炎症性肠病（Inflammatory bowel disease, IBD）会造成胃肠道持续性炎症，全球患病率和严重程度显着增加。随着我国的工业化建设加快，我国的炎症性肠病患者数量也在快速增加。肠道环境中大量的活性氧和活性氮（Reactive oxygen and nitrogen species, RONS）的存在导致局部氧化还原稳态的破坏和大肠杆菌的异常扩张，这是IBD的主要原因。因此，清除过量的RONS，抑制大肠杆菌的增殖已成为治疗IBD的有效方法。而现阶段临床上用于治疗炎症性肠病的药物疗效并不佳，容易引起一定程度的不良反应，因此，开发新型的抗IBD药物具有重要意义。

普鲁士蓝不仅作为性能卓越的蓝色着色剂被广泛应用，还在临床医学中展现出显著解毒和ROS清除功效。2016年，顾宁教授课题组发现普鲁士蓝纳米颗粒（Prussian blue nanoparticles, PBNPs）具有类过氧化氢酶（Catalase, CAT）、类过氧化物酶（Peroxidase, POD）和类超氧化物歧化酶（Superoxide dismutase, SOD）等多种类酶活性，具有十分高效的活性氧（Reactive oxygen species, ROS）清除能力，因此普鲁士蓝也应该具有优异的IBD治疗效果。钨元素作为过渡金属中的一员，可以通过置换兼性厌氧菌中的关键呼吸酶-钼辅酶中的钼原子从而阻断其厌氧呼吸。同时，钨元素本身是一个多价态的金属元素，含有丰富的氧化还原电对，可以通过电子转移来清除ROS。得益于普鲁士蓝纳米酶具有丰富的孔隙和易于掺杂的特性，钨元素可以被掺杂进普鲁士蓝的晶界中，从而合成出新型纳米酶，用于治疗IBD。

在本研究中，首先通过一锅水热法，利用氯化钨水解产生盐酸创造酸性环境，从而使聚乙烯吡咯烷酮（Polyvinyl pyrrolidone, PVP）具有一定的还原性，在铁氰化钾（K₃[Fe(CN)₆]）体系中，Fe³⁺通过还原反应转化为Fe²⁺，随后Fe³⁺/Fe²⁺氧化还原对与CN⁻配体发生协同配位作用，自组装形成钨元素修饰的普鲁士蓝（W-PB）纳米结构。

通过多维度表征技术联用策略，系统评估了纳米颗粒的物理化学特性：采用高分辨透射电子显微镜（High resolution-transmission electron microscope, HR-TEM）、透射电字显微镜（Transmission electron microscope, TEM）、扫描电子显微镜（Scanned electron microscope, SEM）等观察进行形貌学解析，动态光散射系统（Dynamic light scattering, DLS）测定流体力学直径及Zeta电位，结合X射线衍射（X-ray diffraction, XRD）进行晶体学结构解析，还通过X射线光电子能谱（X-ray photoelectron spectroscopy, XPS）探究主要元素的化学态以及相对占比。综合分析表明，所得纳米晶呈现典型立方体形貌，粒径尺寸约为200 nm。DLS测试显示W-PB纳米颗粒的流体力学直径为（197±1.17）nm（多分散指数PDI=0.18），该数值与HRTEM统计获得的投影直径约为200 nm左右在误差范围内高度吻合，这种纳米尺度一致性验证了样品在液相环境中的单分散特性。Zeta电位显示纳米粒的平均电位在-21.05 mV，XRD和XPS数据表明合成的钨元素被掺杂在普鲁士蓝纳米粒的晶界中，并且在清除ROS前后，钨元素发生了价态的变化。溶液实验表明W-PB纳米粒是一种优良的过氧化氢酶，能催化分解羟基自由基、超氧阴离子和2,2-diphenyl-1-picrylhydrazyl（DPPH）自由基等种类的活性氧和活性氮。

随后，我们就纳米粒在细胞水平上的安全性以及清除ROS和抗菌效果进行验证。结果表明W-PB纳米粒具有优异的生物相容性，可以很好地保护RAW264.7细胞免受ROS引起的细胞凋亡，抑制STING通路的激活，降低M1型巨噬细胞的比例，增加M2型巨噬细胞的比例最终导致促炎细胞因子减少。除此之外，W-PB纳米酶还能阻断肠杆菌的硝酸盐呼吸从而抑制其增殖。

为了实现W-PB对结肠的精确递送，我们采用海藻酸钠和壳聚糖通过静电交联的策略包裹W-PB形成凝胶。进一步研究发现，该凝胶在弱碱性肠道中响应性释放W-PB，通过取代钼辅因子中的钼，触发钨离子的释放，抑制IBD模型小鼠结肠部位大肠杆菌的生长。此外，W-PB还能清除过量的RONS，保护肠细胞。

总的来说，W-PB凝胶能够通过重新编程肠道菌群和减轻氧化应激，在右旋糖酐硫酸钠（Dextran sulfate sodium, DSS）诱导的结肠炎小鼠模型中具有显著的治疗效果，能够为IBD的治疗及相关口服药物的研发提供新思路。

Inflammatory bowel disease (IBD) can cause persistent inflammation in the gastrointestinal tract. It has become a disorder with rapidly increasing global prevalence and severity. Owing to the advancement of the industrialization process, the risk of IBD is also increasing in China. The heightened accumulation of reactive oxygen and nitrogen species (RONS) within the intestinal milieu induces disturbance in the intestinal redox equilibrium and disproportionate proliferation of Escherichia coli, constituting the primary pathogenic mechanism underlying IBD development. Therefore, clearing excess RONS and inhibiting the proliferation of Escherichia coli have become effective methods for treating IBD. Current therapeutic agents for IBD exhibit suboptimal clinical efficacy in practice, with concomitant safety concerns arising from treatment-related adverse reactions. Therefore, the development of new anti-IBD drugs is of great significance.

Prussian blue is an excellent blue fuel and detoxification drug, which shows a nano-enzyme activity. In 2016, Professor Gu Ning's research group reported highly efficient reactive oxygen species (ROS) clearing property of the prussian blue nanoparticles (PBNPs) as a result of the enzymatic activities of the PBNPs, such as catalase, peroxidase and superoxide dismutase. It was therefore hypothesized that the prussian blue may also have anti-IBD effects. Tungsten, a member of transition metals, can block anaerobic respiration in facultative anaerobic bacteria by replacing the molybdenum atom in the key respiratory enzyme molybdenum coenzyme. Additionally, tungsten itself is a multivalent metal element containing abundant redox pairs, which can be eliminated through electron transfer. Owing to the rich pores and easy doping properties of Prussian blue nano-enzymes, tungsten element can be doped into the grain boundaries of the Prussian blue, thereby synthesizing new nano-enzymes for the treatment of IBD.

To investigate our hypothesis, firstly, a one pot hydrothermal method was used to create an acidic environment by hydrolyzing tungsten chloride to produce hydrochloric acid, making the polyvinylpyrrolidone (PVP) reducible. The iron ions in potassium ferrocyanide were reduced, and Fe³⁺/Fe²⁺coordinated with CN ^- to form tungsten doped Prussian blue (W-PB) nanoparticles. Subsequent structural characterization revealed uniformly dispersed cubic particles with an average edge length of 200 nm. Dynamic light scattering (DLS) measurements indicated a hydrodynamic diameter of 197 nm for W-PB nanoparticles, aligning closely with Transmission electron microscopy (TEM) observations. Surface charge analysis via zeta potential quantification demonstrated a mean value of -21.05 mV, confirming colloidal stability. X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) data showed that the synthesized tungsten element was doped in the grain boundaries of PBNPs, and the valence state of tungsten changed before and after ROS removal.

The solution experiment showed that W-PB were not only able to catalyze the decomposition of hydrogen peroxide (H₂O₂) to produce oxygen, but could also catalyze the decomposition of ROS such as hydroxyl radicals (·OH) and superoxide anions (O₂-). In addition, we also investigated its catalytic ability for RONS, and the results showed that W-PB can also decompose 2,2-diphenyl-1-picrylhydrazyl (DPPH).

Subsequent cellular-level evaluations confirmed their biosafety while demonstrating dual functionality: ROS neutralization and antimicrobial activity. Experimental data demonstrated the nanoparticles' superior biocompatibility, effectively shielding RAW264.7 macrophages from ROS-mediated apoptosis. Mechanistically, W-PB nanoparticles suppressed STING pathway activation, thereby changing the macrophage phenotypes from the pro-inflammatory M1 to an anti-inflammatory M2 subtype. This immunomodulatory effects culminated in a marked cytokine profile shift, characterized by reduced pro-inflammatory factors (e.g., tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6]) and elevated anti-inflammatory mediators (e.g., interleukin-10 [IL-10]). In addition, W-PB nano-enzyme particles can also block the nitrate respiration process of the Escherichia coli, thereby inhibiting E. coli reproduction.

Considering the complex gastrointestinal environment, colon-targeting agents are highly unstable in vivo and prone to be disintegrated, resulting in poor bioavailability and therapeutic outcome. In order to achieve precise colonic delivery of W-PB, a sodium alginate/chitosan hydrogel was prepared via electrostatic interaction and used for W-PB encapsulation. In vivo evaluation using dextran sulfate sodium (DSS)-induced IBD mice revealed that this hydrogel could released W-PB in response in the weakly alkaline environment of the intestine and colon. The released W-PB then displaced molybdenum in the molybdenum cofactor, triggering tungsten ion release, which suppressed E. coli growth in the colon of the IBD mice. Additionally, W-PB could also scavenged excess RONS and protect the intestinal cells, all of which resulted in alleviated IBD symptoms (weight loss, shortened colon lengths, hematochezia, etc.) of the IBD mice.

In summary, this study designed a multifunctional nano-enzyme that specifically inhibits the abnormal expansion of E. coli during colitis and clears excess RONS in the intestine, achieving long-term precise regulation of intestinal homeostasis. In order to achieve the precise delivery of W-PB to the colon, we used alginate and chitosan to wrap W-PB to form gel through the strategy of electrostatic cross-linking. The hydrogel could release W-PB in response to the weakly alkaline environment of the intestinal tract, and triggers the release of tungsten ions by replacing molybdenum in the molybdenum cofactor, thus inhibiting the growth of E. coli. In addition, W-PB can also clear excess RONS and protect intestinal cells. In general, W-PB gel can provide significant therapeutic effects for DSS-induced colitis and new ideas for oral drug treatment of IBD by reprogramming intestinal flora and reducing oxidative stress.