298 Botanicals reduce osteoarthritic-like inflammation influencing the endocannabinoid system in vitro
Authors: Ghiselli Federico , Majer Roberta , Piva Andrea , Grilli Ester 
Published: September 2024

Abstract
Osteoarthritis (OA) is a prevalent joint disease in dogs. Botanicals (BOT) interacting with the endocannabinoid system (ECS) can reduce pain, inflammation, and other OA-related processes, and could become potential treatments for pets. This study investigates various BOT in vitro for their potential to reduce OA-like inflammation, oxidative stress, and apoptosis, and influence the expression of ECS enzymes or receptors. Capsicum oleoresin (10% capsaicinoids) at 100 ppm, cinnamaldehyde at 5 ppm, turmeric extract (95% curcuminoids) at 10 ppm, and eugenol at 10 ppm were selected for their documented ability to interact with the ECS and used as treatments. The efficacy of these BOT was assessed by pre-treating human chondrosarcoma cells (SW1353 - ATCC HTB-94) with BOT for 2 h before exposure to interleukin (IL)1β and tumor necrosis factor (TNF) α for 24 h. Following the challenge, the cells and supernatants were collected. Then, qPCR on selected markers and IL8 quantification with ELISA were conducted. Additionally, to assess antioxidant and anti-apoptotic capabilities, BOT-treated cells were exposed to menadione for 1 h to measure reactive oxygen species (ROS) and for 6 h to induce apoptosis and evaluate chondrocyte viability. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons (n = 3; P &lt; 0.05) and reported in this work compared against the positive control. Under inflammatory conditions, SW1353 cells exhibited a significant increase in IL6 (234-fold; P = 0.0333) and IL8 (4,500-fold; P = 0.0395) expression, and MMP1 (13-fold, P = 0.0052), MMP3 (40-fold; P = 0.0474), and MMP13 (26-fold; P = 0.0260) expression. Among the different BOTs, only cinnamaldehyde effectively countered IL6 and IL8 upregulation, reducing their expression to 0 for IL6 and by 4,400-fold for IL8 (P = 0.0086). Additionally, it reduced MMP3 by 40-fold (P = 0.0242) and MMP13 by 24-fold (P = 0.0275), while eugenol significantly reduced MMP3 by 36-fold (P = 0.0250). Only cinnamaldehyde significantly decreased IL8 secretion by 86% (P &lt; 0.0001), and all tested botanicals reduced chondrocyte apoptosis (P &lt; 0.0001). Eugenol and turmeric extract exhibited significant antioxidant effects, reducing ROS levels by 70% compared with the positive control (P &lt; 0.05). Concerning ECS response to the inflammatory challenge, MAGL levels increased by 4.2-fold (P = 0.0253), with cinnamaldehyde significantly reducing its mRNA expression by 4-fold (P = 0.0078). PPAR-γ expression showed a numerical decrease of 0.7-fold under inflammatory conditions, but cinnamaldehyde and capsicum oleoresin upregulated its mRNA expression by 2.3-fold (P = 0.0162) and 1.3-fold (P = 0.0006), respectively. Cinnamaldehyde also reduced DAGLα expression by 0.3-fold (P = 0.0079), and upregulated CBR2 and NRF2 mRNA expression by 7-fold (P &lt; 0.0001) and 1.6-fold (P = 0.0295) respectively, while other botanicals had no effects. In conclusion, the tested BOTs displayed promising properties to mitigate OA in vitro, with cinnamaldehyde proving to be the most promising one. These findings suggest the potential of BOT active on ECS as a novel treatment for canine OA, prompting further in vivo research for comprehensive characterization.