Therapeutic Effects and Mechanisms of Emodin on Dextran Sulfate Sodium-Induced Ulcerative Colitis in Mice
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摘要:
目的 探讨大黄素（emodin，EMO）对右旋糖酐硫酸钠（dextran sodium sulfate，DSS）诱导小鼠溃疡性结肠炎（ulcerative colitis，UC）的治疗作用及机制。 方法 DSS诱导UC小鼠模型，检测体重、结肠长度和组织病理学变化；酶联免疫吸附测定（enzyme-linked immunosorbent assay，ELISA）检测肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β，IL-1β）、白细胞介素-6（interleukin-6，IL-6）、白细胞介素-10（interleukin-10，IL-10）及髓过氧化物酶（myeloperoxidase，MPO）的水平；通过蛋白质免疫印迹（Western blot）分析Toll样受体4（toll-like receptor 4，TLR4）/髓样分化因子88（myeloid differentiation factor 88，MyD88）/核因子κB（nuclear factor-kappa B，NF-κB）信号通路相关蛋白的表达；利用流式细胞术评估辅助性T细胞17（t helper 17，Th17）与调节性T细胞（regulatory t cell，Treg）的比例；16S rDNA测序评估肠道菌群组成。 结果 与正常组相比，DSS组小鼠体重显著下降、结肠长度缩短并出现明显组织学损伤（P < 0.001）。EMO干预尤其是高剂量组可剂量依赖性改善体重和结肠损伤（P < 0.05）。ELISA显示，EMO降低TNF-α、IL-1β、IL-6并升高IL-10水平（P < 0.05）；Western blot提示EMO抑制TLR4/MyD88/NF-κB通路异常激活并恢复IκBα表达（P < 0.05）；流式检测发现EMO降低Th17比例、升高Treg比例（P < 0.05）；菌群分析显示EMO改善DSS诱导的菌群失调，增加有益菌相对丰度（P < 0.05）。 结论 EMO通过调控TLR4/MyD88/NF-κB通路、恢复Th17/Treg平衡及维持菌群稳态，有效改善DSS诱导的UC炎症反应和肠道损伤，为其作为UC潜在治疗药物提供理论依据。 -
关键词:
- 大黄素 /
- 右旋糖酐硫酸钠 /
- 溃疡性结肠炎 /
- TLR4/MyD88/NF-κB通路 /
- 肠道菌群 /
- Th17/Treg平衡
Abstract:Objective To investigate the therapeutic effects and mechanisms of emodin （EMO） on dextran sulfate （DSS）-induced ulcerative colitis （UC） in mice. Methods DSS induced UC mouse model, detection of body weight, colon length and histopathological changes. Enzyme-linked immunosorbent assay （ELISA） was used to measure tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β）, interleukin-6 （IL-6）, interleukin-10 （IL-10）, and myeloperoxidase （MPO） levels. Western blot analysis examined the expression of Toll-like receptor 4 （TLR4）/myeloid differentiation factor 88 （MyD88）/nuclear factor κB （NF-κB） signaling pathway-related proteins. Flow cytometry assessed the ratio of helper T cells 17 （Th17） to regulatory T cells （Treg）. Additionally, 16S rDNA sequencing was employed to evaluate gut microbiota composition. Results Compared with the normal group, DSS-treated mice exhibited significant weight loss, shortened colon length, and marked histological damage （P < 0.001）. EMO intervention, particularly at high doses, demonstrated dose-dependent improvements in body weight and colon injury （P < 0.05）. ELISA analysis showed EMO reduced TNF-α, IL-1β, and IL-6 levels while increasing IL-10 （P < 0.05）. Western blot results indicated EMO inhibited abnormal activation of the TLR4/MyD88/NF-κB pathway and restored IκB. Conclusion EMO effectively mitigates DSS-induced ulcerative colitis （UC） inflammation and intestinal damage by regulating the TLR4/MyD88/NF-κB pathway, restoring Th17/Treg balance, and maintaining microbial homeostasis, providing theoretical support for its potential as a UC therapeutic agent. -
Key words:
- Emodin /
- Dextran sulfate sodium /
- Ulcerative colitis /
- TLR4/MyD88/NF-κB pathway /
- Gut microbiota /
- Th17/Treg balance
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图 1 EMO治疗对DSS诱导小鼠体重和结肠长度的影响（x̄±s，n=3）
A：不同处理组小鼠体重变化趋势图；B：不同处理组小鼠结肠外观形态；C：不同处理组小鼠结肠长度定量分析。与Control组相比，**P < 0.01，***P < 0.001；与DSS组相比，#P < 0.05，###P < 0.001。
Figure 1. Effects of EMO treatment on body weight and colon length in DSS induced mice（x̄±s，n=3）
图 2 不同处理组小鼠结肠组织病理学变化（H&E染色，×100）
黑色箭头指示隐窝结构（肠腺）；红色箭头指示炎性细胞聚集区域；蓝色箭头标记固有层内特定结构；红/黑色三角形箭头标注上皮细胞层。
Figure 2. Histopathological changes in colonic tissues of mice from different groups （H&E staining，×100）
图 3 不同处理组小鼠结肠组织中炎症因子及MPO活性水平变化（$\bar x \pm s $，n = 8）
A：各组IL-1β含量比较；B：各组TNF-α含量比较；C：各组IL-6含量比较；D：各组MPO含量比较；E：各组IL-10含量比较。与Control组相比，*P < 0.05；与DSS组相比，#P < 0.05。
Figure 3. Changes in inflammatory cytokines and MPO activity in colonic tissues of mice from different groups （$\bar x \pm s $，n = 8）
图 4 EMO对结肠炎小鼠TLR4/MyD88/NF-κB通路相关蛋白表达的影响
A：各组蛋白电泳图；B：各个基因p-IκBα、IκBα、TLR-4、MyD88、p65蛋白统计学分析。与Control组相比，*P < 0.05；与DSS组相比，#P < 0.05。
Figure 4. Effect of EMO on the expression of TLR4/MyD88/NF-κB pathway-related proteins in colonic tissues of mice with colitis
图 5 不同处理组小鼠肠道菌群在门和属水平的相对丰度分布
A：小鼠肠道菌群在门水平的相对丰度分布；B：小鼠肠道菌群在属水平的相对丰度分布。
Figure 5. Relative abundance distribution of gut microbiota at the phylum and genus levels in different groups of mice
图 6 各组小鼠肠道菌群α多样性指数比较
A：各组observed species指数分析比较；B：各组chao指数分析比较；C：各组ace指数比较；D：各组Shannon指数分析比较；E：各组Simpson指数分析比较。与Control组相比，###P < 0.001；与DSS组相比，*P < 0.05，**P < 0.01。
Figure 6. Comparison of gut microbiota α-diversity indices among groups of mice
图 7 肠道菌群β多样性PCoA分析
Figure 7. Principal coordinates analysis （PCoA） of gut microbiota β-diversity
表 1 各组Th17和Tregs细胞比例（$\bar x \pm s $，%，n = 8）
Table 1. Proportions of Th17 and Treg cells in each group （$\bar x \pm s $，%，n = 8）
组别 Th17 Tregs Control 0.33 ± 0.03 4.17 ± 0.26 DSS 0.52 ± 0.03* 3.60 ± 0.29* SASP 0.81 ± 0.02* 2.19 ± 0.30* L-EMO 0.64 ± 0.02# 3.54 ± 0.27# M-EMO 0.53 ± 0.02# 3.74 ± 0.14# H-EMO 0.50 ± 0.04# 3.90 ± 0.24# F值 38.72 25.83 P值 <0.001 <0.001 注：与Control组相比，*P < 0.05；与DSS组相比，#P < 0.05。 
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表 2 各组小鼠肠道菌群在属水平的相对丰度变化（$\bar x \pm s $，%，n = 8）
Table 2. Relative abundance of gut microbiota at the genus level in each group of mice （$\bar x \pm s $，%，n = 8）
组别 Muribaculaceae Muribaculaceae Lactobacillus Bacteroides Control 0.29 ± 0.05 0.005 ± 0.001 0.22 ± 0.03 0.030 ± 0.005 DSS 0.19 ± 0.03* 0.150 ± 0.020* 0.06 ± 0.02* 0.180 ± 0.030* SASP 0.38 ± 0.06# 0.015 ± 0.005# 0.17 ± 0.03# 0.061 ± 0.010# L-EMO 0.55 ± 0.08# 0.006 ± 0.003# 0.12 ± 0.02# 0.063 ± 0.011# M-EMO 0.45 ± 0.07# 0.006 ± 0.001# 0.21 ± 0.03# 0.025 ± 0.002# H-EMO 0.46 ± 0.07# 0.001 ± 0.001# 0.17 ± 0.03# 0.071 ± 0.015# F值 42.18 38.92 25.64 31.07 P值 <0.001 <0.001 <0.001 <0.001 与Control组相比，*P < 0.05；与DSS组相比，#P < 0.05。
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