【作者】 关爽；

【导师】 邓旭明；

【作者基本信息】 吉林大学 ， 基础兽医学， 2011， 博士

【摘要】 红景天苷（salidroside）是传统中药红景天中最重要的有效成份之一,临床上具有抗疲劳,抗氧化,保肝,改善血液循环等多种药理功能。为了提高红景天苷的药用价值,进一步扩大该药在临床上的应用范围,本文对红景天苷的抗炎作用进行了深入研究。首先,利用脂多糖（LPS）刺激小鼠RAW 264.7单核-巨噬细胞建立的体外炎症反应模型,研究红景天苷的体外抗炎活性,并借助细胞内第二信使和炎性信号转导通路平台进一步探索它的分子抗炎作用机制。在此基础上,构建LPS所致小鼠急性肺损伤模型和内毒素血症模型,检测红景天苷对两种动物模型的保护和治疗作用。LPS是革兰氏阴性菌细胞壁的主要成分,它可以激活巨噬细胞释放多种炎性细胞因子如肿瘤坏死因子α（TNF-α）,白细胞介素1β（IL-1β）和白细胞介素6（IL-6）等及炎性介质如一氧化氮（NO）和前列腺素E₂（PGE₂）等,从而形成瀑布效应,导致多种炎症疾病发生,甚至死亡。因此,LPS激活的巨噬细胞已被广泛用于评价各种化学物质的抗炎作用。本论文首先通过建立LPS诱导的小鼠单核-巨噬细胞RAW 264.7的体外炎症模型,研究了不同浓度红景天苷对RAW 264.7细胞的细胞因子TNF-α,IL-1β和IL-6及炎性介质NO和PGE₂分泌的影响。结果显示,红景天苷抑制了TNF-α、IL-6、IL-1β的分泌,这与其mRNA的表达结果一致,并呈现剂量依赖性。另外,红景天苷还能抑制NO、PGE₂的合成。以上数据表明,在体外实验中,红景天苷能够通过调节细胞因子和炎性介质的分泌而发挥抗炎作用。我们又进一步对LPS诱导细胞因子及炎性介质产生的机制进行了深入研究,细胞第二信使[Ca²⁺]_i、cAMP/cGMP通路和最常见的两条炎性信号转导通路NF-κB和MAPKs起了关键作用。本实验通过用LPS刺激小鼠的RAW 264.7细胞,测定红景天苷对第二信使[Ca²⁺]_i、cAMP/cGMP信号通路、NF-κB和MAPKs信号转导通路的影响。结果显示,红景天苷显著抑制了LPS刺激的小鼠RAW 264.7巨噬细胞[Ca²⁺]_i浓度、NF-κB的活力和ERK、p38 MAPKs蛋白表达,作用呈剂量依赖方式,同时升高了cAMP浓度,但对cGMP无影响。这说明,红景天苷通过调控[Ca²⁺]_i、cAMP、NF-κB、ERK和p38 MAPKs通路而抑制细胞因子TNF-α、IL-6、IL-1β分泌和炎性介质NO、PGE₂合成。在体外实验的基础上,我们进一步验证了红景天苷在体内的抗炎作用。通过LPS诱导建立了急性炎症肺损伤模型,来研究预注射红景天苷（120 mg/kg）对急性肺损伤的作用。通过收集肺组织以测定湿干重（W/D）比率、髓过氧化物酶（MPO）活性和观察病理组织学的变化,制备支气管肺泡灌洗液（BALF）以检测蛋白浓度、炎性细胞因子浓度。结果显示预服用红景天苷能显著地降低小鼠肺的干湿重的比率、支气管肺泡灌洗液中蛋白浓度、炎性细胞数量和MPO的活性,抑制了TNF-α、IL-6和IL-1β的分泌,病理切片表明红景天苷明显地减弱LPS导致的肺组织损伤。表明红景天苷对LPS诱导的急性炎症肺损伤具有保护作用。在LPS诱导的小鼠内毒素血症模型中,我们研究了红景天苷对小鼠内毒血症的预防和治疗作用及对小鼠血清中细胞因子TNF-α,IL-1β,IL-6分泌的影响。实验结果显示,红景天苷通过对小鼠血清炎性细胞因子的抑制作用,提高了内毒血症小鼠的生存率,对小鼠内毒素血症产生较好的预防和治疗作用。更多还原

【Abstract】 Salidroside is a phenylpropanoid glycoside extracted from Rhodiola rosea and regarded as the most important bioactive component. It has been reported to have various pharmacological properties including anti-aging, anticancer, hepatoprotective, antivirus and antioxidative effects. In this paper, we study the anti-inflammatory activity of salidroside deeply. We built in vitro inflammatory model by LPS-stimulated murine RAW 264.7 macrophages, and further explored anti- inflammatory molecular mechanism of salidrosie by cellular second messenger and inflammatory signal transduction pathway. Meanwhile, we studied the effects of salidroside on LPS-induced murine ALI and endotoxic shock.LPS-mediated activation of macrophages leads to the production of various cytokines such as tumour necrosis factor-α（TNF-α）, interleukin-1β（IL-1β）, interleukin-6 （IL-6） and inflammation mediators such as nitric oxide （NO） and prostaglandin E2 （PGE₂）. The production of these cytokines may result in the systemic inflammatory response syndrome （SIRS）, severe tissue damage, and septic shock. So lipopolyssacharide （LPS）-activated macrophages have typically been used to evaluate the anti-inflammatory effects of various materials. First, we investigated the effect of different concentrations of salidroside on cytokine TNF-α, IL-1β, IL-6. The result showed that salidroside inhibited TNF-α, IL-6 and IL-1βsecretion level in a dose-dependent manner, consistent with cytokine mRNA expression. Salidroside also inhibited NO and PGE₂ synthesis in LPS-stimulated murine RAW 264.7 macrophages by inhibiting iNOS and COX-2 protein expression, cytosolic free Ca²⁺ ([Ca²⁺]_i), and increasing cAMP level.NF-κB and MAPKs are known as important targets for anti-inflammatory molecular mechanism. In order to study anti-infalmmatory molecular mechanism, we further investigated the effect of salidroside on NF-κB and MAPKs signal transduction pathways in LPS-stimulated murine RAW264.7 macrophages. The result showed that salidroside inhibited NF-κB activity, ERK1/2 and p38 protein phosphorylation in a dose-dependent manner. It suggested that salidroside inhibited cytokine TNF-α, IL-1β, IL-6, NO and PGE₂ secretion by regulating both NF-κB and ERK and p38 MAPKs pathways.We further studied in vivo ant-inflammatory of salidroside. In this study, we established a mouse model of LPS-induced inflammatory lung injury and investigated the effect of salidroside （120 mg/kg） on acute lung injury eight hours after LPS challenge. We prepared bronchoalveolar lavage fluid （BALF） for measuring protein concentrations, cytokine levels and collected lungs for assaying wet-to-dry weight （W/D） ratios, myeloperoxidase （MPO） activity, histological change. We found that the preadministration of salidroside significantly decreases the W/D ratio of lungs, protein concentrations and the number of total cells, neutrophils, macrophages and leukomonocytes, and histologic analysis indicates that salidroside significantly attenuates tissue injury. Furthermore, salidroside significantly increases LPS-induced lung MPO activity, consistent with its effects on neutrophil infiltration. In addition, salidroside also inhibits the production of TNF-α, L-6, and IL-1β. The results showed that salidroside had a protective effect on LPS-induced inflammatory lung injury in mice.We investigated the effect of different concentrations of salidroside on preventive and therapeutic effect in LPS-induced endotoxic shock mice, and the effect on cytokine TNF-α, IL-1β, IL-6 production in murine serum. The result showed that salidroside significantly improved murine survival rate and decreased TNF-α, IL-1βand IL-6 level in serum. It suggested that salidroside improved murine survival rate through regulating the level of cytokines.更多还原