【作者】 陈志宝；

【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】 一些抗生素除了具有抗菌作用外,还被报道在体外具有调节免疫应答的作用。沃尼妙林是新一代广谱的截短侧耳素类半合成抗生素,对革兰氏阴性菌、革兰氏阳性菌、厌氧菌、支原体以及螺旋体均有效。临床上常被用来治疗猪的肠道疾病和急性多关节炎,以及猪和禽类的地方性肺炎。也被用在人类医学中耐药支原体感染的治疗。但目前该药仅作为抗菌剂在临床上应用,为了充分发挥沃尼妙林的药用价值,进一步扩大该药在临床上的应用范围,本研究利用LPS刺激小鼠RAW 264.7单核-巨噬细胞建立的体外炎症反应,研究沃尼妙林的体外抗炎活性,并在此基础上借助炎性信号传导通路平台进一步探索它的分子抗炎作用机制。同时构建LPS所致小鼠急性肺损伤(ALI)模型和内毒素性休克模型,检测沃尼妙林对两种动物模型的保护作用。巨噬细胞在特异性和非特异性炎症过程中都起到决定性作用。用脂多糖(LPS)活化巨噬细胞常用于评价众多药物的抗炎活性。LPS活化的巨噬细胞会分泌多种细胞因子,例如肿瘤坏死因子α(TNF-α),白细胞介素1p(IL-1p),白细胞介素6(IL-6)和白细胞介素10(IL-10)等；还可产生多种炎性介质,如一氧化氮(NO)和前列腺素E2(PGE2)等。大量产生这些细胞因子和炎性介质能够导致全身性炎症反应综合症(SIRS)、严重的组织损伤,甚至内毒素性休克。因此,本试验首先通过建立LPS诱导的小鼠单核-巨噬细胞RAW 264.7的体外炎症模型,研究了不同浓度沃尼妙林对RAW 264.7细胞的细胞因子TNF-α、IL-1β,IL-6和IL-10及炎性介质NO和PGE2分泌的影响。结果显示沃尼妙林剂量依赖的抑制了TNF-α、IL-6的分泌,提高了IL-10的水平,抑制了NO和PGE2的合成,但对IL-1β的作用不显著。这表明,沃尼妙林有可能通过调节细胞因子和炎性介质的分泌而发挥抗炎作用。LPS诱导细胞因子及炎性介质产生的机制已被深入研究,其中最常见的两条信号传导通路NF-κB和MAPKs起了关键作用。因为它们常被作为抗炎分子机制的重要靶位。本实验通过用LPS刺激小鼠的RAW 264.7细胞,测定沃尼妙林对NF-κB和MAPKs信号传导通路的影响,从而探究沃尼妙林抗炎分子机制。结果显示沃尼妙林显著抑制了LPS刺激的小鼠RAW 264.7巨噬细胞NF-κB的活力,同时显著抑制了LPS刺激的小鼠RAW 264.7巨噬细胞ERK、JNK和p38 MAPKs蛋白表达,作用呈剂量依赖方式。这说明,沃尼妙林通过调控NF-κB、ERK、JNK和p38 MAPKs通路而抑制细胞因子TNF-α、IL-6和炎性介质NO、PGE2合成。为了对沃尼妙林的临床疗效提供客观的评价指标,我们进一步验证了沃尼妙林在体内的抗炎作用。通过LPS诱导建立了急性炎症肺损伤模型,来研究预服用沃尼妙林(100 mg/kg)对急性肺损伤的作用。制备支气管肺泡灌洗液以检测蛋白浓度、细胞因子水平以及超氧化物歧化酶(SOD)的活性；收集肺组织以测定湿干重(W/D)比率、髓过氧化物酶(MPO)活性、炎性细胞因子mRNA的表达和观察组织学的变化。结果显示,预服用沃尼妙林显著的降低了肺的W/D比率、蛋白浓度和总白细胞数以及中性粒细胞数、巨噬细胞数、淋巴细胞数,并且组织学分析表明沃尼妙林明显的减弱了组织损伤。此外,沃尼妙林还显著的增大了支气管肺泡灌洗液中的SOD的活性,同时很好的减弱了肺中MPO的活性,这与对中性粒细胞浸润的影响一致。而且沃尼妙林还抑制了TNF-α、IL-6和IL-1β等炎性细胞因子的产生,这个结果与肺组织中mRNA的表达结果一致。这些结果表明,沃尼妙林对LPS诱导的急性炎症肺损伤小鼠模型具有保护作用。还通过构建LPS诱导的小鼠内毒素性休克模型,研究了不同浓度沃尼妙林对小鼠的预防和治疗作用及对小鼠血清中早期细胞因子TNF-α,IL-1β,IL-6和IL-10合成的影响。实验结果显示,沃尼妙林有效的改善了内毒素性休克小鼠的生存率；显著的降低了小鼠血清中TNF-α、IL-6的合成,提高了IL-10的水平,但对IL-1β作用不显著。表明沃尼妙林通过调节细胞因子水平提高了LPS所致内毒血症小鼠的生存率。本研究为沃尼妙林在临床上的合理应用提供了理论依据,提高其在兽医临床上的应用价值,同时使得该药物对由于单核-巨噬细胞等的过度激活导致的过度炎症反应,以及SIRS和MODS的防治具有重要意义。更多还原

【Abstract】 Some antibacterial agents were shown to regulate immune and inflammatory responses, in addition to their antibacterial activity. Valnemulin is a broad-spectrum, pleuromutilin antibiotic; its broad-spectrum activity targets Gram-negative and Gram-positive bacteria, anaerobic bacteria, Mycoplasma and spirochaetes. It is used in veterinary medicine to treat enteric diseases and acute polyarthritis in pigs, and enzootic pneumonia in pigs and poultry. Valnemulin has also been used in human medicine to treat patients with mycoplasma infections that were resistant to conventional treatments. However, valnemulin is widely used only as antibacterial clinic. In order to fully educe clinical value and expand applied scope of valnemulin, we built in vitro inflammatory model by LPS-stimulated murine RAW 264.7 macrophages, and further explored anti-inflammatory molecular mechanism of valnemulin by inflammatory signal transduction pathway. Meanwhile, we studied the effects of valnemulin on LPS-induced murine ALI and endotoxic shock.Macrophages play a crucial role in both the specific and nonspecific inflammatory processes. Therefore, lipopolyssacharide (LPS)-activated macrophages have typically been used to evaluate the anti-inflammatory effects of various materials. 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 interleukin-10 (IL-10), 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. first, we investigated the effect of different concentrations of valnemulin on cytokine TNF-α. IL-1β, IL-6 and IL-10. inflammatory mediator NO and PGE2 secretions in LPS-stimulated murine RAW 264.7 macrophages. The result showed that valnemulin inhibited TNF-α, IL-6 and IL-1βsecretion and increased IL-10 level in a dose-dependent manner, and valnemulin also inhibited NO and PGE2 synthesis, but had no significant effect on IL-1β.These indicated valnemulin may educe anti-inflammatory effect through regulating the secretion of cytokines and inflammatory mediators in inflammatory process.The mechanism by which LPS induces the production of cytokines and inflammatory mediators has been intensively investigated. Among these. MAPKs and NF-κB pathways may play an essential role. 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 valnemulin on NF-κB and MAPKs signal transduction pathways in LPS-stimulated murine RAW264.7 macrophages. The result showed that valnemulin inhibited NF-κB activity, ERK, JNK and p38 protein phosphorylation in a dose-dependent manner. It suggested that valnemulin inhibited cytokine TNF-α, IL-6, NO and PGE2 secretion by regulating both NF-κB and ERK, JNK and p38 MAPKs pathways.To provide objective evaluation index on clinical therapeutic of valnemulin, we further studied in vivo ant-inflammatory of valnemulin. In this study, we established a mouse model of LPS-induced inflammatory lung injury and investigated the effect of valnemulin (100 mg/kg) on acute lung injury eight hours after LPS challenge. We prepared bronchoalveolar lavage fluid (BALF) for measuring protein concentrations, cytokine levels and superoxidase dismutase (SOD) activity, and collected lungs for assaying wet-to-dry weight (W/D) ratios, myeloperoxidase (MPO) activity, cytokine mRNA expression and histological change. We found that the preadministration of valnemulin 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 valnemulin significantly attenuates tissue injury. Furthermore, valnemulin significantly increases LPS-induced SOD activity in BALF. and decreases lung MPO activity as well, consistent with its effects on neutrophil infiltration. In addition, valnemulin also inhibits the production of TNF-α, L-6, and IL-1β, which is consistent with mRNA expression in lung. The results showed that valnemulin had a protective effect on LPS-induced inflammatory lung injury in mice.We investigated the effect of different concentrations of valnemulin on preventive and therapeutic effect in LPS-induced endotoxic shock mice, and the effect on cytokine TNF-α, IL-1β, IL-6 and IL-10 production in murine serum. The result showed that valnemulin significantly improved murine survival rate and decreased TNF-αand IL-6, increased IL-10 level in serum, but had little effect on IL-1β. It suggested that valnemulin improved murine survival rate through regulating the level of cytokines.The study will provide foundation for reasonable application of valnemulin in clinic and improve clinical value, and it may be of importance as a therapeutics in treatment of excessive inflammatory reaction, SIRS and MODS in macrophages.更多还原