IADR Abstract Archives
Chronic Capsaicin Stimulus Changes the Expression of Cannabinoids-receptors in Odontoblast-like-cells
Objectives: Determine changes in the cannabinoids receptors expression induced after chronic capsaicin stimulation in human Odontoblast-like-cells (OLC).
Methods: Human OLC differentiated from dental pulp stem cells were cultured in 6-well plates at a density of 150,000 cells/well in DMEM supplemented with 10% SFB and TGF-b. After 24 hours, cells were stimulated chronically with 150 nM capsaicin for 3, 5 and 7 days. Cells were lysed at each time point and the total RNA was extracted. Messenger RNA of CB1 (cannabinoid receptor type-1), CB2 (cannabinoid receptor type 2)and transient receptor potential vanilloid 1 (TRPV1, capsaicin receptor) were analyzed by a SYBR green relative quantitation RT-qPCR. β-actin and dentin matrix protein 1 (DMP-1) were used as reference genes and differentiated unstimulated cells were used as controls. Simultaneously, OLC cultured on glass coverslips at a density of 5,000 cells/well were stimulated with 150 nM capsaicin andthen analyzed by immunofluorescence to CB1 and CB2 receptors.
Results: Amplicons for CB1, CB2 and TRPV1 mRNA were detected with different patterns of band intensity after PCR. mRNA for CB1 and CB2 receptors increase significantly after chronic capsaicin stimulation with a peak at day 3 (40- and 130-fold change regarding unstimulated cells respectively), but remain high at 5 and 7 days post-stimulation. Capsaicin stimulation also induced a 600-fold up-regulation of their TRPV-1 receptor after 5 days of stimulation. There was no change in cell survival by the chronic stimulus, and immunofluorescence signal of receptors slightly increased in treated cultures.
Conclusions: TRPV1 mediated capsaicin chronic stimulation induces both auto- and CB receptors upregulation indicating a close relationship that could reinforce the positive role of cannabinoids during chronic pain and the putative anti-nociceptive activity in dental pain.
Methods: Human OLC differentiated from dental pulp stem cells were cultured in 6-well plates at a density of 150,000 cells/well in DMEM supplemented with 10% SFB and TGF-b. After 24 hours, cells were stimulated chronically with 150 nM capsaicin for 3, 5 and 7 days. Cells were lysed at each time point and the total RNA was extracted. Messenger RNA of CB1 (cannabinoid receptor type-1), CB2 (cannabinoid receptor type 2)and transient receptor potential vanilloid 1 (TRPV1, capsaicin receptor) were analyzed by a SYBR green relative quantitation RT-qPCR. β-actin and dentin matrix protein 1 (DMP-1) were used as reference genes and differentiated unstimulated cells were used as controls. Simultaneously, OLC cultured on glass coverslips at a density of 5,000 cells/well were stimulated with 150 nM capsaicin andthen analyzed by immunofluorescence to CB1 and CB2 receptors.
Results: Amplicons for CB1, CB2 and TRPV1 mRNA were detected with different patterns of band intensity after PCR. mRNA for CB1 and CB2 receptors increase significantly after chronic capsaicin stimulation with a peak at day 3 (40- and 130-fold change regarding unstimulated cells respectively), but remain high at 5 and 7 days post-stimulation. Capsaicin stimulation also induced a 600-fold up-regulation of their TRPV-1 receptor after 5 days of stimulation. There was no change in cell survival by the chronic stimulus, and immunofluorescence signal of receptors slightly increased in treated cultures.
Conclusions: TRPV1 mediated capsaicin chronic stimulation induces both auto- and CB receptors upregulation indicating a close relationship that could reinforce the positive role of cannabinoids during chronic pain and the putative anti-nociceptive activity in dental pain.
