*5.1. In Vitro Studies*

A study on A549 cells has shown that marijuana smoke causes mitochondrial damage, impairing the energetic metabolism of the cell [107]. Furthermore, using the ECV304 cell line, Sarafian et al. demonstrated that even short exposures to marijuana smoke cause an increase in reactive oxygen species (ROS) production capable of inducing necrotic cell death, and the effects seem to be mainly attributed to the gaseous phase—not the particulate phase—of the smoke [108].

However, studies focused on the direct effects of the substances isolated from plants showed controversial results. In a study on multiple human cell lines, including eosinophils and natural killer (NK) cells, both THC and CBD decreased the levels of IL-8, macrophage inflammatory protein (MIP)-1, IFN-γ and TNF-α, thus demonstrating anti-inflammatory properties [109].

The anti-inflammatory effects of cannabinoids were cited in the context of septic lung injury. The highly CB2-selective synthetic cannabinoid HU308 decreased the levels of TNF-α, IL-18, IL-1β, and NLR family pyrin domain containing 3 (NLRP3) in RAW264.7 macrophages in lipopolysaccharide (LPS)-induced inflammation, effects which were also observed in an in vivo animal model of acute lung injury (ALI) in the same study [110].

Additional studies revealed a more complex interaction between cannabinoids and the immune system. In a study on macrophages, lung fibroblasts and epithelial cells, Muthumalage et al. showed that CBD reduced the levels of IL-8, monocyte chemoattractant protein (MCP)-1 and nuclear factor-kappa B (NF-κB) activity when they were increased by an LPS-induced inflammatory state [111]. However, when co-administered with dexamethasone, CBD demonstrates antagonistic effects, possibly due to receptor competitivity and pharmacological interactions in the signaling pathways [111,112].

Cannabinoids have also demonstrated anti-angiogenic properties, with possible applications in inflammation and cancer. The synthetic cannabinoids arachidonyl-2′ chloroethylamide (ACEA) and JWH-133 are selective CB1 and, respectively, CB2 agonists which inhibit the LPS-induced production and release of vascular endothelial growth factors A and C, angiopoietins 1 and 2, and IL-6 from human lung macrophages [113].
