Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine Production of Senior Horses

Shelley Turner; Virginia Day Barker; Amanda A. Adams

doi:10.1016/j.jevs.2021.103668

Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine Production of Senior Horses

Shelley Turner, Virginia Day Barker, Amanda A. Adams

Veterinary Science

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Cannabis sativa L. contains cannabidiol (CBD), a compound that has many anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in vitro efficacy as an anti-inflammatory agent. Heparinized blood was collected via jugular venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell counter was used to evaluate viability after incubation. PBMCs were stained intracellularly for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2, TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA (significance P < .05). Viability of PBMCs with CBD was completed to determine cytotoxicity. The dilution of CBD that did not affect cell viability was 4 µg/mL (P<0.05). CBD at 4 µg/mL significantly reduced production of IFN-γ and TNF-α (P < .05). RT-PCR results for TNFα and IFNγ at 4 µg/mL showed a reduction compared with the positive control and IL-10 showed a similar reduction at 2 µg/mL and 4 µg/mL. RT-PCR gene expression results showed significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4 µg/mL reduced in vitro production of inflammatory cytokines from senior horses. This in vitro study supports further investigation of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic inflammation in the horse.

Original language	English
Article number	103668
Journal	Journal of Equine Veterinary Science
Volume	103
DOIs	https://doi.org/10.1016/j.jevs.2021.103668
State	Published - Aug 2021

Bibliographical note

Funding Information:
The authors would like to thank the farm staff at the University of Kentucky's C. Oran Little Farm (Versailles, KY) for their assistance in the daily care of the research horses and the laboratory team for their assistance with the sample collection. In addition, we thank AgTech Scientific (Paris, KY) for their funding of this project. Conflict of interest statement: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The funder of the study, Agtech Scientific, had no involvement in the study design, data collection, analysis, interpretation of the results, the writing of this article, nor the submission of this article for publication.

Publisher Copyright:
© 2021

Keywords

Cannabidiol
Cannabis sativa L
Equine
Inflammation

ASJC Scopus subject areas

Equine

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10.1016/j.jevs.2021.103668

Cite this

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title = "Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine Production of Senior Horses",

abstract = "Cannabis sativa L. contains cannabidiol (CBD), a compound that has many anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in vitro efficacy as an anti-inflammatory agent. Heparinized blood was collected via jugular venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell counter was used to evaluate viability after incubation. PBMCs were stained intracellularly for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2, TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA (significance P < .05). Viability of PBMCs with CBD was completed to determine cytotoxicity. The dilution of CBD that did not affect cell viability was 4 µg/mL (P<0.05). CBD at 4 µg/mL significantly reduced production of IFN-γ and TNF-α (P < .05). RT-PCR results for TNFα and IFNγ at 4 µg/mL showed a reduction compared with the positive control and IL-10 showed a similar reduction at 2 µg/mL and 4 µg/mL. RT-PCR gene expression results showed significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4 µg/mL reduced in vitro production of inflammatory cytokines from senior horses. This in vitro study supports further investigation of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic inflammation in the horse.",

keywords = "Cannabidiol, Cannabis sativa L, Equine, Inflammation",

author = "Shelley Turner and Barker, {Virginia Day} and Adams, {Amanda A.}",

note = "Funding Information: The authors would like to thank the farm staff at the University of Kentucky's C. Oran Little Farm (Versailles, KY) for their assistance in the daily care of the research horses and the laboratory team for their assistance with the sample collection. In addition, we thank AgTech Scientific (Paris, KY) for their funding of this project. Conflict of interest statement: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The funder of the study, Agtech Scientific, had no involvement in the study design, data collection, analysis, interpretation of the results, the writing of this article, nor the submission of this article for publication. Publisher Copyright: {\textcopyright} 2021",

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AU - Adams, Amanda A.

N1 - Funding Information: The authors would like to thank the farm staff at the University of Kentucky's C. Oran Little Farm (Versailles, KY) for their assistance in the daily care of the research horses and the laboratory team for their assistance with the sample collection. In addition, we thank AgTech Scientific (Paris, KY) for their funding of this project. Conflict of interest statement: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The funder of the study, Agtech Scientific, had no involvement in the study design, data collection, analysis, interpretation of the results, the writing of this article, nor the submission of this article for publication. Publisher Copyright: © 2021

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N2 - Cannabis sativa L. contains cannabidiol (CBD), a compound that has many anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in vitro efficacy as an anti-inflammatory agent. Heparinized blood was collected via jugular venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell counter was used to evaluate viability after incubation. PBMCs were stained intracellularly for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2, TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA (significance P < .05). Viability of PBMCs with CBD was completed to determine cytotoxicity. The dilution of CBD that did not affect cell viability was 4 µg/mL (P<0.05). CBD at 4 µg/mL significantly reduced production of IFN-γ and TNF-α (P < .05). RT-PCR results for TNFα and IFNγ at 4 µg/mL showed a reduction compared with the positive control and IL-10 showed a similar reduction at 2 µg/mL and 4 µg/mL. RT-PCR gene expression results showed significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4 µg/mL reduced in vitro production of inflammatory cytokines from senior horses. This in vitro study supports further investigation of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic inflammation in the horse.

AB - Cannabis sativa L. contains cannabidiol (CBD), a compound that has many anti-inflammatory properties. In this study, 99.9% CBD powder was used to determine its in vitro efficacy as an anti-inflammatory agent. Heparinized blood was collected via jugular venipuncture from senior horses. PBMCs were isolated then incubated for 24 hours with increasing dilutions of CBD dissolved in DMSO. PBMCs were stimulated the last 4 hours of incubation with PMA/IO and Brefeldin A. A Vicell counter was used to evaluate viability after incubation. PBMCs were stained intracellularly for IFNγ and TNFα then analyzed via flow cytometry. RT-PCR was used to analyze samples for gene expression. Five equine-specific intron-spanning primers/probes used are: CB1, CB2, TNFα, IFNγ, IL-10, and Beta-glucuronidase. Data was analyzed using RM One-way ANOVA (significance P < .05). Viability of PBMCs with CBD was completed to determine cytotoxicity. The dilution of CBD that did not affect cell viability was 4 µg/mL (P<0.05). CBD at 4 µg/mL significantly reduced production of IFN-γ and TNF-α (P < .05). RT-PCR results for TNFα and IFNγ at 4 µg/mL showed a reduction compared with the positive control and IL-10 showed a similar reduction at 2 µg/mL and 4 µg/mL. RT-PCR gene expression results showed significance for 10 μg/mL CBD in CB1 and CB2. CBD at 4 µg/mL reduced in vitro production of inflammatory cytokines from senior horses. This in vitro study supports further investigation of CBD to determine if it may be effective as an anti-inflammatory treatment for chronic inflammation in the horse.

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Effects of Cannabidiol on the In Vitro Lymphocyte Pro-Inflammatory Cytokine Production of Senior Horses

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

Access to Document

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