炎症部位定植水凝胶益生菌治疗炎性肠病的效果观察

doi:10.3969/j.issn.1674-8115.2024.07.009

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (7): 883-890.doi: 10.3969/j.issn.1674-8115.2024.07.009

• 论著 · 临床研究 • 上一篇

炎症部位定植水凝胶益生菌治疗炎性肠病的效果观察

徐唯¹^,²(), 李蒙², 王昊泽², 崔凯²(), 肖泽宇¹^,²()

^1.上海大学生命科学学院，上海 200444
^2.上海交通大学医学院药理学与化学生物学系，上海 200025

收稿日期:2024-02-06 接受日期:2024-03-25 出版日期:2024-07-28 发布日期:2024-07-28
通讯作者: 崔凯,肖泽宇 E-mail:2648798108@qq.com;CuiKai101102@163.com;zxiao@sjtu.edu.cn
作者简介:徐　唯（1998—），女，硕士生；电子信箱： 2648798108@qq.com。第一联系人：#为共同通信作者。
基金资助:
国家重点研发计划(2020YFA0210800);国家自然科学基金国际（地区）合作项目(52161160307)

Observation on the effect of hydrogel probiotics colonized in inflammatory sites in the treatment of inflammatory bowel disease

XU Wei¹^,²(), LI Meng², WANG Haoze², CUI Kai²(), XIAO Zeyu¹^,²()

^1.School of Life Sciences, Shanghai University, Shanghai 200444, China
^2.Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

Received:2024-02-06 Accepted:2024-03-25 Online:2024-07-28 Published:2024-07-28
Contact: CUI Kai,XIAO Zeyu E-mail:2648798108@qq.com;CuiKai101102@163.com;zxiao@sjtu.edu.cn
Supported by:
National Key R&D Program of China(2020YFA0210800);International (regional) Cooperation Project of National Natural Science Foundation of China(52161160307)

摘要/Abstract

摘要：

目的·构建负载在富含负电羟基的高聚物聚乙烯醇（polyvinyl alcohol，PVA）/海藻酸盐（alginate，ALG）水凝胶（PVA-ALG）上的益生菌（ Escherichia coli Nissle1917，EcN）体系（EcN@PVA-ALG），探究其在结肠炎症部位定植性及其对葡聚糖硫酸钠盐（dextran sulfate sodium salt，DSS）诱导的炎性肠病（inflammatory bowel disease，IBD）的治疗效果。方法·将EcN悬液加入PVA-ALG水凝胶中，过筛离心后得到EcN@PVA-ALG水凝胶益生菌复合物，使用流变仪验证PVA-ALG水凝胶的合成。利用电位仪检测EcN@PVA-ALG的表面电荷，荧光显微镜观察PVA-ALG上的EcN负载情况。通过酶标仪检测EcN@PVA-ALG在600 nm处的吸光度；同时，取EcN@PVA-ALG复合物悬液进行细菌平板计数，检测EcN@PVA-ALG内EcN的生长活力。采用CCK-8法评估EcN@PVA-ALG对HEK293细胞增殖的抑制作用。利用活体成像系统（IVIS）分析PVA-ALG对炎症结肠的富集作用，观察其炎症靶向性能；将EcN负载在PVA-ALG上，再用IVIS观察EcN@PVA-ALG对炎症结肠的富集，观察其在炎症部位的定植能力。建立DSS诱导的IBD小鼠模型，EcN@PVA-ALG组（ n=5）每天灌肠给予1×10 ⁸CFU的EcN@PVA-ALG，连续5 d；另设PVA-ALG组、EcN组、PBS组和健康对照组，每组5只。治疗期间，每天记录小鼠的体质量。治疗结束后处死小鼠，取其结肠组织，测量结肠长度；进行疾病活动指数（disease activity index，DAI）评分；检测炎症细胞因子的水平，包括肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素（interleukin，IL）-6、IL-10和转化生长因子-β（transforming growth factor-β，TGF-β）；通过苏木精-伊红染色（H-E染色）进行结肠组织的病理评估。结果·PVA-ALG和EcN@PVA-ALG均带负电。EcN成功负载于PVA-ALG，并且PVA-ALG不影响EcN的生长活力；同时，PVA-ALG对正常细胞也具有良好的安全性。与健康对照组相比，PVA-ALG对炎症结肠组织具有超过2倍的富集效果。体内外实验发现，负载EcN的EcN@PVA-ALG复合物对炎症组织的富集效果比未加任何修饰的EcN高8倍。EcN@PVA-ALG治疗后，小鼠体质量迅速恢复，DAI的上升被显著抑制，结肠长度与健康小鼠相近，促炎细胞因子TNF-α、IL-6水平降低而抗炎细胞因子IL-10和TGF-β水平升高，结肠组织隐窝结构恢复。结论·相比于未加修饰的EcN，EcN@PVA-ALG促进了EcN在结肠炎症部位的定植，并使其发挥更好的治疗DSS诱导的IBD的功效。

关键词: 益生菌, 水凝胶, 炎性肠病, 炎性定植

Abstract:

Objective ·To construct a probiotic ( Escherichia coli Nissle1917,EcN) system (EcN@PVA-ALG) loaded on polyvinyl alcohol (PVA) / alginate (ALG) hydrogel (PVA-ALG) rich in negative hydroxyl groups, and to explore its colonization in the inflammatory site of colon and its therapeutic effect on dextran sulfate sodium salt (DSS)-induced inflammatory bowel disease (IBD). Methods ·EcN suspension was added to the PVA-ALG hydrogel, and then EcN@PVA-ALG hydrogel probiotic complex was obtained after screening and centrifugation. The synthesis of PVA-ALG hydrogel was verified by rheometer. The surface charge of EcN@PVA-ALG was detected by potentiometer and the load of EcN on PVA-ALG was observed by fluorescence microscope. The absorbance of EcN@PVA-ALG at 600 nm was detected by enzyme labeling instrument. Meanwhile, the bacterial plate count of EcN@PVA-ALG complex suspension was taken to study the growth activity of EcN in EcN@PVA-ALG. The CCK-8 kit was used to assess the inhibitory ability of EcN@PVA-ALG on HEK cell proliferation. In vivo imaging system (IVIS) was used to firstly analyze the enrichment of PVA-ALG on inflammatory colon to study its inflammatory targeting property; then EcN was loaded on PVA-ALG, and IVIS was used to observe the enrichment of EcN@PVA-ALG on inflammatory colon to study its ability to colonize the inflammatory site. To establish the model of IBD mice induced by DSS, EcN@PVA-ALG group ( n=5) was given 1×10 ⁸CFU EcN@PVA-ALG every day for 5 d, and PVA-ALG group, EcN group, PBS group and healthy control group with 5 mice were set up. During the treatment, the body mass of the mice was recorded every day. After treatment, the colonic tissue was taken, and the length of colon was measured. The disease activity index (DAI) score was graded. The levels of inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-10 and transforming growth factor-β (TGF-β) were detected, and the pathological evaluation of colonic tissue was made by H-E staining. Results ·Both PVA-ALG and EcN@PVA-ALG were negatively charged. EcN was successfully loaded onto PVA-ALG and PVA-ALG did not affect the growth viability of EcN, which contributed to the subsequent colonization of inflammatory colons. PVA-ALG had a favorable safety profile on normal cells. Compared with healthy controls, PVA-ALG had more than 2-fold enrichment effect on inflammatory colon tissue. In vitro and in vivo experiments revealed that EcN@PVA-ALG complex loaded with EcN had 8 times higher enrichment effect on inflammatory tissue than EcN without any modification. After EcN@PVA-ALG treatment, the body weight of mice recovered rapidly. The increase of DAI was significantly inhibited. The length of colon was similar to that of healthy mice. The levels of TNF- α and IL-6 decreased, while the levels of IL-10 and TGF- β increased. The crypt structure of colon tissue recovered. Conclusion ·Compared to unmodified EcN, EcN@PVA-ALG promotes the colonization of EcN at inflammatory sites of colon and allows it to exert better efficacy on treating DSS-induced IBD.

Key words: probiotics, hydrogel, inflammatory bowel disease, inflammatory colonization

中图分类号:

R574.4

徐唯, 李蒙, 王昊泽, 崔凯, 肖泽宇. 炎症部位定植水凝胶益生菌治疗炎性肠病的效果观察[J]. 上海交通大学学报（医学版）, 2024, 44(7): 883-890.

XU Wei, LI Meng, WANG Haoze, CUI Kai, XIAO Zeyu. Observation on the effect of hydrogel probiotics colonized in inflammatory sites in the treatment of inflammatory bowel disease[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(7): 883-890.

图/表 6

图1 EcN@PVA-ALG的制备与表征Note： A. Rheological properties of PVA-ALG hydrogels. B. Fluorescence image of EcN. C. EcN@PVA-ALG image in brightfield. D. Image of EcN@PVA-ALG in fluorescence field.

Fig 1 Preparation and characterization of EcN@PVA-ALG

图2 EcN@PVA-ALG的细菌活力与毒性Note： A. D (600 nm) at different time. B. Bacterial enumeration at different time. C. Survival of HEK293 cells under EcN@PVA-ALG treatment.

Fig 2 Bacterial viability and cytotoxicity of EcN@PVA-ALG

图3 PVA-ALG水凝胶在健康与炎症状态下的靶向性Note： A. Ex vivo fluorescence of PVA-ALG hydrogels in different physiological states. B. Quantification of in vitro fluorescence. C. In vivo fluorescence of PVA-ALG hydrogels in different physiological states. D. Quantification of fluorescence in vivo.

Fig 3 Targeting of PVA-ALG hydrogels in health and disease states

图4 EcN及 EcN@PVA-ALG的炎症定植观察Note： A. Fluorescence of EcN and EcN@PVA-ALG on isolated distal colon tissues of IBD mice. B. Quantification of in vitro fluorescence. C. Bacterial enumeration of EcN and EcN@PVA-ALG. D. Fluorescence of EcN and EcN@PVA-ALG on isolated proximal colon tissues of IBD mice. E. Quantification of in vitro fluorescence. F. Bacterial enumeration of EcN and EcN@PVA-ALG. G. Fluorescence of EcN and EcN@PVA-ALG in IBD mice.

Fig 4 Inflammatory colonization of EcN and EcN@PVA-ALG

图5 EcN@PVA-ALG治疗 IBD的效果观察Note： A. Change of mouse body weight. B. Changes of DAI in mice. C. Change of colon length. D?G.Levels of TNF-α, IL-6, IL-10 and TGF-β.

Fig 5 Effect of EcN@PVA-ALG on IBD

图6 结肠组织病理染色评估（ H-E染色）

Fig 6 Pathological evaluation of colonic tissue (H-E staining)

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炎症部位定植水凝胶益生菌治疗炎性肠病的效果观察

Observation on the effect of hydrogel probiotics colonized in inflammatory sites in the treatment of inflammatory bowel disease

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