化浊解毒方促进骨髓间充质干细胞归巢及对溃疡性结肠炎治疗作用的研究
Study on the function of Huazhuo Jiedu Decoction in promoting the homing of bone marrow mesenchymal stem cells and contributing to the treatment of ulcerative colitis.
作者信息
Ma Yumei, Li Yongzhang, Zhang Shuo, Liu Zongxiu, Du Lipeng, Zhang Xiaoyan, Jia Xuemei, Yang Qian
机构信息
Hebei Province Hospital of Chinese Medicine Research Center, Hebei, China.
Hebei Key Laboratory of Integrated Chinese and Western Medicine for Gastroenterology Research, Hebei, China.
出版信息
Heliyon. 2023 Jul 28;9(8):e18802. doi: 10.1016/j.heliyon.2023.e18802. eCollection 2023 Aug.
OBJECTIVE
To study the function of Huazhuo Jiedu Decoction (HZJD) in promoting the homing of bone marrow mesenchymal stem cells (BMSCs) and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis.
METHODS
Bone mesenchymal stem cells derived from mice were isolated and cultured, osteogenic and adipogenic assays to study the differentiation ability of BMSCs, and flow cytometry was used to detect the surface marker of the third generation cells. 30 mice were selected and divided into blank group, model group, HZJD group, BMSCs group, and HZJD combined with BMSCs group. Mouse colon length, body weight, and DAI score were used to assess efficacy. The levels of IL-6, IL-1β, TNF-α, and IFN-γ in serum were measured by ELISA. BMSCs transfected with GFP were used to mark the homing of BMSCs in mice. The BMSCs tagging protein CD90+/CD29+ was detected by immunofluorescence. H&E staining detects damage to the colon and the inflammatory response. The expression levels of claudin-2, claudin-4, occludin, and ZO-1 in colon tissues were detected by Western blot.
RESULTS
After subculture, the cell grew with adherence. Flow cytometry showed that the cells were CD73+/CD90+/CD29+/CD45-/CD34-, which belonged to bone mesenchymal stem cells. ELISA showed that the treatment with HZJD and BMSCs suppressed the DSS-induced inflammatory response. BMSCs carrying GFP can be detected in intestinal tissues. Immunofluorescence showed that the HZJD combined with the BMSCs group had more BMSCs homing to the colonic tissue. The results of H&E and Western blot showed that DSS-induced intestinal mucosal damage in UC mice was repaired by HZJD and BMSCs, and the abnormal tight junction proteins claudin-2, claudin-4, occludin, and ZO-1 were normalized.
CONCLUSION
HZJD has a therapeutic effect on ulcerative colitis by promoting the migration of BMSCs to ulcers of the colon and contributing to the reconstruction of the intestinal mucosal barrier in ulcerative colitis.
目的
研究化浊解毒方（HZJD）促进骨髓间充质干细胞（BMSCs）归巢及有助于溃疡性结肠炎肠黏膜屏障重建的作用。
方法
分离培养小鼠来源的骨髓间充质干细胞，进行成骨和成脂实验以研究BMSCs的分化能力，采用流式细胞术检测第三代细胞的表面标志物。选取30只小鼠，分为空白组、模型组、HZJD组、BMSCs组和HZJD联合BMSCs组。采用小鼠结肠长度、体重和疾病活动指数（DAI）评分评估疗效。用酶联免疫吸附测定（ELISA）法检测血清中白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）和干扰素-γ（IFN-γ）的水平。用绿色荧光蛋白（GFP）转染的BMSCs标记小鼠体内BMSCs的归巢情况。通过免疫荧光检测BMSCs标记蛋白CD90+/CD29+。苏木精-伊红（H&E）染色检测结肠损伤及炎症反应。采用蛋白质免疫印迹法检测结肠组织中紧密连接蛋白claudin-2、claudin-4、闭合蛋白（occludin）和紧密连接蛋白1（ZO-1）的表达水平。
结果
传代培养后，细胞贴壁生长。流式细胞术显示细胞为CD73+/CD90+/CD29+/CD45-/CD34-，属于骨髓间充质干细胞。ELISA结果显示，HZJD和BMSCs治疗可抑制葡聚糖硫酸钠（DSS）诱导的炎症反应。在肠道组织中可检测到携带GFP的BMSCs。免疫荧光显示，HZJD联合BMSCs组有更多BMSCs归巢至结肠组织。H&E和蛋白质免疫印迹结果显示，HZJD和BMSCs修复了DSS诱导的溃疡性结肠炎（UC）小鼠的肠黏膜损伤，使异常的紧密连接蛋白claudin-2、claudin-4、occludin和ZO-1恢复正常。
结论
HZJD通过促进BMSCs向结肠溃疡部位迁移，有助于溃疡性结肠炎肠黏膜屏障重建，对溃疡性结肠炎具有治疗作用。
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