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

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.