Ursodeoxycholic acid attenuates the expression of proinflammatory cytokines in periodontal cells

Abstract

Background: Ursodeoxycholic acid (UDCA) is one of the first-line therapeutic medications used in treatment of cholestatic liver disease. Considering that periodontitis is epidemiologically linked to liver diseases, the question arises weather UDCA holds anti-inflammatory properties on periodontal health. Herein, we provide information that support anti-inflammatory effects of UDCA on three different periodontium-related cell types.

Methods: Gingival fibroblasts and the oral human squamous carcinoma cell line HSC-2 were exposed to interleukin (IL)1β and tumor necrosis factor (TNF)α with and without UDCA. Murine RAW 264.7 macrophages were incubated with sterile-filtered human saliva also in the presence of UDCA. The expression of inflammatory cytokines was measured by reverse transcription-polymerase chain reaction. Immunoassay was applied to detect the production of IL6. Immunostaining was performed for the p65 subunit to further support the anti-inflammatory role of UDCA.

Results: We report here that UDCA significantly reduced the IL1β and TNFα-induced expression of IL1, IL6, and IL8 in gingival fibroblasts and the HSC-2 cell line. In RAW 264.7 macrophages, UDCA attenuated the expression of IL1α, IL1β, and IL6 that was increased by saliva. Immunoassay confirmed the capacity of UDCA to reduce inflammation-induced production of IL6 in gingival fibroblasts, HSC-2 and RAW 264.7 cells. Immunostaining revealed the blocking of nuclear translocation of p65 in gingival fibroblasts.

Conclusions: Taken together, UDCA can attenuate the provoked expression of inflammatory cytokines in oral fibroblasts, oral human squamous carcinoma cells and macrophages in vitro. These data support the hypothesis that patients with cholestatic liver disease might benefit from UDCA with respect to periodontal health.

Keywords: in vitro; inflammation; liver cirrhosis; mouth; periodontitis; ursodeoxycholic acid.

Figure 1

UDCA decreased the viability of oral fibroblasts at millimolar concentrations. Human gingival fibroblasts were exposed to increasing concentrations of UDCA for 24 hours before the production of formazan crystals was determined. Data show the production of formazan crystals normalized to untreated controls

Figure 2

UDCA decreased the expression of proinflammatory cytokines in oral fibroblasts. Human gingival fibroblasts were exposed to IL1β and TNFα for 1 hour followed by the addition of UDCA for 3 hours. Relative gene expression for (A) IL1, (B) IL6, and (C) IL8 was determined by RT‐PCR. The dot blots show the data of independent experiments. Statistical analysis was based on Wilcoxon matched‐pairs signed rank test

Figure 3

UDCA diminished the nuclear translocation of p65 in oral fibroblasts exposed to IL1β and TNFα. Human gingival fibroblasts were left untreated (wo; without) or exposed to IL1β and TNFα for 20 minutes followed by the addition of UDCA for 1 hour. Alexa 488 labeling appearing in green detected the NFkB p65 primary antibody. Note the nuclear translocation of the p65 subunit by the inflammatory cytokines and the concomitant suppression by UDCA. Pictures are taken at 20× magnification

Figure 4

UDCA decreased the expression of proinflammatory cytokines of oral HSC‐2 cells. Human HSC‐2 cells were exposed to IL1β and TNFα for 1 hour followed by the addition of UDCA for 3 hours. Relative gene expression for (A) IL1, (B) IL6, and (C) IL8 was determined by RT‐PCR. The dot blots show the data of independent experiments. Statistical analysis was based on Wilcoxon matched‐pairs signed rank test

Figure 5

UDCA reduced the expression of proinflammatory cytokines of RAW 264.7 cells. Mouse RAW 264.7 macrophages were exposed to the human saliva for 1 hour followed by the addition of UDCA for 3 hours. Relative gene expression for (A) IL1α, (B) IL1β, and (C) IL8 was determined by RT‐PCR. The dot blots show the data of independent experiments. Statistical analysis was based on Wilcoxon matched‐pairs signed rank test