In Vitro Model of Neuroinflammation: Efficacy of Cannabigerol, a Non-Psychoactive Cannabinoid

Abstract

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.

Keywords: Cannabis sativa; cannabigerol; neuroinflammation; oxidative stress; phytocannabinoid.

Figure 1

(A) Cell viability in NSC-34 motor neurons exposed to different CBG concentrations. All CBG doses increased cell viability compared to the control (CTR). (B) Cell viability in NSC-34 exposed to the medium of LPS-stimulated macrophages and in cells pre-treated with CBG. The exposure to the cell culture medium of LPS-stimulated macrophages reduced cell viability, but the pre-treatment with CBG partially restored it. The experiments were performed in triplicate. ** p < 0.01 NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 cells pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages; **** p < 0.0001 NSC-34 treated with the medium of LPS stimulated macrophages vs. control.

Figure 2

CBG pre-treatment was able to inhibit apoptosis induced by the medium of LPS-stimulated macrophages in NSC-34 motor neurons. (A) Immunocytochemistry showed that NSC-34 cells treated with the medium of LPS-stimulated macrophages expressed Bax but not Bcl-2. CBG pre-treatment abolished Bax expression and restored those of Bcl-2. (B) The treatment with the medium of LPS-stimulated macrophages induced caspase 3 activation in NSC-34 cells, but CBG pre-treatment reduced its expression. (C) Quantitative analysis of positive staining. The experiments were repeated three times. **** p < 0.0001, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages, NSC-34 treated with the medium of LPS stimulated macrophages vs. control; Scale bar: 50 µm.

Figure 3

CBG pre-treatment was able to reduce the levels of pro-inflammatory cytokines in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. Immunocytochemistry with the quantitative analysis of positive staining showed that the treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of the pro-inflammatory cytokines IL-1β, TNF-α and IFN-γ. CBG pre-treatment reduced the protein levels of the pro-inflammatory cytokines. The immunocytochemical assays were repeated three times. **** p < 0.0001, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages, NSC-34 treated with the medium of LPS stimulated macrophages vs. control; Scale bar: 50 µm.

Figure 4

CBG pre-treatment was able to reduce the levels of PPARγ in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of PPARγ, but CBG pre-treatment reduced its levels. The immunocytochemical assays were repeated three times. **** p < 0.0001, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 cells pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. control; Scale bar: 50 µm.

Figure 5

CBG pre-treatment was able to reduce the levels of oxidative stress markers nitrotyrosine, iNOS and SOD1 in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. The treatment with the medium of LPS-stimulated RAW 264.7 macrophages induced the expression of nitrotyrosine, iNOS and SOD1, but CBG pre-treatment reduced their levels. The immunocytochemical assays were repeated three times. **** p < 0.0001, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 cells pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. control; Scale bar: 50 µm.

Figure 6

CBG pre-treatment was able to restore Nrf-2 expression in NSC-34 cells treated with the medium of LPS-stimulated RAW 264.7 macrophages. Control cells and NSC-34 cells treated with CBG alone expressed Nrf-2. On the contrary, the treatment with the medium of LPS-stimulated RAW 264.7 macrophages abolished its expression, but CBG pre-treatment restored its levels. The immunocytochemical assays were repeated three times. **** p < 0.0001, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. NSC-34 cells pre-treated with CBG and then exposed to the medium of LPS stimulated macrophages, NSC-34 cells treated with the medium of LPS stimulated macrophages vs. control; Scale bar: 50 µm.