Dysfunctional inflammation is a major cause of severe diseases in dairy cows, such as coliform mastitis, leading to diminished milk production, negatively impacting cow well-being, and possibly resulting in death, even with medical intervention. Endocannabinoids (EC) are potent lipid-based mediators, that modulate inflammation in humans and rodents to via stimulation of shared and dedicated cannabinoid receptors-1 and -2 (CB1/2). Closely related to the cyclooxygenase derived oxylipids targeted by non-steroidal anti-inflammatory drugs (NSAID) in dairy cattle, endocannabinoids have gotten very little attention regarding involvement in dairy inflammation. To address this gap, we determined EC and oxylipid concentrations in plasma of dairy cows suffering from naturally occurring acute coliform mastitis. We showed that concentrations of one of the most thoroughly studied EC, arachidonoylethanolamide (AEA), increased after medical intervention with an NSAID, only among those cows that died of the infection. We were also able to show that among those cows that died of the infection, the NSAID flunixin meglumine was not able to reduce isoprostane (IsoP) production, the gold-standard biomarker of oxidative stress. Elevated AEA was demonstrated in several human and rodent inflammatory models in response to NSAID treatment and associated with analgesic effects of the drug. However, AEA also induces ROS production and time dependent pro-inflammatory signaling in immune cells. Thus, we set out to establish the presence of the EC system (ECS) in bovine aortic endothelial cells and the effect of elevated AEA during endotoxin challenge on endothelial barrier and cell function. We found that ECS related genes are expressed in bovine aortic endothelial cells and expression is affected by endotoxin challenge. We also determined that addition of AEA further reduced endothelial barrier integrity of endothelial cells pre-treated with endotoxin, via CB1 mediated mitochondrial dysfunction, oxidative stress, and activation of apoptotic signaling. We showed for the first time that EC concentrations are affected by coliform mastitis and that increased plasma AEA concentrations following NSAID treatment may contribute to the death of the animal. Our work suggests that even though AEA may possess anti-inflammatory and analgesic effects in other cell types, increased concentrations of the EC have detrimental effects on endothelial cells pre-treated with endotoxin. The focus of future studies should be determining the timing dependent effects of increased AEA, as studies with concurrent endotoxin and AEA treatment show anti-inflammatory effects of the EC. Additionally, the usage of ECS targeted drugs in combination or without NSAIDs should be investigated, as we showed that antagonism of CB1 resulted in improved endothelial function during endotoxin challenge.
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