RESUMEN
ABSTRACT Odontoblasts and gingival fibroblasts play essential roles in the physiological and pathological processes of dental tissue. Cannabinoid receptors (CB1 and CB2) are involved in analgesia by modulating the función of calcium channels that inhibit the synthesis of some neurotransmitters. A better understanding of the physiology of these receptors would provide the possibility of using them as therapeutic targets in controlling dental pain. The aim of this study was to evaluate the presence and activity of cannabinoid receptors in human odontoblast-like cells (OLC) and human gingival fibroblasts (HGF). CB1 and CB2 transcription was analyzed by real-time PCR, proteins were detected by immunofluorescence, and functional cannabinoid receptors were evaluated by measuring intracellular calcium concentration after stimulation with cannabidiol (CBD) and pre-treatment with a CB1 antagonist, a CB2 inverse agonist and a TRPV1 antagonist. Transcripts for CB1 and CB2 were found in both odontoblasts and gingival fibroblasts. Cannabidiol induced an increase in [Ca2+]i in both cells types, but surprisingly, pre-treatment with selective cannabinoid antagonists attenuated this effect, suggesting a functional communication between specific cannabinoid receptors and other CBD target receptors. In conclusion, human odontoblasts and gingival fibroblasts express functional CB1 and CB2 cannabinoid receptors, which could be modulated to improve the treatment of pain or dental sensitivity.
RESUMEN Los odontoblastos y los fibroblastos gingivales desempeñan funciones esenciales en los procesos fisiológicos y patológicos de los tejidos dentales. Los receptores cannabinoides (CB1 y CB2) participan en la analgesia mediante la modulación de la función de canales de calcio que inhiben la síntesis de algunos neurotransmisores. Un mejor conocimiento de su fisiología abre la posibilidad de utilizar estos receptores como dianas terapéuticas en el control del dolor dental. Este trabajo tuvo como objetivo evaluar la presencia y la actividad de los receptores cannabinoides en células humanas similares a los odontoblastos (OLC) y en fibroblastos gingivales humanos (HGF). Se analizó la transcripción de CB1 y CB2 por PCR en tiempo real, la detección de las proteínas por inmunofluorescencia y se evaluaron los receptores cannabinoides funcionales midiendo las concentraciones de calcio intracelular, tras la estimulación con cannabidiol (CBD) y el pretratamiento con un antagonista de CB1, un agonista inverso de CB2 y un antagonista de TRPV1. Se encontraron mensajeros para CB1 y CB2 tanto en odontoblastos como en fibroblastos gingivales. El cannabidiol indujo un aumento de la [Ca2+]i en ambos tipos de células, pero sorprendentemente el pretratamiento con antagonistas cannabinoides selectivos atenuó este efecto, lo que sugiere una comunicación funcional entre receptores cannabinoides específicos y otros receptores diana del CBD. En conclusión, los odontoblastos humanos y los fibroblastos gingivales expresan receptores cannabinoides CB1 y CB2 funcionales, que podrían ser modulados para mejorar el tratamiento del dolor o la sensibilidad dental.
RESUMEN
Objective To investigate the effects of spinal cannabinoid type 2 receptor (CB2R) and microglia activation on hyperalgesia in neuropathic pain mice. Methods Male C57/BL mice were randomly divided into six groups: sham, spinal nerve ligation (SNL), SNL＋CB2R agonist AM1241 (SNL＋AM1241), SNL＋microglia inhibitor minocycline (SNL＋minocycline), SNL＋small interfering RNA (siRNA) targeting CB2R (SNL＋siRNA), and SNL＋siRNA＋minocycline groups. A neuropathic pain mouse model was established by SNL. The expression levels of spinal CB2R and microglia-specific protein ionized calcium-binding adapter molecule 1 (IBA-1) were determined by Western blotting, mechanical pain thresholds were measured by Von Frey, spinal microglia activation was observed by IBA-1 immunofluorescence, and the expression levels of inflammatory factors in spinal cord dialysate were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Electrophysiology was applied to observe the effect of CB2R agonist on spontaneous inhibitory postsynaptic current (sIPSC) in the spinal dorsal horn. Results Compared with the sham group, the expression of CB2R in spinal cord was significantly decreased in the SNL group (P＜0.012 5), the pain threshold was significantly reduced (P＜0.016 7), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P＜0.008 3), and the mRNA expression levels of tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 were significantly increased (all P＜0.008 3). After intrathecal injection of CB2R agonist AM1241 or microglial inhibitor minocycline, compared with the SNL group, the pain thresholds of mice were significantly increased in the SNL＋AM1241 and SNL＋minocycline groups (both P＜0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly decreased (both P＜0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly decreased (all P＜0.008 3). After targeted interfering CB2R expression by siRNA, compared with the SNL group, the pain threshold was significantly decreased in the SNL＋siRNA group (P＜0.008 3), the fluorescence quantification and protein expression of IBA-1 were significantly increased (both P＜0.008 3), and the mRNA expression levels of TNF-α, IL-1β and IL-6 were significantly increased (all P＜0.008 3); while intrathecal injection of minocycline significantly reversed the above changes (all P＜0.008 3). Intervention in vitro of AM1241 could significantly enhance the frequency and amplitude of sIPSC in the spinal dorsal horn (both P＜0.05), while continuous treatment with minocycline inhibited the enhancement effects of AM1241 on sIPSC. Conclusion CB2R can reduce the neuroinflammatory responses and enhance the inhibitory electrical activity in the spinal cord by inhibiting spinal microglia activation, thereby alleviating hyperalgesia of neuropathic pain in mice.