响
目的：观察淫羊藿苷对卵蛋白诱导大鼠哮喘性气道炎症的影响。
方法：(1)72只雄性SD大鼠随机分为6组,每组12只,分别是正常对照组(PBS)、模型组(OVA)、阳性对照组(OVA+地塞米松组)和OVA+淫羊藿苷低、中、高剂量组(5、10、20mg/kg-1);(2)采用卵蛋白(OVA)致敏和反复激发大鼠建立哮喘模型；(3)采用HE染色观察淫羊藿苷对OVA诱导的大鼠肺组织炎症浸润的干预作用及病理学评分的影响；(4)观察6组外周血总细胞数、嗜酸性粒细胞和巨噬细胞计数情况；(5)采用酶联免疫吸附测定法检测血清中IL-4、IFN-γ、IL-13和TNF-α的含量；(6)取各组大鼠肺泡灌洗液,采用酶联免疫吸附测定法检测肺泡灌洗液中IL-4和IFN-γ的含量。
结果：(1)淫羊藿苷对大鼠哮喘模型肺组织病理学和炎症分级评分影响的结果显示：OVA+淫羊藿苷高剂量组支气管、血管和周围肺组织有炎性细胞浸润,支气管上皮可见部分损伤,与模型组相比较,支气管上皮和血管周围的炎症有所减轻。模型组大鼠肺组织炎症评分明显高于正常对照组, OVA+淫羊藿苷高剂量组与模型组相比较,炎症评分降低,差异有显著性(P<0.05)；(2)淫羊藿苷对大鼠哮喘模型外周血细胞计数的影响结果显示：模型组外周血总细胞、嗜酸性粒细胞和巨噬细胞与正常对照组相比较均显著增加,OVA+淫羊藿苷高剂量组对嗜酸性粒细胞和巨噬细胞抑制明显,差异有显著性(P<0.05)；(3)淫羊藿苷对大鼠哮喘模型外周血细胞因子影响的结果显示：模型组大鼠血清IL-4、IL-13和TNF-α水平与正常对照组相比较均显著增加,血清IFN-γ水平显著降低。OVA+淫羊藿苷高、中剂量组对哮喘大鼠血清IL-4、IL-13和TNF-α水平抑制明显,差异具有显著性(P<0.05),淫羊藿苷高剂量组能够增加大鼠哮喘模型血清IFN-γ的表达水平,差异有显著性(P<0.05)；(4)淫羊藿苷对大鼠哮喘模型肺泡灌洗液中IL-4和IFN-γ表达影响的结果显示：模型组大鼠肺泡灌洗液中IL-4表达明显高于正常对照组；OVA+淫羊藿苷高剂量组与哮喘模型组相比较,大鼠肺泡灌洗液中IL-4表达明显降低,差异有显著性(P<0.05),哮喘模型组大鼠肺泡灌洗液中IFN-γ表达明显低于正常对照组,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺泡灌洗液中IFN-γ表达有所升高,差异有显著性(P<0.05)。
结论：(1)淫羊藿苷具有改善哮喘大鼠肺组织炎症的作用；(2)淫羊藿苷具有抑制哮喘大鼠外周血嗜酸性粒细胞和巨噬细胞聚集的作用；(3)淫羊藿苷具有抑制哮喘大鼠血清和肺泡灌洗液中炎症细胞因子的作用。
第二部分
淫羊藿苷对大鼠哮喘性气道炎症影响的分子机制研究
目的：观察淫羊藿苷对卵蛋白诱导大鼠Th1/Th2细胞因子失衡表达的影响及机理探讨。
方法：(1)60只雄性SD大鼠随机分为正常对照组(PBS)、模型组(OVA诱导)、地塞米松组、OVA+淫羊藿苷低、中、高剂量组(5、10、20mg/kg-1);(2)采用卵蛋白(OVA)致敏和激发大鼠建立哮喘模型；(3)采用ELISA法观察淫羊藿苷对各组大鼠肺组织IL-4和IFN-γ的含量的影响；(4)采用免疫组化染色观察淫羊藿苷对各组大鼠肺组织T-bet和GATA-3的干预作用；(5)采用Realtime RT-PCR法观察淫羊藿苷对各组大鼠肺组织和脾脏淋巴细胞T-bet和GATA-3 mRNA表达的干预作用；(6)采用Western blot法观察淫羊藿苷对各组大鼠肺组织T-bet、GATA-3和NF-kB p65蛋白表达的干预作用。
结果：(1)各组大鼠肺组织ELISA结果显示,OVA+淫羊藿苷中、高剂量组与哮喘模型组相比较,大鼠肺组织IL-4表达明显降低,差异有显著性(P<0.05),大鼠肺组织IFN-γ表达有升高趋势,但是无统计学意义(P>0.05)；(2)各组大鼠肺组织GATA-3和T-bet免疫组化染色结果显示,OVA+淫羊藿苷高剂量组GATA-3染色明显减少,T-bet染色没有明显减少；(3)各组大鼠肺组织和脾脏淋巴细胞T-bet和GATA-3 mRNA表达结果显示,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺组织中T-bet和GATA-3 mRNA表达明显降低,差异有显著性(P<0.05)；哮喘模型组大鼠脾脏淋巴细胞中GATA-3和T-betmRNA表达明显高于正常对照组,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺组织中GATA-3 mRNA表达明显降低,差异有显著性(P<0.05), T-bet mRNA表达有所降低,但是差异无显著性(P>0.05)；(4)各组大鼠肺组织T-bet和GATA-3蛋白表达的结果显示,模型组T-bet和GATA-3表达与PBS对照组相比较显著增加,淫羊藿苷能够抑制GATA-3蛋白的增加,而对T-bet的抑制作用不明显；(5)各组大鼠肺组织NF-kB p65免疫组化染色结果显示,OVA+淫羊藿苷高剂量组的支气管、血管黏膜下和周围肺组织有炎性细胞浸润,NF-kB p65着色细胞较少,与模型组相比较,NF-kB p65阳性表达较少；(6)各组大鼠肺组织NF-kB p65蛋白检测结果显示,淫羊藿苷治疗组的p65总蛋白的表达减少,胞浆p65蛋白表达增加。
结论：(1)淫羊藿苷能够调节哮喘大鼠肺组织Th1/Th2相关细胞因子表达失衡；(2)淫羊藿苷能够调节哮喘大鼠肺组织和脾脏淋巴细胞中Th1/Th2相关转录因子(T-bet/GATA-3)表达失衡；(3)淫羊藿苷具有抑制哮喘大鼠肺组织NF-kB p65蛋白激活的作用。
第三部分
淫羊藿苷调控内毒素诱导炎症反应的体内及体外实验研究
目的：本研究旨在探讨淫羊藿苷在抑制LPS诱导的体内和体外炎症反应中的作用及信号转导机制。
方法：(1)肺组织HE染色观察淫羊藿苷对LPS诱导的急性炎症的影响；(2)检测肺组织髓过氧化物酶(MPO)观察淫羊藿苷对LPS诱导的炎症反应的影响；(3)ELISA法观察淫羊藿苷对各组小鼠血清细胞因子的抑制作用(TNF-α, PGE2和NO); (4) Real-time RT-PCR观察淫羊藿苷对各组小鼠肺组织TNF-α、iNOS和COX-2mRNA表达的影响；(5) Western blot观察淫羊藿苷对PI3K/AKT的激活作用；(6)共聚焦和EMSA观察淫羊藿苷对NF-kB p65激活的抑制作用。结果：淫羊藿苷(20 mg/kg-1)干预后,能抑制LPS诱导的小鼠肺组织TNF-α、IL-6和COX-2 mRNA表达的增加；淫羊藿苷能够降低过氧化物酶(MPO)活性；在RAW 264.7细胞中,淫羊藿苷对LPS的细胞毒作用具有保护作用；免疫印迹和共聚焦显微镜分析表明,淫羊藿苷预处理RAW 264.7巨噬细胞后,能够减少p65核转位；淫羊藿苷能够激活PI3K/AKT信号通路。
结论：(1)淫羊藿苷具有抑制LPS诱导的小鼠肺组织炎症反应的作用；(2)淫羊藿苷能够抑制LPS诱导的小鼠肺组织炎症因子基因表达；(3)淫羊藿苷能够抑制p65从胞浆进入细胞核；(4)淫羊藿苷能够激活PI3K/AKT信号通路。
partⅠ
Effects 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.
PartⅡ
Effects 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.
PartⅢ
Icariin regulation of allergic inflammation induced by endotoxin in vivo and in vitro experimental study
Objective: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.
淫羊藿苷对卵蛋白诱导大鼠哮喘性气道炎症的影响
目的：观察淫羊藿苷对卵蛋白诱导大鼠哮喘性气道炎症的影响。
方法：(1)72只雄性SD大鼠随机分为6组,每组12只,分别是正常对照组(PBS)、模型组(OVA)、阳性对照组(OVA+地塞米松组)和OVA+淫羊藿苷低、中、高剂量组(5、10、20mg/kg-1);(2)采用卵蛋白(OVA)致敏和反复激发大鼠建立哮喘模型；(3)采用HE染色观察淫羊藿苷对OVA诱导的大鼠肺组织炎症浸润的干预作用及病理学评分的影响；(4)观察6组外周血总细胞数、嗜酸性粒细胞和巨噬细胞计数情况；(5)采用酶联免疫吸附测定法检测血清中IL-4、IFN-γ、IL-13和TNF-α的含量；(6)取各组大鼠肺泡灌洗液,采用酶联免疫吸附测定法检测肺泡灌洗液中IL-4和IFN-γ的含量。
结果：(1)淫羊藿苷对大鼠哮喘模型肺组织病理学和炎症分级评分影响的结果显示：OVA+淫羊藿苷高剂量组支气管、血管和周围肺组织有炎性细胞浸润,支气管上皮可见部分损伤,与模型组相比较,支气管上皮和血管周围的炎症有所减轻。模型组大鼠肺组织炎症评分明显高于正常对照组, OVA+淫羊藿苷高剂量组与模型组相比较,炎症评分降低,差异有显著性(P<0.05)；(2)淫羊藿苷对大鼠哮喘模型外周血细胞计数的影响结果显示：模型组外周血总细胞、嗜酸性粒细胞和巨噬细胞与正常对照组相比较均显著增加,OVA+淫羊藿苷高剂量组对嗜酸性粒细胞和巨噬细胞抑制明显,差异有显著性(P<0.05)；(3)淫羊藿苷对大鼠哮喘模型外周血细胞因子影响的结果显示：模型组大鼠血清IL-4、IL-13和TNF-α水平与正常对照组相比较均显著增加,血清IFN-γ水平显著降低。OVA+淫羊藿苷高、中剂量组对哮喘大鼠血清IL-4、IL-13和TNF-α水平抑制明显,差异具有显著性(P<0.05),淫羊藿苷高剂量组能够增加大鼠哮喘模型血清IFN-γ的表达水平,差异有显著性(P<0.05)；(4)淫羊藿苷对大鼠哮喘模型肺泡灌洗液中IL-4和IFN-γ表达影响的结果显示：模型组大鼠肺泡灌洗液中IL-4表达明显高于正常对照组；OVA+淫羊藿苷高剂量组与哮喘模型组相比较,大鼠肺泡灌洗液中IL-4表达明显降低,差异有显著性(P<0.05),哮喘模型组大鼠肺泡灌洗液中IFN-γ表达明显低于正常对照组,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺泡灌洗液中IFN-γ表达有所升高,差异有显著性(P<0.05)。
结论：(1)淫羊藿苷具有改善哮喘大鼠肺组织炎症的作用；(2)淫羊藿苷具有抑制哮喘大鼠外周血嗜酸性粒细胞和巨噬细胞聚集的作用；(3)淫羊藿苷具有抑制哮喘大鼠血清和肺泡灌洗液中炎症细胞因子的作用。
第二部分
淫羊藿苷对大鼠哮喘性气道炎症影响的分子机制研究
目的：观察淫羊藿苷对卵蛋白诱导大鼠Th1/Th2细胞因子失衡表达的影响及机理探讨。
方法：(1)60只雄性SD大鼠随机分为正常对照组(PBS)、模型组(OVA诱导)、地塞米松组、OVA+淫羊藿苷低、中、高剂量组(5、10、20mg/kg-1);(2)采用卵蛋白(OVA)致敏和激发大鼠建立哮喘模型；(3)采用ELISA法观察淫羊藿苷对各组大鼠肺组织IL-4和IFN-γ的含量的影响；(4)采用免疫组化染色观察淫羊藿苷对各组大鼠肺组织T-bet和GATA-3的干预作用；(5)采用Realtime RT-PCR法观察淫羊藿苷对各组大鼠肺组织和脾脏淋巴细胞T-bet和GATA-3 mRNA表达的干预作用；(6)采用Western blot法观察淫羊藿苷对各组大鼠肺组织T-bet、GATA-3和NF-kB p65蛋白表达的干预作用。
结果：(1)各组大鼠肺组织ELISA结果显示,OVA+淫羊藿苷中、高剂量组与哮喘模型组相比较,大鼠肺组织IL-4表达明显降低,差异有显著性(P<0.05),大鼠肺组织IFN-γ表达有升高趋势,但是无统计学意义(P>0.05)；(2)各组大鼠肺组织GATA-3和T-bet免疫组化染色结果显示,OVA+淫羊藿苷高剂量组GATA-3染色明显减少,T-bet染色没有明显减少；(3)各组大鼠肺组织和脾脏淋巴细胞T-bet和GATA-3 mRNA表达结果显示,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺组织中T-bet和GATA-3 mRNA表达明显降低,差异有显著性(P<0.05)；哮喘模型组大鼠脾脏淋巴细胞中GATA-3和T-betmRNA表达明显高于正常对照组,淫羊藿苷治疗组与哮喘模型组相比较,大鼠肺组织中GATA-3 mRNA表达明显降低,差异有显著性(P<0.05), T-bet mRNA表达有所降低,但是差异无显著性(P>0.05)；(4)各组大鼠肺组织T-bet和GATA-3蛋白表达的结果显示,模型组T-bet和GATA-3表达与PBS对照组相比较显著增加,淫羊藿苷能够抑制GATA-3蛋白的增加,而对T-bet的抑制作用不明显；(5)各组大鼠肺组织NF-kB p65免疫组化染色结果显示,OVA+淫羊藿苷高剂量组的支气管、血管黏膜下和周围肺组织有炎性细胞浸润,NF-kB p65着色细胞较少,与模型组相比较,NF-kB p65阳性表达较少；(6)各组大鼠肺组织NF-kB p65蛋白检测结果显示,淫羊藿苷治疗组的p65总蛋白的表达减少,胞浆p65蛋白表达增加。
结论：(1)淫羊藿苷能够调节哮喘大鼠肺组织Th1/Th2相关细胞因子表达失衡；(2)淫羊藿苷能够调节哮喘大鼠肺组织和脾脏淋巴细胞中Th1/Th2相关转录因子(T-bet/GATA-3)表达失衡；(3)淫羊藿苷具有抑制哮喘大鼠肺组织NF-kB p65蛋白激活的作用。
第三部分
淫羊藿苷调控内毒素诱导炎症反应的体内及体外实验研究
目的：本研究旨在探讨淫羊藿苷在抑制LPS诱导的体内和体外炎症反应中的作用及信号转导机制。
方法：(1)肺组织HE染色观察淫羊藿苷对LPS诱导的急性炎症的影响；(2)检测肺组织髓过氧化物酶(MPO)观察淫羊藿苷对LPS诱导的炎症反应的影响；(3)ELISA法观察淫羊藿苷对各组小鼠血清细胞因子的抑制作用(TNF-α, PGE2和NO); (4) Real-time RT-PCR观察淫羊藿苷对各组小鼠肺组织TNF-α、iNOS和COX-2mRNA表达的影响；(5) Western blot观察淫羊藿苷对PI3K/AKT的激活作用；(6)共聚焦和EMSA观察淫羊藿苷对NF-kB p65激活的抑制作用。结果：淫羊藿苷(20 mg/kg-1)干预后,能抑制LPS诱导的小鼠肺组织TNF-α、IL-6和COX-2 mRNA表达的增加；淫羊藿苷能够降低过氧化物酶(MPO)活性；在RAW 264.7细胞中,淫羊藿苷对LPS的细胞毒作用具有保护作用；免疫印迹和共聚焦显微镜分析表明,淫羊藿苷预处理RAW 264.7巨噬细胞后,能够减少p65核转位；淫羊藿苷能够激活PI3K/AKT信号通路。
结论：(1)淫羊藿苷具有抑制LPS诱导的小鼠肺组织炎症反应的作用；(2)淫羊藿苷能够抑制LPS诱导的小鼠肺组织炎症因子基因表达；(3)淫羊藿苷能够抑制p65从胞浆进入细胞核；(4)淫羊藿苷能够激活PI3K/AKT信号通路。
partⅠ
Effects 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.
PartⅡ
Effects 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.
PartⅢ
Icariin regulation of allergic inflammation induced by endotoxin in vivo and in vitro experimental study
Objective: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.
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