*3.5. TRP Receptors*

It has been shown that CBD can also a ffect redox balance and inflammation by modulating mammalian transient receptor potential (TRP) channels [72,73]. CBD activates vanilloid receptors (TRPV), directly or indirectly, by increasing the level of endogenous AEA, which is one of the endogenous TRPV1 agonists [64]. CBD, as a TRPV1 receptor agonist, binds to it and causes desensitization, leading to "paradoxical analgesic activity" similar to that of capsaicin [26]. It has been suggested that there is a relationship between molecular signaling of TRPV1 and oxidative stress [74] because ROS and lipid peroxidation products can regulate the physiological activity of TRPV1 by oxidizing its thiol groups [75]. Consequently, CBD not only activates TRP through a direct agonist-receptor interaction, but also by lowering the level of oxidative stress. In addition, CBD activates other vanilloid receptors such as TRPV2 and the potential ankarin protein 1 receptor subtype (TRPA1), while antagonizing the TRP-8 receptor (TRPM8) [72]. CBD has also been shown to stimulate calcium ions in transfected

HEK-293 cells via TRPV3 [76] and regulate calcium ion homeostasis in immune and inflammatory cells mainly via TRP channels, which is important for proliferation and pro-inflammatory related cytokine secretion [77]. In addition, Ca2+ ions control the activation of several transcription factors (e.g., NFAT) that regulate the expression of various cytokines, such as IL-2, IL-4 and IFNγ, which a ffect cellular inflammatory responses [78].

Regardless of the direct e ffect of CBD on TRP receptors, increasing the level of AEA, as a full TRPV1 agonist, also a ffects the activation of TRP receptors and negatively regulates the 2-arachidonoylglycerol (2-AG) metabolism [79]. It has been shown that both AEA and 2-AG can be synthesized in the plasma membrane. However, the degradation of phosphatidylinositol by phospholipase C results in the formation of a diacylglycerol precursor, whose hydrolysis (through diacylglycerol lipase activity, DAGL) allows the formation of 2-AG [80]. However, activation of DAGL α and DAGLβ requires GSH. Additionally, these enzymes are sensitive to Ca2+ ions [81]. TRPV1 agonists, such as capsaicin and AEA, have been shown to inhibit 2-AG synthesis in striatal neurons of C57BL/6 mice by glutathione-dependent pathways, since DAGL is stimulated by GSH [82]. In addition, the interaction between AEA and 2-AG has been shown to disappear after inactivation of TRPV1 channels. This suggests that the negative effect of AEA on 2-AG metabolism can be mimicked by stimulation of TRPV1 channels. Therefore, AEA and 2-AG interactions require redox balance, due to the participation of GSH in 2-AG synthesis. In summary, CBD modifies TRPV1 receptor activation through reducing oxidative stress as well as biosynthesis of 2-AG.

## *3.6. PPAR*γ *Receptor*

CBD is an agonist of the PPARγ receptor, which is a member of the nuclear receptor superfamily of ligand-inducible transcription factors [52]. PPARγ, an ubiquitin E3 ligase, has been shown to interact directly with NFκB. The interaction occurs between the ligand-binding domain of PPARγ and the Rel homology domain region of the p65 subunit of NFκB. Lys48-linked polyubiquitin of the ligand-binding domain of PPARγ is responsible for proteosomal degradation of p65 [83]. In this way, PPARγ participates in the modulation of inflammation by inducing ubiquitination proteosomal degradation of p65, which causes inhibition of pro-inflammatory gene expression, such as cyclooxygenase (COX2) and some pro-inflammatory mediators such as TNFα, IL-1β, and IL-6, as well as inhibition of NFκB-mediated inflammatory signaling [84]. For this reason, PPARγ agonists can play an anti-inflammatory role by inhibiting the NFκB-mediated transcription of downstream genes [84]. This molecular mechanism is mediated by β-catenin and glycogen synthase kinase 3 beta (GSK-3β). β-catenin attenuates transcription of pro-inflammatory genes by inhibiting NFκB [85,86]. On the other hand, GSK-3β is decreased by PPARγ stimulation [87].

PPARγ cooperates also with another transcription factor, Nrf2, which controls the expression of genes encoding cytoprotective proteins, particularly antioxidant proteins [28,88]. PPARγ may bind to specific elements in the promoter region of genes it regulates, including Nrf2, catalase (CAT), glutathione *S*-transferase (GST), heme-oxygenase-1 (HO-1), and manganese-dependent superoxide dismutase (Mn-SOD). In contrast, Nrf2 can regulate PPARγ expression by binding to the PPARγ promoter in the sequence of antioxidant response elements (ARE) that are located in the -784/-764 and -916 regions of the PPARγ promoter [89,90]. The reduction in PPARγ expression in Nrf2 knockout mice provides confirmation of this regulation [91].

Acting through the PPARγ receptor, CBD demonstrates anti-inflammatory and antioxidant properties. In addition, direct CBD activity is enhanced by the action of AEA and 2-AG, which are also PPARγ agonists and whose levels are elevated by CBD [92]. It has been found that stimulation of PPAR α and reduction of oxidative stress by CBD prevents amyloid β-induced neuronal death by increasing the levels of Wnt/β-catenin [84]. However, there are no data on the interaction between CBD and other PPAR subtypes (PPAR α, β, δ). It is known that the endocannabinoids AEA (whose biosynthesis is stimulated by CBD) and 2-AG can activate PPARγ [92]. AEA activates PPAR α, while the 2-AG derivative 15-hydroxyyeicosatetraenoic acid glyceryl ester increases the transcriptional activity of PPARα [92]. In summary, CBD demonstrates anti-inflammatory activity and antioxidant effects by activating PPARs, either directly or indirectly.

## *3.7. GPR Receptors*

GPR55, which is strongly expressed in the nervous and immune systems as well as in other tissues, is a G-protein coupled receptor [93]. Activation of GPR55 increases the intracellular level of calcium ions [94]. CBD is a GPR55 antagonist and can modulate neuronal Ca2+ levels depending on the excitability of cells [95]. CBD antagonism is manifested as an anticonvulsant effect [96]. Because CBD increases endocannabinoid expression, it can also indirectly affect inflammation and redox balance via these molecules [58]. In addition, GPR55 knockout mice have been shown to have high levels of anti-inflammatory interleukins (IL-4, IL-10, and IFN-γ) [97], while high expression of GPR55 reduces ROS production [98]. Therefore, the organism's response to CBD depends on whether direct or indirect effects dominate.

CBD has also been shown to be an inverse agonist of other GPR receptors, including GPR3, GPR6 and GPR12. It reduces β-arestinin 2 levels and cAMP accumulation in amyloid plaque formation in the development of Alzheimer's disease, in a concentration-dependent manner [98]. In addition, one of the neuropharmacological effects of CBD is its reducing effect on hippocampal synaptosomes mediated by its interaction with GPR3 [99]. It has also been suggested that the effect of CBD on these orphan receptors represents a new therapeutic approach in diseases such as Alzheimer's disease, Parkinson's disease, cancer, and infertility [100].
