School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, China.
First Clinical medical college, Guangzhou University of Chinese Medicine, China.
J Ethnopharmacol. 2022 Dec 5;299:115652. doi: 10.1016/j.jep.2022.115652. Epub 2022 Aug 28.
Dahuang Mudan decoction (DMD) is a classic prescription for treating intestinal carbuncle from Zhang Zhongjing's "Essentials of the Golden Chamber" in the Han Dynasty. Recent studies also prove that DMD has a therapeutic effect on ulcerative colitis (UC), but its mechanism is still unclear.
In this study, we aim to assess the therapeutic effect of DMD on DSS-induced chronic colitis in mice and deeply expound its underlying regulative mechanism.
The efficacy of DMD on mice with 2% DSS-induced chronic colitis was examined by changes in mouse body weight, DAI score, colon length changes, peripheral blood white blood cells (WBC) and red blood cells (RBC) counts, and hemoglobin (HGB) content, using mesalazine as a positive control. A small animal imaging system observed the FITC-Dextran fluorescence distribution in mice, and the contents of IL-22 and IL-17A in colon tissue homogenate supernatant and LPS in peripheral blood were detected by ELISA. Fluorescence in situ molecular hybridization and bacterial culture were used to investigate bacterial infiltration in intestinal mucosa and bacterial translocation in mesenteric lymph nodes and spleen. Mice immune function was further evaluated by analyzing the changes in spleen index, thymus index, and the ratio of peripheral blood granulocytes, monocytes, and lymphocytes. Meanwhile, the proportion of NCR group 3 innate lymphoid cells (ILC3), NCRILC3, and IL-22ILC3 in colonic lamina propria lymphocytes of mice was detected by flow cytometry. The contents of effectors IL-22, IL-17A, and GM-CSF were detected by RT-PCR. We use cell scratching to determine the effect of DMD conditioned medium on the migration of Caco-2 cells by establishing an in vitro model of MNK-3 conditioned medium (CM) intervening Caco-2 cells. RT-PCR and WB detect the expression of tight junction ZO-1, Occludin, and Claudin-1.
DMD restored the body weight, colon length, peripheral blood RBC numbers, and HGB content of chronic colitis mice and reduced peripheral blood WBC and colon inflammatory cell infiltration. Moreover, DMD decreased LPS content in serum, bacterial infiltration of colonic mucosa, and bacterial translocation in spleen and mesenteric lymph nodes. Simultaneously, DMD intensified the expression of ZO-1, Occludin, and Claudin-1, the ratio of NCRILC3 and IL-22ILC3, and decreased the proportion of NCRILC3. In vitro studies also confirmed that the conditioned medium of DMD promoted the migration of Caco-2 cells and the expression of tight junction proteins.
Our results confirm that DMD improves inflammation and restores intestinal epithelial function in mice with chronic colitis, and the mechanism may be related to regulating ILC3 function.
大黄牡丹汤（DMD）是汉代张仲景《金匮要略》中治疗肠痈的经典方剂。最近的研究也证明 DMD 对溃疡性结肠炎（UC）有治疗作用，但作用机制尚不清楚。
本研究旨在评估 DMD 对 DSS 诱导的慢性结肠炎小鼠的治疗作用，并深入阐述其潜在的调节机制。
采用 2% DSS 诱导的慢性结肠炎小鼠模型，观察 DMD 对小鼠体质量、DAI 评分、结肠长度变化、外周血白细胞（WBC）和红细胞（RBC）计数及血红蛋白（HGB）含量的影响，并以美沙拉嗪为阳性对照。小动物成像系统观察 FITC-Dextran 荧光在小鼠体内的分布情况，ELISA 检测结肠组织匀浆上清液中 IL-22 和 IL-17A 的含量及外周血中 LPS 的含量。荧光原位分子杂交和细菌培养用于检测肠道黏膜中细菌浸润和肠系膜淋巴结及脾脏中细菌易位。通过分析脾指数、胸腺指数以及外周血粒细胞、单核细胞和淋巴细胞的比值，进一步评估小鼠的免疫功能。同时，采用流式细胞术检测小鼠结肠固有层淋巴细胞中 NCR 组 3 固有淋巴细胞（ILC3）、NCRILC3 和 IL-22ILC3 的比例。采用 RT-PCR 检测效应因子 IL-22、IL-17A 和 GM-CSF 的含量。采用细胞划痕实验建立 MNK-3 条件培养基（CM）干预 Caco-2 细胞的体外模型，观察 DMD 条件培养基对 Caco-2 细胞迁移的影响。采用 RT-PCR 和 WB 检测紧密连接蛋白 ZO-1、Occludin 和 Claudin-1 的表达。
DMD 恢复了慢性结肠炎小鼠的体质量、结肠长度、外周血 RBC 计数和 HGB 含量，减少了外周血 WBC 和结肠炎症细胞浸润。此外，DMD 降低了血清中 LPS 含量、结肠黏膜中细菌浸润以及脾脏和肠系膜淋巴结中的细菌易位。同时，DMD 增强了 ZO-1、Occludin 和 Claudin-1 的表达、NCRILC3 和 IL-22ILC3 的比例，降低了 NCRILC3 的比例。体外研究也证实，DMD 促进了 Caco-2 细胞的迁移和紧密连接蛋白的表达。
本研究证实 DMD 可改善慢性结肠炎小鼠的炎症反应，恢复肠道上皮功能，其机制可能与调节 ILC3 功能有关。
