Abstract:

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.

Key words: probiotic-derived metabolites, indole-3-lactic acid, oral care products, immunomodulation, macrophages, inflammation, oral microbiota