背景:肠屏障功能障碍是溃疡性结肠炎(UC)复发和难治性的重要因素。促进第3组固有淋巴样细胞(ILC3s)与肠道菌群之间的相互作用是粘膜修复的有价值的策略。芍药汤(PD)是临床上常用的治疗UC的复方,但其确切机制尚不清楚。
目的:我们旨在研究PD对葡聚糖硫酸钠(DSS)所致慢性结肠炎肠黏膜损伤的保护作用。以及阐明其潜在的机制。
方法:用2%DSS诱导C57BL/6小鼠慢性结肠炎,分为4组:对照组,模型组,PD低剂量(4g/kg),和高剂量(8μg/kg)组。根据体重变化评估PD治疗慢性结肠炎小鼠的有效性,结肠长度,结肠病理组织评分,炎症因子IL-6和IL-1β的mRNA水平。肠上皮紧密连接蛋白(ZO-1和Occludin)的表达,使用免疫荧光和RT-PCR观察IL-22和MUC2。此外,流式细胞术检测结肠中ILC3和天然细胞毒性受体(NCR)+ILC3的比例。此外,UHPLC-QE-MS用于鉴定PD的化学成分,网络药理学用于预测UC中PD干预的潜在途径。随后,MNK-3细胞(ILC3体外细胞系)和NCM460细胞用于验证网络药理学结果。最后,使用体外发酵和16SrDNA技术探索了PD对UC肠道菌群的影响。
结果:结果显示,PD可显著恢复慢性结肠炎小鼠的体重和结肠长度,同时还能减少结肠炎症细胞浸润和IL-6和IL-1β的表达。此外,PD显著促进MUC2、ZO-1、Occludin、和IL-22,以及增加ILC3和NCR+ILC3的比例。UHPLC-QE-MS分析鉴定出443种PD成分,和网络药理学表明PD可以靶向芳烃受体(AHR)信号通路,MNK-3细胞和体外发酵实验证实了这一点。此外,MNK-3条件培养基(CM)增加了NCM460细胞中ZO-1和Occludin的表达。此外,16SrDNA结果表明,PD促进了乳杆菌的丰度,从而通过激活ILC3中的AHR信号促进粘膜损伤修复。
结论:总之,我们的研究表明,PD通过调节肠道菌群与ILC3的相互作用来修复慢性结肠炎的肠黏膜损伤,其具体机制与AHR信号通路的激活有关。
BACKGROUND: Intestinal barrier dysfunction is a significant contributor to the recurrence and refractory of ulcerative colitis (UC). Promoting the interaction between group 3 innate lymphoid cells (ILC3s) and gut flora is a valuable strategy for mucosal repair. Paeoniae decoction (PD) is a compound commonly used in clinical treatment of UC, but its exact mechanism remains unclear.
OBJECTIVE: We aimed to investigate the protective effect of PD on intestinal mucosal injury induced by dextran sulfate sodium (DSS) in chronic colitis, as well as to elucidate its potential mechanism.
METHODS: C57BL/6 mice were induced with chronic colitis by 2 % DSS and divided into four groups: control group, model group, PD low dose (4 g/kg), and high dose (8 g/kg) group. The effectiveness of PD in treating chronic colitis mice was evaluated based on changes in body weight, colon length, colon pathological tissue scores, and the mRNA levels of inflammatory factors IL-6 and IL-1β. The expressions of intestinal epithelial tight junction proteins (ZO-1 and Occludin), IL-22, and MUC2 were observed using immunofluorescence and RT-PCR. Additionally, the proportion of ILC3 and natural cytotoxicity receptor (NCR)+ ILC3 in the colon were detected using flow cytometry. Furthermore, UHPLC-QE-MS was utilized to identify chemical components of PD and network pharmacology was employed to predict potential pathways for PD intervention in UC. Subsequently, MNK-3 cells (ILC3 in vitro cell line) and NCM460 cells were used to verify the network pharmacology results. Finally, the effects of PD on UC gut flora have been explored using in vitro fermentation and 16S rDNA techniques.
RESULTS: The results showed that PD significantly restored body weight and colon length in mice with chronic colitis, while also reducing colon inflammatory cell infiltration and the expression of IL-6 and IL-1β. Additionally, PD notably promoted the expression of MUC2, ZO-1, Occludin, and IL-22, as well as increasing the ratio of ILC3 and NCR+ILC3. UHPLC-QE-MS analysis identified 443 components of PD, and network pharmacology suggested that PD could target the aryl hydrocarbon receptor (AHR) signaling pathway, which was confirmed by MNK-3 cells and in vitro fermentation experiments. Furthermore, MNK-3-conditioned medium (CM) increased the expression of ZO-1 and Occludin in NCM460 cells. In addition, 16S rDNA results indicated that PD promoted the abundance of Lactobacillales, thus contributing to mucosal damage repair by activating the AHR signal in ILC3s.
CONCLUSIONS: In summary, our study demonstrates that PD repairs intestinal mucosal damage in chronic colitis by regulating the interaction of gut flora with ILC3, and the specific mechanism is related to the activation of AHR signaling pathway.