Gallic Acid Ameliorates the Inflammatory State of Periodontal Ligament Stem Cells and Promotes Pro-Osteodifferentiation Capabilities of Inflammatory Stem Cell-Derived Exosomes

Abstract

The slow proliferation rate and poor osteodifferentiation ability of inflammatory periodontal membrane stem cells extracted from periodontitis tissues (i-PDLSCs) account for poor efficiency in treating inflammatory bone loss. Exosomes reportedly have inducible and relatively stable components, allowing them to promote inflammatory bone repair, but obtaining i-PDLSCs exosomes with the ability to promote osteodifferentiation is challenging. In the present study, i-PDLSCs were extracted from periodontal membrane tissues of patients with severe periodontitis, and in vitro induction with gallic acid (GA) significantly promoted the proliferative activity of i-PDLSCs at a concentration of 10 mM, with TC₀ of 11.057 mM and TC₅₀ of 67.56 mM for i-PDLSCs. After mRNA sequencing, we found that GA could alleviate oxidative stress in i-PDLSCs and increase its mitochondrial membrane potential and glucose aerobic metabolism level, thus promoting the osteodifferentiation of i-PDLSCs. After exosomes of i-PDLSCs after GA induction (i-EXO-GA) were isolated by differential centrifugation, we found that 200 ug/mL of i-EXO-GA could remarkably promote the osteodifferentiation of i-PDLSCs. Overall, our results suggest that GA induction can enhance the proliferation and osteodifferentiation in primary cultures of i-PDLSCs in vitro, mediated by alleviating oxidative stress and glycometabolism levels in cells, which further influences the osteodifferentiation-promoting ability of i-EXO-GA. Overall, we provide a viable cell and exosome induction culture method for treating inflammatory alveolar defects associated with periodontitis.

Keywords: aerobic glucose metabolism; exosomes; gallic acid; osteodifferentiation; oxidative stress; periodontal membrane stem cells; periodontitis.

Figure 1

The effect of GA on the of PDLSCs proliferation detected by MTT. (A) The OD value ratio of i-PDLSCs-GA to i-PDLSCs; (B) The OD value ratio of i-PDLSCs-GA to h-PDLSCs; (C) The micrographs of i-PDLSCs-GA.

Figure 2

The effect of GA-induced culture by mRNA-Seq. (A) Heat map of differentially expressed genes; (B) GO enrichment analysis of differentially expressed genes; (C) KEGG enrichment analysis of DEGs; (D) ELISA detection of protein expression levels of differentially expressed genes, ns: p > 0.05, *, **, ***: compared with h-PDLSCs group p < 0.05, p < 0.01, p < 0.001, #, ##, ###: compared with i-PDLSCs group p < 0.05, p < 0.01, p < 0.001.

Figure 3

The effects of GA-induced culture on mitochondrial membrane potential and oxidative stress levels. (A(i)) fluorescence image of mitochondrial membrane potential JC-1 staining, with a scale of 10 μm; (A(ii)) fluorescence ratio quantitative analysis of mitochondrial membrane potential JC-1 staining, *: p < 0.05, **: p < 0.01; (B) ELISA detection of oxidative stress indexes MDA and SOD, *: p < 0.05 compared with h-PDLSCs group, #: p < 0.05 compared with i-PDLSCs group.

Figure 4

The effects of GA-induced culture on glycometabolism. (A) Determination of ATP production and lactic acid content; (B) ELISA detection of PFK-1 and PK enzyme activity; (C,D) RT-qPCR detection of CS, IDH, NDUFB-3, and SDHB. *: p < 0.05 compared with h-PDLSCs group, #: p < 0.05 compared with i-PDLSCs group.

Figure 5

The effects of GA-induced culture on the level of osteodifferentiation. (A) Quantitative analysis of ALP staining and staining intensity; (B) Alizarin red staining and quantitative analysis of staining intensity. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001; (C) RT-qPCR detection of osteodifferentiation-related genes on days 0, 7, 14, and 21. ns: p > 0.05, #: h-PDLSCs-CCCP group compared with h-PDLSCs group p < 0.05; ##: h-PDLSCs-CCCP group compared with h-PDLSCs group p < 0.01; *: p < 0.05, **: p < 0.01, ***: p < 0.001.

Figure 6

Exosome characterization, osteodifferentiation function detection and concentration screening. (A) BCA protein concentration detection of exosome samples, #: compared with the i-PDLSCs group, p < 0.05, *: compared with the h-PDLSCs group, p < 0.05; (B) Western blot detection of exosome markers; (C) NTA detection of exosome samples; (D) ALP staining and quantitative analysis of staining intensity of i-PDLSCs co-cultured with exosomes on day 7. (E) Alizarin red staining and quantitative analysis of staining intensity of i-PDLSCs co-cultured with exosomes on day 14. (F) RT-qPCR detection of osteodifferentiation-related genes cocultured with i-PDLSCs and exosomes on days 0, 7, 14 and 21. ns: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001.