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| 基于TLR4-Ig融合蛋白探究TLR4信号在溃疡性结肠炎中的关键作用 |
| 钱珂文1,王楚棋2,张淑怡3,李光耀3,邹宜覃1,郑欣亚1,艾泓如1,傅文燕4,雷长海3,胡适1* |
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(1. 海军军医大学(第二军医大学)基础医学院生物医学工程教研室, 上海 200433;
2. 新加坡国立大学药学系, 新加坡 119077;
3. 海军军医大学(第二军医大学)基础医学院生物物理学教研室, 上海 200433;
4. 上海交通大学医学院附属第九人民医院辅助生殖科, 上海 200011
*通信作者) |
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| 摘要: |
| 目的 探究Toll样受体4（TLR4）信号与溃疡性结肠炎（UC）发生、发展的关系。方法 收集UC患者结直肠活检组织样本（n＝63），根据Matts组织病理学分级标准分为1～5级，利用免疫组织化学染色检测TLR4表达情况。利用FreeStyle 293表达系统制备TLR4-Ig融合蛋白。体外培养健康者来源的外周血单个核细胞（PBMC），在培养基中添加100 μg/mL脂多糖诱导炎症模型，并加入100 μg/mL TLR4-Ig阻断TLR4，检测粒细胞-巨噬细胞集落刺激因子、干扰素γ、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-8、IL-10和TNF-α的分泌水平。以葡聚糖硫酸钠（DSS）诱导小鼠急性结肠炎模型，分别采用20 mg/kg TLR4-Ig、40 mg/kg TLR4-Ig、抗生素＋40 mg/kg TLR4-Ig进行干预，取小鼠结肠组织测量结肠长度并进行组织病理学评估。结果 UC患者结肠组织Matts分级越高TLR4高表达的样本比例越高（P＜0.001）。TLR4-Ig融合蛋白能够以高亲和力结合TLR4配体，阻断TLR4信号介导的PBMC活化和炎症因子分泌。TLR4-Ig融合蛋白对TLR4信号的阻断加重了DSS诱导的小鼠急性结肠炎，而抗生素处理则对TLR4信号阻断造成的小鼠急性结肠炎加重有缓解作用。结论 TLR4信号与肠道菌群的信号交流是UC发生早期的重要保护机制，TLR4信号的缺失不利于缓解急性炎症和修复肠黏膜。 |
| 关键词: Toll样受体4 溃疡性结肠炎 抗体融合蛋白 免疫组织化学 葡聚糖硫酸钠 |
| DOI：10.16781/j.CN31-2187/R.20240141 |
| 投稿时间：2024-02-28修订日期：2024-03-28 |
| 基金项目:国家自然科学基金（82322055，82272792，81903140，92169115）. |
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| To study the key role of TLR4 signal in ulcerative colitis using TLR4-Ig fusion protein |
| QIAN Kewen1,WANG Chuqi2,ZHANG Shuyi3,LI Guangyao3,ZOU Yitan1,ZHENG Xinya1,AI Hongru1,FU Wenyan4,LEI Changhai3,HU Shi1* |
(1. Department of Biomedical Engineering, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
2. Department of Pharmacy, National University of Singapore, Singapore 119077, Singapore;
3. Department of Biophysics, College of Basic Medical Sciences, Naval Medical University (Second Military Medical University), Shanghai 200433, China;
4. Department of Assisted Reproduction, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
* Corresponding author) |
| Abstract: |
| Objective To investigate the correlation between Toll-like receptor 4 (TLR4) signal and the development and progression of ulcerative colitis (UC). Methods The colon biopsy samples (n＝63) were collected from UC patients and were divided into Matts’ histological grade 1-5. The TLR4 expression level was detected by immunohistochemistry. TLR4-immunoglobulin (Ig) fusion protein was prepared using FreeStyle 293 expression system. Peripheral blood mononuclear cells (PBMCs) from healthy individuals were cultured in vitro, 100 μg/mL lipopolysaccharide was added to induce inflammation, and 100 μg/mL TLR4-Ig was added to block TLR4. Then the secretion levels of 10 different inflammatory cytokines, including granulocyte-macrophage colony-stimulating factor, interferon γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, and tumor necrosis factor α, were detected. The acute colitis mouse model was induced by dextran sulfate sodium (DSS), and was intervened with 20 mg/kg TLR4-Ig, 40 mg/kg TLR4-Ig or antibiotics＋40 mg/kg TLR4-Ig, respectively. Then the colon length of mice was measured and histopathological evaluation was performed. Results The expression level of TLR4 in colon tissues of UC patients was positively correlated with Matts’ histological grades (P＜0.001). TLR4-Ig fusion protein could bind TLR4 ligands with high affinity, blocking the activation of PBMCs and the secretion of inflammatory cytokines mediated by TLR4 signal. The blocking of TLR4 signal by TLR4-Ig fusion protein aggravated DSS-induced acute colitis in mice, and treatment with antibiotics alleviated the aggravation of the colitis caused by TLR4 signal blocking. Conclusion The communication between TLR4 signal and intestinal flora is an important protective mechanism at the early stage of UC. The absence of TLR4 signal is not conducive to relieving acute inflammation and repairing intestinal mucosa. |
| Key words: Toll-like receptor 4 ulcerative colitis antibody fusion protein immunohistochemistry dextran sulfate sodium |
