Cannabidiol (CBD) Prevents Palmitic Acid-Induced Drop in Mitochondrial Membrane Potential

Exposure of macrophages and microglia cells to the saturated palmitic acid (PA) leads to reduction in the mitochondrial membrane potential ( m ∆Ψ ), shrinkage of the cells and apoptosis. Here we show that the Cannabis component Cannabidiol (CBD) rescues both macrophages and microglia cells from the detrimental effects of PA. CBD prevents the shrinkage in cell size and the reduction in m ∆Ψ caused by PA. The protective effect of CBD on the macrophage mitochondria is important for sustaining the macrophage population even under the immunosuppressed conditions caused by this drug. To a similar extent, the antagonistic effect of CBD on PA-mediated microglia cytotoxicity is important for its role in neuroprotection.


Introduction
Cannabidiol (CBD) is known to exert strong anti-inflammatory and anti-nociceptive properties [1]- [6], besides its well-known anti-microbial activities [7]. In addition, CBD has been shown to be neuroprotective and possess anti-oxidative activities [8]. These promiscuous properties have made CBD a broad-spectrum attractive drug for the treatment of a wide range of diseases such as autoimmune diseases, rheumatoid arthritis, multiple sclerosis and diabetes, and for the alleviation of cancer-associated and other disease-associated pain [6] [9]. In addition, How to cite this paper: Gallily, R., Yekhtin, Z., Tarshis [13]. CBD also alters microglial gene expression in response to lipopolysaccharides, with a prominent elevation of Nrf2-regulated genes involved in oxidant defense and redox signaling [14].
Palmitic acid (PA) leads to apoptosis of macrophages in a CD36-dependent manner [15] [16]. It causes apoptosis through generation of ROS by the NADPH oxidase subunit NOX2 [15]. PA also activates microglia cells, leading to their increased secretion of pro-inflammatory cytokines, ROS and NO· production, which ultimately trigger bystanding neuronal death [17].
Here we show that CBD rescues macrophages and microglia cells from the cytotoxic effects of PA. Of particular interest is the antagonistic effect of CBD on the PA-mediated drop in mitochondrial membrane potential.

Cell Culture
RAW 264.7 macrophage cell line and BV-2 microglial cell lines were grown in DMEM supplemented with 10% heat-inactivated FCS, and incubated at 37˚C in a humidified atmosphere containing 5% CO 2 .

Treatments
RAW 264.7 or BV-2 cells were incubated with 75 -150 μM PA in the absence or presence of 1 -5 μg/ml CBD for 24 hrs. 30 minutes before the end of incubation, the cells were exposed to 10 μM Red MitoTracker. Immediately thereafter, the cultures were inspected by confocal microscopy (Nikon A1 HD25). The relative red intensity was calculated by ImageJ measurements divided by number of cells.
Control cells received the respective vehicles.

Statistics
The results are presented as average ± standard error. p-values were calculated from two-tail Student's t-tests. A p-value equal or below 0.05 was considered sta-

Cannabidiol (CBD) Prevents Palmitic Acid (PA)-Induced Drop in Mitochondrial Membrane Potential in Macrophages
Palmitic acid (PA) has previously been shown to induce apoptosis of macrophages [15] [16]. Here we show that a 24 hrs incubation with palmitic acid leads to strong reduction in mitochondrial membrane potential (   showed that macrophages co-treated with CBD and PA had only a 40% ± 5% reduction in MitoTracker staining in comparison to control macrophages ( Figure   1; compare G-H with A-C). Compared to PA-treated macrophages, there was a 3-fold increase in MitoTracker staining in the presence of CBD. CBD also prevented the rounding up of macrophages (Figure 1 and Figure 2), suggesting for a pro-survival effect. The CBD protection was more profound when the macrophages were treated with 75 μM PA (Figure 1), than when treated with 150 μM PA ( Figure 2). Macrophages treated with CBD alone showed similar Mito-Tracker staining as control cells (Figure 3(B) and Figure 4).

CBD Prevents PA-Mediated Cytotoxicity of Microglia Cells
Palmitic acid has previously been shown to reduce the viability of microglia cells [18]. This is also demonstrated here by exposing BV-2 microglia cells to 75 μM PA for 24 hrs resulting in a 55% ± 5% reduction in cell viability ( Figure 5 compare D-F to A-C; and Figure 6). Simultaneous exposure of microglia cells to both 75 μM PA and 5 μg/ml CBD prevented the cytotoxic effect of PA ( Figure 5 compare G-I with D-F; and Figure 6), suggesting for a protective role for CBD.

Discussion
Cannabidiol (CBD) has repeatedly been shown to exert immunosuppressive effects in vivo [1]     caused by PA. A recent study has observed a similar protection of hepatocytes from PA-induced apoptosis by CBD [19]. These authors observed that PA re- duced the mitochondrial membrane potential and increased the mitochondrial reactive oxygen species production in hepatocytes, both effects antagonized by CBD [19]. Also the neuroprotective activity of CBD has been related to its effects on mitochondria [20], where CBD under pathophysiological conditions prevented apoptosis via restoration of Ca 2+ homeostasis. CBD interacts with Ca 2+ TRP channels [6], which might account for this effect. The pro-survival effect of CBD on macrophages observed in the present study is intriguing in light of its ability to suppress various macrophage functions. This characteristic of CBD is important for sustaining the macrophage population even under immunosuppressed conditions. Similarly, the pro-survival effect of CBD on microglia is important for neuroprotection. In both macrophages and microglia cells, there is a correlation between the documented reduction in ROS and NO· production caused by CBD and the preservation of mitochondrial function described in the present study. It is thus likely that the anti-oxidant properties of CBD contribute to the survival of both macrophages and microglia cells.

Conflicts of Interest
The authors declare no conflicts of interest.