紫锥菊多糖抗炎作用及其对炎症信号转导通路的调控

Herbal Medicine Echinacea is widely used in the anti-inflammatory and immune of human, livestock and poultry, but there is no report about its anti-inflammatory and immunoregulatory mechanism in China. Bacterial diseases are common diseases of livestock and poultry, whose pathogenesis is closely related with endotoxemia of LPS.The pre-research of the applicant manifested the Echinacea polysaccharide EPS not only can stimulate the normal cell of RAW264.7 to produce TNF-α, also can apparently reduce expression dose of TNF-αowing to LPS-stimulated RAW264.7. It preliminary confirmed that the anti-inflammatory mechanism of EPS is related with NF-κB transduction pathway. But It is still an unknown which transduced elements regulate this spatial-temporal expression, and how the interaction of these factors affect the expression of the various regulator genes in the signal transduction pathway. This study intends to build model in vitro that EPS reduces RAW264.7 cell damage after LPS stimulated. The real-time PCR,western blot, immunohistochemistry are used to research the effect of Echinacea polysaccharide to main cell factors and inflammatory signal transduction pathway NF-κB/MAPK; the model of acute lung injury in mice is established in vivo by LPS stimulation to detect the protection of Echinacea polysaccharide to Animal model. The anti-inflammatory and immunoregulatory mechanism of Echinacea polysaccharide is clarified to provide theory evidence for its clinical application.

广泛用于人及畜禽"抗炎与免疫"草药紫锥菊，国内外少见其抗炎与免疫作用机理的研究。细菌病是畜禽常见病，其发病机制与LPS内毒素血症密不可分。申请人前期研究表明紫锥菊多糖EPS不仅可以刺激正常RAW264.7产生TNF-α，而且可以下调由LPS刺激RAW264.7产生的大量TNF-α等，初步证实EPS的抗炎作用与NF-κB信号通路有关。但它们的时空表达是被哪些转导因子所调控，这些因子之间的相互作用如何影响转导通路各调控基因的表达，还是一个未知领域。本研究体外试验拟以EPS对LPS介导RAW264.7细胞损伤为研究模型，采用荧光定量PCR、免疫蛋白印迹、免疫组化等方法，研究EPS对主要细胞因子及炎性信号传导通路NF-κB/MAPK的影响；体内试验拟建立LPS介导小鼠急性肺损伤模型，检测EPS对动物模型的炎性保护作用。证实EPS的抗炎作用并阐明其作用机制,为紫锥菊的临床应用提供理论依据。

紫锥菊多糖（EPS）作为紫锥菊提取物中主要成分之一，其对急性肺损伤的作用目前尚未见报道。本课题通过动物模型和细胞模型评估EPS在脂多糖（LPS）诱导小鼠ALI中的保护作用及其可能的作用机制。动物试验：将BALB/C小鼠随机分为6组，即Control组、EPS组、LPS组、LPS+EPS高剂量组、LPS+EPS中剂量组、LPS+EPS低剂量组，每组6只。Control和LPS组于试验前3d开始，每天每只灌胃100μL PBS；EPS组和LPS+EPS高、中、低剂量组，于试验前3d，每天每只灌胃100μL EPS溶液（浓度分别为100mg/kg、50mg/kg和25mg/kg）。干冰麻醉40sec，LPS和LPS+EPS高、中、低剂量组小鼠，鼻腔吸入LPS（6mg/kg），50μL/只；Control、EPS组，鼻腔吸入PBS，50μL/只。鼻腔吸入6h后收集样本。通过肺组织病理学观察，肺湿干重比，BALF中蛋白浓度变化，MPO活性，细胞因子、mRNA及蛋白表达水平。细胞试验：以小鼠肺泡巨噬细胞RAW264.7作为模型细胞，使用不同浓度的EPS和LPS先后作用于细胞，检测细胞活性，ALP活性，通过ELISA法、RT-PCR和Western Blot检测炎症因子的分泌、mRNA以及相关通路蛋白的表达。结果：（1）以6mg/kg剂量鼻腔吸入LPS导致肺组织病理损伤明显，肺湿干重比显著升高（P<0.05），BALF中TNF-α和IL-1β等促炎因子显著升高（P<0.01），肺组织中MPO活性和NO浓度在6h时显著升高（P<0.01）。（2）高剂量EPS对小鼠进行灌胃，能够显著减轻LPS对肺组织的损伤，显著抑制LPS诱导的BALF中IL-1β、IL-6、TNF-α和IL-8的分泌增加，能够显著抑制肺组织IL-1β、IL-6、TNF-α、TLR4和iNOS mRNA的表达水平。（3）RAW264.7细胞经EPS（100 μg/mL）处理后，能够显著抑制IL-1β、IL-6、IL-8、TNF-α、NO的分泌及IL-1β、IL-6、TNF-α、TLR4和iNOS的mRNA表达水平，从而抑制RAW264.7细胞NF-κB和MAPK信号通路的激活及转导；同时，EPS还能有效抑制p65、ERK、JNK、p38的磷酸化和IκB-α的降解，以抑制NF-κB的激活和MAPK信号转导。