肠道菌群代谢产物调控巨噬细胞极化参与坏死性小肠结肠炎的分子机制

郎兴; 李静涛; 马新生; 魏建新; 高于光

肠道菌群代谢产物调控巨噬细胞极化参与坏死性小肠结肠炎的分子机制

Molecular mechanism of intestinal flora metabolites regulating the polarization of macrophages and participate in necrotizing enterocolitis

摘要

摘要:
目的： 探讨肠道菌群代谢产物吲哚丙酸（indole 3–propionic acid，IPA）调控巨噬细胞极化参与坏死性小肠结肠炎（NEC）的分子机制。
方法： 取C57小鼠，随机分为3组：Sham + vehicle组，NEC + vehicle组，NEC + IPA组。构建NEC小鼠模型，以20mg/kg IPA剂量对NEC + IPA组小鼠进行灌胃，Sham + vehicle组和NEC + vehicle组小鼠灌同等体积0.9%氯化钠溶液。在第14天观察各组小鼠死亡率，HE染色病理情况和病理评分，iNOS/F4/80免疫荧光双标染色观察M1型巨噬细胞数，CD206/F4/80免疫荧光双标染色观察M2型巨噬细胞数。Western blotting法观察Toll样受体4（TLR4）、信号转导及转录激活蛋白6（STAT6），CD16/32等蛋白表达情况。ELISA法检测血清白细胞介素（IL）–1β、肿瘤坏死因α、IL–6、IL–10水平。
结果： NEC + IPA组小鼠较NEC + vehicle组肠组织HE病理损伤明显减轻，死亡率和病理评分降低（P﹤0.01）。免疫荧光结果显示NEC + IPA组小鼠较NEC + vehicle组iNOS/F4/80即M1型巨噬细胞数降低（P﹤0.01），CD206/F4/80即M2型巨噬细胞数升高（P﹤0.01）。Western blotting结果显示NEC + IPA组小鼠较NEC + vehicle组TLR4和CD16/32蛋白表达水平降低（P﹤0.01），而STAT6蛋白表达水平升高（P﹤0.01）。ELISA结果显示NEC + IPA组小鼠与NEC + vehicle组小鼠的IL–1β、肿瘤坏死因α、IL–6和IL–10表达水平组间差异无统计学意义（P ＞ 0.05），但与Sham + vehicle组比较差异均有统计学意义（P ＜ 0.05 ~ P ＜ 0.01）。
结论： 肠道菌群代谢产物IPA通过TLR4/STAT6信号通路，调控巨噬细胞向M2抗炎方向极化减轻NEC肠道损伤。

Abstract:
Objective To investigate the molecular mechanism by which the intestinal microbiota metabolite indole 3-propionic acid (IPA) regulates macrophage polarization in necrotizing enterocolitis (NEC).
Methods C57 mice were randomly divided into three groups: Sham + vehicle group, NEC + vehicle group, and NEC + IPA group. A NEC mouse model was established by administering 20 mg/kg IPA via gastric intubation to the NEC + IPA group, while the Sham + vehicle and NEC + vehicle groups received an equal volume of 0.9% sodium chloride solution. On day 14, mortality rates, HE-stained pathological findings, and pathological scores were observed in all groups. iNOS/F4/80 immunofluorescence double staining was used to quantify M1-type macrophages, while CD206/F4/80 immunofluorescence double staining was employed to quantify M2-type macrophages. Western blotting was performed to detect the expression levels of Toll-like receptor 4 (TLR4), signal transducer and transcriptional activator 6 (STAT6), and CD16/32. ELISA was used to measure serum interleukin (IL)-1β, tumor necrosis factor-α, IL-6, and IL-10 levels.
Results Compared with the NEC + vehicle group, the NEC + IPA group exhibited significantly reduced HE pathological damage in intestinal tissues, lower mortality rates, and improved pathological scores (P ＜ 0.01). Immunofluorescence results showed that the number of iNOS/F4/80 (i.e., M1-type macrophages) was reduced in the NEC + IPA group compared to the NEC + vehicle group (P ＜ 0.01), while the number of CD206/F4/80 (i.e., M2-type macrophages) was increased (P ＜ 0.01). Western blotting results indicated that the protein expression levels of TLR4 and CD16/32 were decreased in the NEC + IPA group compared to the NEC + vehicle group (P ＜ 0.01), whereas the protein expression level of STAT6 was elevated (P ＜ 0.01). ELISA results demonstrated that there were no statistically significant differences in the expression levels of IL-1β, tumor necrosis factor-α, IL-6, and IL-10 between the NEC + IPA group and the NEC + vehicle group (P ＞ 0.05), but all differences were statistically significant compared to the Sham + vehicle group (P ＜ 0.05 to P ＜ 0.01).
Conclusions The intestinal microbiota metabolite IPA regulates macrophage polarization toward the M2 anti-inflammatory direction through the TLR4/STAT6 signaling pathway, thereby alleviating NEC-induced intestinal injury.

HTML全文

参考文献(29)

施引文献

资源附件(0)