Inflammation and oxidative stress play main roles in neurodegeneration. Interestingly, different natural compounds may be able to exert neuroprotective actions against inflammation and oxidative stress, protecting from neuronal cell loss. Among these natural sources, Cannabis sativa represents a reservoir of compounds exerting beneficial properties, including cannabigerol (CBG), whose antioxidant properties have already been demonstrated in macrophages. Here, we aimed to evaluate the ability of CBG to protect NSC-34 motor neurons against the toxicity induced from the medium of LPS-stimulated RAW 264.7 macrophages. Using MTT assay, we observed that CBG pre-treatment was able to reduce the loss of cell viability induced by the medium of LPS-stimulated macrophages in NSC-34 cells. Indeed, CBG pre-treatment inhibited apoptosis, as shown by the reduction of caspase 3 activation and Bax expression, while Bcl-2 levels increased. Furthermore, CBG pre-treatment counteracted not only inflammation, as demonstrated by the reduction of IL-1β, TNF-α, IFN-γ and PPARγ protein levels assessed by immunocytochemistry, but also oxidative stress in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7. Indeed, immunocytochemistry showed that CBG pre-treatment reduced nitrotyrosine, SOD1 and iNOS protein levels and restored Nrf-2 levels. All together, these results indicated the neuroprotective effects of CBG, that may be a potential treatment against neuroinflammation and oxidative stress.
Since ancient times, Cannabis sativa has been known for its medicinal and psychotropic effects. This plant was discovered to be a reservoir of compounds exerting beneficial properties. Until now about 120 cannabinoids have been isolated from Cannabis sativa, including Δ9-tetrahydrocannabinol (Δ9-THC), responsible for the psychotropic effect associated with Cannabis consumption, cannabidiol (CBD) and cannabigerol (CBG) . Other than their psychotropic effects, cannabinoids showed anti-oxidant and anti-inflammatory properties leading to neuroprotection [2,3,4].
Among cannabinoids, CBD is one of the most studied, and its protective effects on different aspects of human health, including in neurodegenerative disorders, are well known [5,6]. On the contrary, our knowledge about CBG, another non-psychoactive phytocannabinoid, is limited, even if the few studies published on CBG showed promising results and encourage the deepening of its effects on human health. We have already demonstrated the CBG antioxidant properties in RAW 264.7 macrophages stimulated with hydrogen peroxide (H2O2) . Also anti-inflammatory and neuroprotective effects were reported for CBG and its derivatives in vitro and in vivo in neurodegenerative disease models [8,9,10,11,12].
The anti-oxidant and anti-inflammatory actions of CBG are particularly interesting taking into account that both inflammation and oxidative stress play pivotal roles in neurodegeneration [13,14,15]. Indeed, both these processes lead to neuronal cell death, then triggering and amplifying degeneration [16,17]. It is important to consider that the two pathophysiological processes are tightly correlated and influence each other. It is more relevant for the brain that is particularly sensitive to oxidative stress . Indeed, inflammatory cells can produce reactive species at the site of inflammation causing oxidative stress, while reactive oxygen/nitrogen species may initiate an intracellular signaling cascade that induces the expression of pro-inflammatory genes [15,19]. Given the interdependence of these processes, a compound able to act against both inflammation and oxidative stress may be a promising strategy in the treatment of neurodegenerative disorders.
Different cell types participate in the inflammatory process, including macrophages. They act in order to maintain homeostasis and are directly involved in neuroinflammation, when also circulating monocytes are recruited from the periphery and enter into the central nervous system, contributing to the inflammatory process. For these reasons these cells play a pivotal role in central nervous system diseases, such as autoimmune and neurodegenerative diseases .
We have already shown the anti-oxidant capacity of CBG in macrophages and in this work, we aimed to deepen our knowledge on CBG properties analyzing its beneficial effects in an in vitro model of neuroinflammation. Specifically, we evaluated whether CBG was able to counteract the toxicity induced in NSC-34 motor neurons by the cell culture medium of (lipopolysaccharide) LPS-stimulated RAW 264.7 macrophages, focusing our attention on the evaluation of CBG anti-inflammatory and anti-oxidant capacities.
2.1. CBG Increased Cell Viability in NSC-34 Motor Neurons
In order to evaluate the effects on cell viability of different concentrations of CBG, NSC-34 motor neurons were incubated 24h with the following CBG doses: 1, 2.5, 5, 7.5, 10, 12.5, 15 and 20 µM. We observed with all doses an increase in cell viability compared to the control, even if the difference was not statistically significant. In particular, for doses from 2.5 to 7.5 µM we observed about a 20% increase in cell viability compared to the control. For higher concentrations, cell proliferation decreased even if it was still higher compared to the control (Figure 1A). We included in our analysis also NSC-34 cells incubated with similar concentrations of dimethyl sulfoxide (DMSO) and no cytotoxicity was observed. On the bases of these results, we decided to perform the other experiments with CBG 7.5 µM, because with this concentration we observed the highest increase in cell viability, even if not significant.