Lipopolysaccharide mediates interleukin-6 release from periodontal ligament cells by promoting hyaluronan receptor CD44 transfer to nuclear

doi:10.3877/cma.j.issn.1674-1366.2023.05.004

Abstract

Abstract:

Objective

To investigate the molecular mechanism of inflammatory response in human periodontal ligament cells (hPDLCs) under inflammatory conditions.

Methods

Vimentin and keratin were identified by immunofluorescence. hPDLCs were stimulated with different concentrations (0, 0.1, 10, 100 μmol/L) of lipopolysaccharide (LPS) , and apoptosis was detected by flow cytometry. The expression of hyaluronan receptor CD44 in cell culture supernatant was detected by enzyme-linked immune sorbent assay (ELISA) after the LPS stimulation. The expression of CD44 and matrix metalloproteinase (MMP) -14 in the cytoplasm and nucleus of hPDLCs in inflammatory environment was detected by ELISA, Western blot and cell immunofluorescence. The expression of inflammatory factors was detected by Cytometric Bead Array (CBA) . Furthermore, MMP-14 and Importin β inhibitor were added in the inflammatory environment, and the expression of CD44 and interleukin-6 (IL-6) mRNA was detected by ELISA and RT-PCR, respectively.

Results

LPS did not affect the apoptosis of hPDLCs at different concentrations (0.1, 1, 10, 100 μmol/L) . After 4 h stimulation with 1 μmol/L LPS, the fluorescence intensity of MMP-14 in hPDLCs significantly increased (233.75) , while that in control group was 107.53, and the difference was statistically significant (t = 10.10, P<0.001) . CD44 fluorescence intensity was 106.7 in the LPS group and 84.58 in the control group, and the difference was statistically significant (t = 3.13, P = 0.02) . MMP-14 inhibitor could significantly inhibit the expression of CD44 in cell culture supernatant, and the expression of CD44 in the LPS+MMP-14 inhibitor group was significantly lower than that in the LPS group, and the difference was statistically significant (t = 5.03, P = 0.001) . LPS stimulation of hPDLCs increased the expression of CD44 and Importin β in the nucleus, and the difference was statistically significant (P<0.001) . After LPS stimulation, the expression of CD44 and Importin β in the nucleus was also up-regulated. LPS increased the expression of IL-6, and the difference was statistically significant (P<0.001) , and the expression of IL-6 mRNA in the cells treated with LPS plus Importin β inhibitor was significantly lower than that in the LPS treatment group. The differences were statistically significant (t = 16.79, P<0.001) .

Conclusions

LPS can up-regulate the expression of CD44, Importin β and IL-6 in hPDLCs. MMP-14 and Importin β inhibitor can inhibit the nuclear translocation of CD44 and the up-regulated expression of IL-6 in inflammatory environment. These results suggested that MMP-14 and CD44 participated in the inflammatory response of hPDLCs under inflammatory conditions, and CD44 can transfer to the nucleus and mediate the release of IL-6.

Key words: Periodontitis, Human periodontal ligament cells, Lipopolysaccharide, Hyaluronan receptors, CD44, Interleukin-6

Yanqun Xu, Ping Li, Xing Yang, Hui Xue. Lipopolysaccharide mediates interleukin-6 release from periodontal ligament cells by promoting hyaluronan receptor CD44 transfer to nuclear[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2023, 17(05): 335-344.

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References 23

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