为了揭示益生菌衍生代谢产物吲哚-3-乳酸（ILA）在口腔护理产品中的免疫调节作用及机制，同时明确其在口腔微生态中的调节效应与实际应用可行性。本研究以牙龈卟啉单胞菌脂多糖（Pg-LPS，1 μg/mL）刺激的人牙龈成纤维细胞（HGFs）和佛波酯（PMA）诱导THP-1单核细胞分化的巨噬细胞为体外模型，使用80，160和320 μmol/L的ILA处理上述细胞。同时使用不同终浓度的ILA分别培养HGFs和巨噬细胞48 h，用于考察ILA的细胞毒性。采用CCK-8法检测细胞活力，ELISA法检测炎症因子及基质金属蛋白酶（MMPs）分泌，Western blot法分析NF-κB信号通路相关蛋白磷酸化水平，中性红吞噬实验评估巨噬细胞吞噬功能，流式细胞术及RT-qPCR检测巨噬细胞极化标志物表达。建立口腔常见共生菌与致病菌共培养模型，采用80，160和320 μmol/L的ILA进行干预。同时测定ILA在牙膏基质中的稳定性。结果显示，ILA浓度≤320 μmol/L时对HGFs和巨噬细胞无显著毒性（P>0.05）。Pg-LPS刺激后，HGFs活力降低，IL-1β、IL-6、IL-8、TNF-α及MMP-1、MMP-3、MMP-9分泌增加，NF-κB p65磷酸化水平升高而IκBα磷酸化水平降低（P<0.05）；巨噬细胞吞噬功能增强且CD86+M1型比例及iNOS、CD86 mRNA表达上调（P<0.05）。ILA干预可剂量依赖性恢复HGFs活力，抑制上述炎症因子及MMPs分泌，调控NF-κB通路活性（P<0.05）；同时降低巨噬细胞过度吞噬功能及M1型极化水平（P<0.05），但对巨噬细胞活力及M2型标志物无显著影响（P>0.05）。ILA浓度≤320 μmol/L时对口腔有益共生菌（唾液链球菌、植物乳杆菌）无显著毒性（P>0.05），且可显著抑制牙龈卟啉单胞菌、伴放线放线杆菌等牙周致病菌的增殖（P<0.05）；此外，ILA在牙膏基质中4 ℃、25 ℃条件下储存3个月保留率均高于90%，具有良好的稳定性。综上，ILA兼具抗炎、免疫调节、微生态调控三重功能，且在口腔护理产品基质中稳定性良好，本研究为ILA作为功能性成分在口腔护理产品中的开发与应用提供了全面的体外实验与应用基础依据。
This study aims to elucidate the immunomodulatory effects and underlying mechanisms of the probiotic-derived metabolite indole-3-lactic acid （ILA） in oral care products, and to clarify its regulatory effect on the oral microecology as well as its feasibility for practical application. Human gingival fibroblasts （HGFs） stimulated by Porphyromonas gingivalis lipopolysaccharide （Pg-LPS, 1 μg/mL） and macrophages differentiated from phorbol myristate acetate （PMA） - induced THP-1 monocytes were employed as in vitro models. These cells were treated with ILA at concentrations of 80, 160, and 320 μmol/L. Meanwhile, HGFs and macrophages were incubated with different final concentrations of ILA for 48 h to evaluate the cytotoxicity of ILA. Cell viability was detected by CCK-8 assay; the secretion of pro-inflammatory cytokines and matrix metalloproteinases （MMPs） was measured by ELISA; the phosphorylation levels of proteins related to the NF-κB signaling pathway were analyzed by Western blotting; the phagocytic capacity of macrophages was assessed by a neutral red phagocytosis assay; and the expression of macrophage polarization markers was detected by flow cytometry and reverse transcription-quantitative polymerase chain reaction （RT-qPCR）. A co-culture model of common oral commensal and pathogenic bacteria was established, and interventions were performed with ILA at concentrations of 80, 160 and 320 μmol/L. Meanwhile, the stability of a ILA in toothpaste matrix was determined. The results show that ILA exhibits no significant toxicity to HGFs and macrophages at concentrations≤320 μmol/L （P>0.05）. After Pg-LPS stimulation, HGF viability decreases, the secretion of IL-1β, IL-6, IL-8, TNF-α, MMP-1, MMP-3, and MMP-9 increases, and the phosphorylation level of NF-κB p65 increases while that of IκBα decreases （P<0.05）; the phagocytic activity of macrophages is enhanced, and the proportion of CD86+ M1-type macrophages as well as the mRNA expression of iNOS and CD86 are upregulated （P<0.05）. ILA intervention restores HGF viability in a dose-dependent manner, inhibits the secretion of the aforementioned inflammatory factors and MMPs, and regulates the activity of the NF-κB pathway （P<0.05）; meanwhile, it reduces the excessive phagocytic capacity and M1 polarization level of macrophages （P<0.05）, but exerts no significant effect on macrophage viability or M2-type markers （P>0.05）. ILA at concentrations≤320 μmol/L shows no significant toxicity to beneficial oral commensal bacteria （Streptococcus salivarius and Lactobacillus plantarum） （P>0.05）, but significantly inhibits the proliferation of periodontal pathogenic bacteria such as Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans （P<0.05）. In addition, the retention rate of ILA in the toothpaste matrix is higher than 90% after 3 months of storage at 4 and 25 ℃, indicating good stability. In conclusion, ILA possesses the triple functions of anti-inflammation, immunomodulation and microecological regulation, and exhibits good stability in the oral care product matrices. This study provides a comprehensive in vitro experimental and application basis for the development and industrial application of ILA as a functional ingredient in oral care products.
