PSII-11 Effects of Cannabidiol on Viability and Inflammatory Response in Bovine Primary Ruminal Epithelial Cells

Abstract

Abstract Phytochemicals derived from hemp (Cannabis sativa) have been noted for their possible beneficial effects, including immunomodulation. Cannabidiol (CBD) may be a promising target as a therapy for gastrointestinal inflammation; however, the effects have not been explored in ruminants. The objective of this study was to investigate immunomodulatory properties of CBD in primary ruminal epithelial cells (REC). REC were isolated from ruminal papillae of yearling Holstein steers (n = 7). A preliminary experiment was performed to determine cytotoxicity of CBD using alamarBlue. Cells were exposed to either 10 or 50 µM CBD for 6 or 24 h. CBD at 10 µM did not affect viability. Growth was inhibited by over 60% at 50 µM for 24 h (P < 0.05), and therefore, 10 µM was selected for use. Two experiments, each 24h in duration, were conducted to explore various responses to CBD when inflammation was induced in REC with lipopolysaccharide (LPS). In Experiment 1, LPS was added prior to CBD exposure, simulating a therapy for established inflammation. In Experiment 2, LPS was added after CBD exposure, simulating a preventative strategy. Treatment groups for both experiments are described in Table 1. Total RNA was extracted from REC and relative gene expression of cytokines TNF, IL1B, IL6, CXCL8 and CXCL10 was evaluated using real time qPCR. Expression was normalized to the geometric mean of two stable housekeeping genes, GAPDH and STX5. All statistical analyses were performed using the lme4 and emmeans packages of R with exposure treatment as the fixed effect. In Experiment 1, expression of TNF, IL1B and CXCL8 was greater for LPS8 compared with CON by 5-, 29- and 14-fold, respectively, but less than LPS24 (P < 0.05). The LPS24+CBD16 group resulted in an upregulation of IL1B compared with CON, but not to the same magnitude as LPS24 (27- vs 93-fold; P = 0.002). A 3-fold down-regulation of IL6 was observed with the addition of CBD alone compared with CON (P < 0.001). Experiment 1 results suggest that CBD may reduce cytokine transcription during LPS-induced inflammation. For Experiment 2, compared with LPS8, there was less expression of IL6 and CXCL10 in CBD24+LPS8 (P < 0.05). There was a 9-fold upregulation of IL6 for CBD16-LPS8 (P < 0.001), but no difference between CON and CBD16-LPS8 for CXCL10 expression. Compared with CON, CBD24 resulted in a 2- and 6-fold down-regulation of IL6 (P = 0.003) and CXCL10 (P = 0.002). Experiment 2 results suggest that IL6 expression was influenced by CBD, but only while present in the media, while CXCL10 expression was suppressed by CBD, even after its removal. Overall, this study provides evidence of immunomodulation by CBD during a pro-inflammatory response in primary REC in culture.