硝唑尼特调控TLR4信号通路的机制及关键作用靶点研究

Nitazoxanide (NTZ) has been approved for the treatment of various human and animal parasitic infections. In recent years, the anti-bacteria and the anti-viral effects of NTZ were also noted. Given the wide spectrum of pathogens targeted by NTZ, it is believed that NTZ may also possess immunomodulating properties. We hypothesize that TLR4 not only mediates innate immune response, but also involves in the process of anti-inflammatory and anti-infection induced by NTZ. Our previous research results have shown that NTZ has a remarkable anti-inflammatory characteristics which could significantly inhibit the LPS induced IL-6, IL-1β、TNF-α secretion and COX-2 expression. However, the mechanisms that underlie the anti-infection of NTZ have not been well understood, and elucidating the anti-inflammatory and anti-infection relationship is much more difficult. In this project, to explore the mechanisms of NTZ regulation on TLR4 signaling pathway, we will investigate the anti-inflammatory effect of NTZ in vivo and in vitro model, and study the influences of NF-κB and MAPKs on the downstream of TLR4 receptor induced by LPS and virus particles. The target proteins that involved in NTZ-regulated TLR4 signaling pathway will be identified by affinity carriers. Furthermore, the interaction between target proteins and NTZ and the signal network its involved in will be investigated via technologies such as site directed mutagenesis, co-immunoprecipitation, and so on. This project will clarify the mechanism of NTZ regulating TLR4 signaling pathway, and establish foundation to discover new drug targets and screen novel anti-infection drugs.

硝唑尼特（NTZ）已被批准用于治疗各类人和动物寄生虫病，近年来研究又发现其还能有效抗多种病原菌和病毒感染，展现出了诱人的开发前景。TLR4介导天然免疫的同时也极有可能参与NTZ抗炎抗感染过程。本团队前期研究发现NTZ能显著性地抑制脂多糖诱导的IL-6、IL-1β、TNF-α分泌以及COX-2表达，具有良好的抗炎特征。然而，至今人们对NTZ抗炎与抗病原体感染的关系仍知之甚少。因此，本项目拟通过研究NTZ在体内外对脂多糖、病毒颗粒介导的炎症反应的调控和TLR4受体下游NF-κB、MAPKs等信号通路的影响，探究NTZ调控TLR4信号通路的机制，并通过亲和载体分离获得与NTZ调控TLR4信号通路相关的靶标蛋白，利用定点突变、免疫共沉淀等技术进一步研究靶标蛋白与NTZ的互作关系以及其参与调控的信号网络。以此阐明NTZ调控TLR4与抗炎抗感染的机制，从而为发现新的药靶和筛选新型抗感染药物奠定基础。

硝唑尼特（nitazoxanide，NTZ）最初仅被批准用于治疗人和动物的各类寄生虫的感染，但后来人们发现硝唑尼特或其主要活性代谢物替唑尼特还具有抗新型冠状病毒、流感病毒等各种病毒的作用。这种广泛的抗病原体生物学活性越来越引起了人们的重视。在前期研究基础上，本课题从天然免疫的角度深入研究了硝唑尼特调控TLR4信号通路的机制和可能的药物作用靶点。研究结果表明：（1）硝唑尼特能抑制LPS诱导的TNF-α、IL-6、IL-1β、NO等细胞炎症因子的转录与释放，也抑制COX-2的激活。这些抗炎作用是替唑尼特通过对LPS诱导的 IкB、IKKα、JNK、p38、ERK、STAT3等炎症相关蛋白激活的抑制，以及对NF-κB p65入核的阻滞产生的。因此，硝唑尼特表现出对TLR4下游信号转导通路NF-κB、MAPKs、STAT3广泛的抑制效应。（2）硝唑尼特对日本乙脑病毒诱导的部分炎症因子也有抑制作用，但其对COX-2的激活却无效。（3）硝唑尼特能显著抑制LPS、病毒诱导的细胞活性氧（ROS）生成，降低细胞氧化应激反应。这可能与硝唑尼特的抗炎作用有关。（4）硝唑尼特或替唑尼特能诱导多种细胞自噬，诱导细胞自噬的机制在于其对PI3K/Akt/mTOR信号通路的抑制。这提示了硝唑尼特或替唑尼特对TLR4的调控作用可能有自噬机制的参与。（5）通过蛋白组学和代谢组学揭示了硝唑尼特的细胞调控网络，其中抗氧化系统和细胞自噬相关的调控在硝唑尼特作用中占重要位置。（6）筛选获得了22个可信的硝唑尼特靶标蛋白，并发现已知靶蛋白NQO1可能不参与TLR4的调控。总之，本研究基本阐释了硝唑尼特调控TLR4通路以抑制炎症的机制，发现了硝唑尼特新的生物作用并筛选获得其作用的蛋白靶点，为更好的利用硝唑尼特抗病原体的潜力提供了理论基础，也为挖掘新的抗感染药物提供了理论基础和新思路。