*6.1. In Vitro Studies*

One of the key elements of the skin inflammatory process is the array of actions performed by the multitude of cytokines that are involved in the response to skin barrier disruption, but also in immunity, apoptosis, and even the development and progression of skin cancer [163–166].

An in vitro experiment in a larger study performed by Karsak et al. investigating the impact of the endocannabinoid system on allergic contact dermatitis (ACD) revealed that HaCaT keratinocytes with contact hypersensitivity conditions induced via polyinosinic:polycytidylic acid express an upregulation of CB1 and a downregulation of CB2 receptors [167]. An ensuing study by Petrosino et al., using the same experimental model, showed that PEA and AEA are upregulated in these conditions, while exogenous PEA causes a decrease of MCP-2 expression [168]. Continuing their previous work on this in vitro model, Petrosino et al. showed that CBD inhibits the production of MCP-2, but also of interleukins 6 and 8 and tumor necrosis factor-alpha (TNF-α) while increasing the endogenous levels of AEA [169].

While they are not considered to have anti-inflammatory effect per se, cannabinoids have demonstrated the ability to inhibit keratinocyte cell proliferation, thus limiting the pro-inflammatory roles of these cells [170]. Wilkinson et al. showed that THC, cannabinol (CBN), CBD and CBG inhibit the proliferation of human papillomavirus (HPV)-16 E6/E7 transformed human skin keratinocytes in an in vitro model for psoriasis [171]. The synthetic CB1 agonist ACEA inhibited the upregulation of keratins K6 and K16 in isolated human skin samples of psoriasis lesions [172].

The cannabinoids' property of decreasing pro-inflammatory cytokine concentrations was also evidenced in an in vitro study performed by Robinson et al. on mononuclear cells from the peripheral blood of dermatomyositis patients treated with ajulemic acid (AJA) [173]. In their paper, the authors cite a significantly decreased concentration of TNF-α, but also of IFN-α and -β in the cells treated with ajulemic acid compared to untreated cells. These findings further indicate that cannabinoids decrease the production of cytokines in inflammatory skin disorders.

A study on SZ95 human sebocyte cultures performed by Oláh et al. demonstrated that CBD can prevent the increase of TNF-α when sebocytes are stimulated with linoleic acid and testosterone in an in vitro acne model [174]. Furthermore, CBD also decreased the expression of IL-1β and IL-6 when the sebocytes were stimulated with lipopolysaccharides, a finding that suggests a potential positive effect of CBD in the treatment of acne vulgaris. The same study identified that the anti-inflammatory effects of CBD are mediated by the Tribbles homolog (TRIB3)-NF- κB pathway via A2a adenosine receptors, yielding important insight into the action mechanisms of cannabinoids.

The anti-inflammatory effects of cannabinoids are also extended to the inflammatory milieu of tumors, as demonstrated in an in vitro study performed by Maor et al. on Kaposi's sarcoma-associated herpesvirus-infected primary human dermal endothelial cells in an in vitro model for Kaposi sarcoma [175]. The authors reported that CBD demonstrates antiproliferative effects by blocking the expression of growth-regulated oncogenes (GRO-α), as well as of the viral G protein-coupled receptor and vascular endothelial growth factor C and its receptor. GRO-α is a chemokine involved in angiogenesis, tumorigenesis and inflammation, suggesting that the inflammatory milieu plays a role in Kaposi cancer

development, and that CBD may also demonstrate anti-tumoral effects through its antiinflammatory properties [176].
