Bioactivities of Novel 20D3 analogues on Human Gingival Epithelium Progenitors
Objectives: This study aimed to investigate the bioactivities of novel 20-hydroxyvitamin D3 (20D3) analogues on human gingival epithelium cells in vitro.
Methods: Novel 20-hydroxyvitamin D3 (20D3) analogues were synthesized and characterized using 1H-NMR and mass spectrometry. Their capability to inhibit inflammation and stimulate the secretion of antimicrobial peptide LL-37 were investigated on human gingival epithelium progenitors (hGEP) cells with 1,25-dihydroxyvitamin D3 (1,25-D3) as control. hGEP cells were assigned to five groups: no treatment, 0.1 nM interleukin(IL)-1β, 0.1 nM IL-1β and 1,25-D3, and 0.1 nM IL-1β and 20D3 analogues for 24 hours. The supernatants were analyzed for IL-6, IL-8, matrix metalloproteinase (MMP)-9 and LL-37 using enzyme-linked immunosorbent assay (ELISA). Gene expression of LL-37 was analyzed using real time polymerase chain reaction (qRT-PCR). T-test was used for group comparison and p<0.05 is considered statistically significant.
Results: Novel 20D3 analogues, specifically 20S-D3, 1,20S-D3, 20S,24R-D3, and 20S,24S-D3, were synthesized and characterized. IL-1β- increased hGEP secretion of IL-6, IL-8 and MMP-9 while decreasing the secretion of LL-37. The 20D3 analogues and 1,25-D3 affected the IL-1β-stimulated hGEP cells differently. All the 20D3 analogues and 1,25-D3 inhibited IL-8 secretion. 1,20S-D3 and 20S,24R-D3 inhibited IL-6 secretion at 100 nM or higher concentration, but 20S-D3 and 20S,24S-D3 inhibited the secretion of IL-6 only at 500 nM. 1,25-D3, 1,20S-D3 and 20S,24S-D3 inhibited the secretion of MMP-9. Moreover, 1,25-D3 increased the secretion of LL-37 at 500 nM; 1,20S-D3 and 20S,24R-D3 increased the secretion of LL-37 at 100 nM and 500 nM. In IL-1β-stimulated hGEP cells, 1,25-D3, 1,20S-D3 and 20S,24R-D3 upregulated the gene expression of LL-37 significantly. Conclusions: The types and concentration of 20D3 analogues affect their bioactivities. Among the analogues tested, 1,20S-D3 shows the best combined anti-inflammatory and antimicrobial effects for treatment of periodontitis. Acknowledgement: This work was financially supported by the UTHSC CORNET Award and NIH grant R21AR063242.