Cannabinoid type‐2 receptor agonist, inverse agonist, and anandamide regulation of inflammatory responses in IL‐1β stimulated primary human periodontal ligament fibroblasts

Abstract

Objective The aim of this study is to understand the role of cannabinoid type 2 receptor (CB2R) during periodontal inflammation and to identify anti‐inflammatory agents for the development of drugs to treat periodontitis (PD). Background Cannabinoid type 2 receptor is found in periodontal tissue at sites of inflammation/infection. Our previous study demonstrated anti‐inflammatory responses in human periodontal ligament fibroblasts (hPDLFs) via CB2R ligands. Methods Anandamide (AEA), HU‐308 (agonist), and SMM‐189 (inverse agonist) were tested for effects on IL‐1β‐stimulated cytokines, chemokines, and angiogenic and vascular markers expressed by hPDLFs using Mesoscale Discovery V‐Plex Kits. Signal transduction pathways (p‐c‐Jun, p‐ERK, p‐p‐38, p‐JNK, p‐CREB, and p‐NF‐kB) were investigated using Cisbio HTRF kits. ACTOne and Tango™ ‐BLA functional assays were used to measure cyclic AMP (cAMP) and β‐arrestin activity. Results IL‐1β stimulated hPDLF production of 18/39 analytes, which were downregulated by the CB2R agonist and the inverse agonist. AEA exhibited pro‐inflammatory and anti‐inflammatory effects. IL‐1β increased phosphoproteins within the first hour except p‐JNK. CB2R ligands attenuated p‐p38 and p‐NFĸB, but a late rise in p‐38 was seen with HU‐308. As p‐ERK levels declined, a significant increase in p‐ERK was observed later in the time course by synthetic CB2R ligands. P‐JNK was significantly affected by SMM‐189 only, while p‐CREB was elevated significantly by CB2R ligands at 180 minutes. HU‐308 affected both cAMP and β‐arrestin pathway. SMM‐189 only stimulated cAMP. Conclusion The findings that CB2R agonist and inverse agonist may potentially regulate inflammation suggest that development of CB2R therapeutics could improve on current treatments for PD and other oral inflammatory pathologies.

Reference gene validation for the relative quantification of cannabinoid receptor expression in human odontoblasts via quantitative polymerase chain reaction

“…1 C–D). 17 , 18 , 19 This analysis demonstrates that for a reference gene, it is not enough to have a low variability in expression; it should also have a similar level of expression (Cq) to the target gene. This explains why β-actin has been discarded as a reference gene in many studies.…”

A critical review of cannabis in medicine and dentistry: A look back and the path forward

“…These studies, along with others (Ossola et al, 2020;Zhang et al, 2020), highlight the important role of the ECS in oral and systemic health. Moreover, the cytokine and chemokine inhibition profiles of HU-308 (agonist) and SMM-189 (inverse agonist) were investigated in activated hPDLFs, and study results showed that CB2R ligands are viable candidates for the development of a new therapeutic intervention for PD (Figure 2) (Abidi et al, 2020). PHARMACODYNAMICS OF CANNABINOIDS…”

“…Therefore, AEA or 2‐AG involvement in gingivitis and PD along with the systemic role of ECS in immunomodulation seems to play a major role. During inflammation, the CB2R are upregulated (Turcotte et al, 2016 ) and in chronic inflammatory disease like PD, the efficacy of endocannabinoids, that is, 2‐AG or AEA in activating the ECS may be insufficient to overcome the proinflammatory feedback loop (dysregulation) (Abidi et al, 2020 ). The limitations of the endogenous ligands may be due to short half‐life, limited efficacy, and reduced potency in chronic inflammation.…”

Potential Mechanisms Underlying Marijuana-Associated Periodontal Tissue Destruction

“…Variant cannabinoids have differing affinities and activation capacities for different CBs, and details are beyond the scope of this review. b AEA and 2-AG are the major endocannabinoids. Proposed minor endocannabinoids have received little attention in the periodontium. c Abidi et al (2018). d Abidi et al (2020). e Jager et al (2020). f No known recent studies. g Qi et al (2021). h Gu et al (2019). i Vasudevan and Stahl (2020).…”

“…While multiple lines of evidence point toward a role of cannabis in the promotion of bacterial dysbiosis at the periodontium (Rabinovich et al 1959;Gu et al 2019;Stahl and Vasudevan 2020) preferable. Indeed, a comparison of the influence of a CB2engaging endocannabinoid (AEA), a CB2 agonist (HU-308), and an inverse agonist (SMM-189) determined that while HU-308 and SMM-189 suppressed the release of multiple inflammatory mediators from IL-1β-stimulated human periodontal ligament fibroblasts, the influence of AEA was more nuanced (Abidi et al 2020). For example, AEA enhanced the IL-1β-induced IFN-γ, IL-6, IL-8, and TNF signals but suppressed MCP-1, CXCL10, and MDC.…”