Effect of Cannabidiol in LPS-Induced Toxicity in Astrocytes: Possible Role for Cannabinoid Type-1 Receptors

Cerebral metabolic abnormalities are common in neurodegenerative diseases. Previous studies have shown that mitochondrial damage alters ATP production and increases reactive oxygen species (ROS) release which may contribute to neurodegeneration. In the present study, we investigated the neuroprotective effects of cannabidiol (CBD), a non-psychoactive component derived from marijuana (Cannabis sativa L.), on astrocytic bioenergetic balance in a primary cell culture model of lipopolysaccharide (LPS)-induced neurotoxicity. Astrocytic metabolic profiling using an extracellular flux analyzer demonstrated that CBD decreases mitochondrial proton leak, increased spare respiratory capacity and coupling efficiency in LPS-stimulated astrocytes. Simultaneously, CBD increased astrocytic glycolytic capacity and glycolysis reserve in a cannabinoid receptor type 1 (CB1)-dependent manner. CBD-restored metabolic changes were correlated with a significant decrease in the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) concentration and reduction of ROS production in LPS-stimulated astrocytes. These results suggest that CBD may inhibit LPS-induced metabolic impairments and inflammation by enhancing astrocytic metabolic glycolysis versus oxidative phosphorylation through its action on CB1 receptors. The present findings suggest CBD as a potential anti-inflammatory treatment in metabolic pathologies and highlight a possible role for the cannabinoidergic system in the modulation of mitochondrial oxidative stress. CBD enhances mitochondrial bioenergetic profile, attenuates proinflammatory cytokines release, and ROS overproduction of astrocytes stimulated by LPS. These effects are not mediated directly by CB1 receptors, while these receptors seem to have a key role in the anti-inflammatory response of the endocannabinoid system on astrocytes, as their specific inhibition by SR141716A led to increased pro-inflammatory cytokines release and ROS production. The graphical abstract is created with BioRender.com.


Introduction
Cannabidiol (CBD) is a non-psychoactive component belonging to a group of 142 phytocannabinoids derived from Cannabis sativa (Ujváry and Hanuš 2016).It was extracted for the first time from Minnesota Wild Hemp in 1940, and its chemical structure was documented 20 years later (Adams et al. 1940;Mechoulam and Shvo 1963).After its discovery, research on CBD's therapeutic potential was accelerated, highlighting its anti-psychotic, anti-epileptic, anxiolytic, and anti-convulsive effects (Chan and Duncan 2021).Recently, studies have demonstrated the potent antioxidant and antiinflammatory properties of CBD in vitro and in vivo (Rajan et al. 2016;Atalay et al. 2020;Kim et al. 2021a).CBD has neuroprotective properties mediated by its effect on the cannabinoid receptors type 2 (CB2) directly and/or its effect on the release of endocannabinoids such as the anandamide.They can also act through CB1 (Izzo et al. 2009;Campos et al. 2016) and other receptors including serotonin 1A (5-HT1A) (Russo et al. 2005), vanilloid receptor 1 (TRPV1) (Gray et al. 2020), adenosine A2A (ADORA2A) (Ribeiro et al. 2012), and peroxisome proliferator-activated receptorgamma (PPARγ) (Sunda and Arowolo 2020).In addition, it has recently been reported that the antioxidant and antiinflammatory effects of CBD are related to its intracellular bioactivity.For example, it has been shown that CBD treatment enhances redox balance directly by acting on redox system components or indirectly by interacting with the PPARs belonging to the redox system (Atalay et al. 2020).
However, CB1 and CB2 receptors remain the primary site of action for CBD and both are expressed on the astrocytic membrane and its mitochondria (Stella 2004;Athanasiou et al. 2007;Busquets-Garcia et al. 2018).Astrocytes are the most abundant glial cells in the brain with several key roles such as the regulation of neuro-inflammation and neuronal metabolism (Scuderi et al. 2013;Acioglu et al. 2021).Also, they play a critical role in maintaining neuronal energetic homeostasis (Hertz et al. 2018).Astrocytes use glycolysis to produce ATP and pyruvate.They synthesize and export glutathione (GSH) precursors, lactate and ketone bodies, and glutamine (Gln) which are used by neurons to generate energy for neurotransmitters synthesis (Hertz et al. 2018).Therefore, in response to increased neuronal energetic demands, astrocytes upregulate their glycolysis and boost their oxidative metabolic capacity through oxidative phosphorylation (OXPHOS) (Rose et al. 2020).
Recently, CBD's effect on astrocytic metabolism has attracted increasing interest regarding its role in the modulation of astrocytic mitochondrial processes such as the regulation of mitochondria-mediated intrinsic apoptosis (McKallip et al. 2006), mitochondrial ferritin concentrations (McKallip et al. 2006), mitochondrial biogenesis (Hao et al. 2015), electron transport chain activity (Valvassori et al. 2013), and intracellular and mitochondrial calcium influx (Ryan et al. 2009;Olivas-Aguirre et al. 2021).On the other hand, mitochondrial metabolic abnormalities are associated with the development of several neurodegenerative disorders, including Parkinson's and Alzheimer's disease (Monzio Compagnoni et al. 2020).With reference to this critical role of astrocytes in neuronal metabolism, we hypothesized that CBD can modulate astrocytic metabolism and inflammatory response against neurotoxicity, via endocannabinoid system activation.For that, in the present study, we investigated the effect of CBD on metabolic aberrations and inflammation in LPS-stimulated primary astrocytes and the implication of CB1 receptor in this neuroprotective response.

Experimental Approach
Primary astrocytes were isolated from the whole brain homogenates of C57BL mice and were then exposed to bacterial endotoxin lipopolysaccharide (LPS; Escherichia coli O26:B6; no.297-473-0, Sigma-Aldrich, Darmstadt, Germany) to induce inflammation.The ability of CBD ≥ 99% (HPLC) (Tocris Bioscience, UK) to regulate the astrocytic mitochondrial bioenergetic profile, inhibit the release of pro-inflammatory cytokines, and modulate intracellular and mitochondrial reactive oxygen species (ROS) production was evaluated in LPS-stimulated astrocytes.CB1 receptor antagonist (SR141716A or Rimonabant hydrochloride, no.0923, Tocris Bioscience, UK) was used to evaluate whether binding of CBD to CB1 receptor is essential for the restoration of LPS-induced ROS production, metabolic aberration, and inflammation in primary astrocytes.

Animal Ethics
The experiments were performed on female Swiss mice, raised in the central animal care facilities of the Faculty of Sciences, Mohammed V University, Rabat, Morocco, under an ethical clearance no.86/609/EC20.The C57BL mice were housed in Plexiglas cages with wood shavings bedding under controlled environmental conditions (12:12 light/dark cycle, 22 ± 2 °C), with free access to food and water.All animal procedures were conducted following the guidelines of the European Council Directive (EU2010/63).Every effort was made to minimize animal suffering, and the study met ethical standards.

Primary Astrocytes Culture
Astrocytes were isolated from C57BL mice brain (postnatal day 0-2) and cultured following the previously described procedure (Robb et al. 2020;Vlachaki et al. 2017), with slight modifications.Briefly, the harvested whole brains were placed on a 35-mm tissue culture dish placed on ice.Brain tissue was then rinsed twice with serum-free medium (SFM, no.MFCD08705553, Sigma-Aldrich, Darmstadt, Germany) previously warmed in the water bath at 37 °C.The meninges were removed from the brain under a stereomicroscope to avoid contamination of the astrocyte culture with fibroblasts and meningeal cells.The brain tissues were transferred to a new 35-mm dish containing 2-ml SFM.Whole brains were cut into small pieces using a scalpel with a sterile blade and transferred to a new sterile 15-ml conical tube containing 5 mL of SFM and 1-ml 10 × Trypsin/DNase solution to the final total volume of 10 ml.The mixture is then placed in the incubator (37 °C, 5% CO 2 ) for 30 min, under stirring every 10 min.After spinning the mixture at 177 G in an oscillating bucket centrifuge for 5 min, the supernatant was removed and the pellet was re-suspended in 10 ml of astrocyte culture medium (ACM, no.A1261301, Gibco, Carlsbad, CA, USA); it is composed from N-2 Supplement, base medium (DMEM), and OneShot fetal bovine serum (FBS) to support an optimal growth of primary astrocytes, retaining their normal and phenotypes.After 5-7 days, the astrocytes were then obtained by removing the microglial cells that developed on the top layer by gently shaking (180 rpm) on an orbital shaker for 30 min.Cells were cultured in Dulbecco's Modified Eagle's Medium (no.30030, Gibco, Carlsbad, CA, USA) containing 25-mM glucose and supplemented with 10% V/V fetal bovine serum (FBS, no.MFCD00132239, Sigma-Aldrich, Darmstadt, Germany), 200-U/ml penicillin-streptomycin, and 8-mM L-glutamine.Cultures were maintained in a humidified incubator at 37 °C with 5% CO 2 .Before experimentation, cells were seeded on poly-L-lysine (PLL; 4 µg/ml) coated plastic ware at an appropriate density.

Cell Treatments
Primary cell-cultured mouse astrocytes were divided into two groups: (1) control and (2) lipopolysaccharide (0.2 µM) groups.LPS was added to the culture medium (free DMEM containing 2.5-mM glucose) and applied for 24 h.The groups were then subdivided into (1) the treatment groups: control groups (physiological saline 0.9%), Saline + CBD, SR, and SR + CBD and (2) experimental groups: LPS + Saline, LPS + CBD, LPS + SR, and LPS + SR + CBD.For that, the vehicle group was transferred into a new culture medium and divided into cannabidiol treatment group (CBD) at 1 µM and CBD + SR141716A at 1 µM treatment group.In the same way, the LPS treatment group, were also divided into CBD treatment group at 1 µM, SR 141716A at 1 µM, and CBD + SR 141716A at 1 µM.The treatments were applied for 12 h before transferring all groups into the PBS for 5 h.One h before the assay, the PBS culture media were exchanged with Seahorse media to prevent the penetration of proton H + into the PBS buffer medium.LPS and SR 141716A were prepared in sterile physiological saline solution (0.9% NaCl), while CBD was dissolved in DMSO (5%) and diluted with saline solution (0.9% NaCl).
Mice primary astrocytes were randomized to each experimental group (Vehicle/Saline; Vehicle/CBD; Vehicle/SR; Vehicle/SR + CBD; LPS/Saline; LPS/CBD; LPS/SR; LPS/ SR + CBD), and all data were collected and analyzed in a double-blind manner.

Metabolic Analysis
The bioenergetic profile of cultured astrocytes was assessed by measuring the metabolic rate, including oxygen consumption rate (OCR) as a measure of mitochondrial function, and extracellular acidification rate (ECAR) as an indicator of glycolysis, using the Seahorse Metabolic Bioanalyzer XFe24 (Agilent; USA), as per the manufacturer's instructions (Agilent Technologies, 2016 5991-7138EN, USA).
Astrocytes were plated in 24 well Agilent Seahorse Culture plates (no.102353-100; Seahorse Bioscience, Agilent) at 4 × 10 4 cells/well, 24 h before treatment.Wells were supplemented with DMEM (10% v/v FBS, 200-U/ml penicillin-streptomycin, 8-mM L-glutamine, and 7.5-mM glucose).Before the assay and 12 h after treatment, cells were washed with Seahorse XF DMEM medium (pH 7.4; Agilent) supplemented with either L-glutamine (2 mM), glucose (2.5 mM), and sodium pyruvate (2.5 mM) for Mito Stress Test, or L-glutamine 2 mM for the Glycolytic Stress Test.Cells were incubated for 1 h in a CO 2 -free incubator at 37 °C.Glycolytic rate and oxidative metabolism were determined as ECAR and OCR assessed on Glycolytic Stress and Mito Stress Test Tests, respectively.

Glycolytic Stress Test
ECAR measurements were assessed by a glycolytic stress test.Briefly, astrocytes in Seahorse XF DMEM medium (2 mM l-glutamine; pH 7.4; Agilent) were supplemented with glucose (10 mM) as an energetic substrate, then with oligomycin (1 µM; an inhibitor of ATP synthase) to stimulate anaerobic glycolysis, and finally with 2-deoxyglucose (2-DG; 50 mM) to inhibit glycolysis pathway.The glycolytic parameters obtained are glycolytic rate considered the highest ECAR measurement after the supply of glucose; glycolytic capacity considered the maximum ECAR measurement after oligomycin supply; the glycolytic reserve is considered the difference between glycolytic capacity and glycolytic rate.

Mito Stress Test
Mitochondrial stress was evaluated by measuring OCR in cells incubated in Seahorse XF DMEM medium (l-glutamine (2 mM); pH 7.4; Agilent) and supplemented with the following drugs: oligomycin (0.5 µM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone and oxidative phosphorylation uncoupler (FCCP; 1 µM), and rotenone (R) with antimycin A (A), a respiratory complex III inhibitor (R/A; 0.5 µM).Mitochondrial respiratory parameters were calculated and evaluated according to the manufacturer's recommendations (Divakaruni et al. 2014).Briefly, basal respiration was calculated as the difference in OCR measurement before oligomycin addition minus the lowest measurement after R/A addition; ATP production was calculated as the difference between basal respiration and the lowest OCR measurement after oligomycin addition; proton leak was calculated as the difference between the lowest OCR measurement after the addition of oligomycin and the lowest OCR measurement after the addition of R/A; maximal respiration was calculated as the maximum OCR measurement after FCCP addition, minus the lowest OCR measurement after R/A addition; spare capacity was calculated as the difference between basal respiration and maximal respiration, and coupling efficiency was expressed as OCR difference between the ATP production rate and basal respiratory rate.

Measuring Cytokine Concentration by Enzyme-Linked Immunosorbent Assay
Culture supernatants from astrocytes of control groups (vehicle) and treated groups with medium-plus CBD, mediumplus LPS at 0.2 µg/mL, or CBD-stimulated group plus SR141716A were collected.Concentration levels of TNFα and IL-6 were determined by ELISA kits (no.ab100747, ab222503 respectively, Abcam, US) following the manufacturer's instruction.

Quantification of Cellular Reactive Oxygen Species
The total intracellular superoxide and hydroxyl radical in cells were detected using Cellular Reactive Oxygen Species Detection Assay Kit (no ab186027, Abcam, US).Control group and experimental group cells were seeded into a 96 well plate at 2 × 10 4 cells per well and incubated for 60 min in redwork solution at 37 °C.The fluorescence signal was examined at Ex/Em = 520/605 nm using a Flexstation II microplate reader.

Statistical Analysis
All analyses were carried out in 4 or 5 biological replicates with three technical replicates.GraphPad 8.0 was used for statistical analysis.The normality of data was determined using the Shapiro-Wilk normality of distribution test.A parametric or non-parametric two-way ANOVA was conducted on data, depending on their distribution.For parametric data, Bonferroni's multiple comparisons were assessed when there was significance.For non-parametric ANOVAs, ranked transformation of data was performed.Subsequently, continuous variables were tested with the Kruskal-Wallis test.In addition, homogeneity of variance across all groups was assessed by Bartlett's test.The level of significance was set at p < 0.05.
The sample size was determined based on literature (Sun et al. 2017) and a pilot study of 12 mice (Robb et al. 2020).This study revealed that a sample size of 4 mice provided 80% power (at a 2-sided alpha level of 5%) to detect at least 25% relative changes between pre-and post-treatment using a paired t-test and assuming a normal distribution.All independent experiments were replicated at least three times.

MTT Test for Toxicity and Dose Selection
The cytotoxicity evaluation of LPS, CBD, and SR141716A and their combined effects on primary astrocytes was conducted using the MTT assay.Primary astrocyte cells were exposed to varying concentrations of LPS (ranging from 0.01 to 1 µM), CBD (ranging from 0.1 to 5 µM), SR141716A (ranging from 0.5 to 2 µM), and a combination of SR141716A and CBD at 1 µM, over a 24-h period.

CBD Enhances Mitochondrial Bioenergetics in LPS-Treated Astrocytes
We utilized the XFe24 Seahorse analyzer to investigate whether CB1 receptors regulate the LPS-induced impact on astrocytic bioenergetic status.The assessment of the bioenergetic profile involved measuring the oxygen consumption rate (OCR) (see Fig. 2A).

CBD Regulates Glucose Metabolism in LPS-Stimulated Astrocytes
We employed the XFe24 Seahorse analyzer to assess extracellular acidification rate (ECAR), investigating the impact of CBD on glucose metabolism while also considering ATP production through the Krebs cycle.An analysis of the glycolysis stress test data using a two-way ANOVA yielded notable findings.There was a significant interaction observed between CBD and LPS treatments regarding glycolytic capacity (Fig. 3C, F (2, 17) = 7.527, p = 0.0046) and glycolytic reserve (Fig. 3D, F (2, 17) = 4.588, p = 0.0255).However, concerning the glycolysis rate (Fig. 3B, F (2, 17) = 1.623, p = 0.2265), independent effects were observed for both CBD and LPS supplemented astrocytes.

CBD Decreases Concentration Levels of TNFα and IL-6 Cytokines in LPS-Treated Astrocytes
To evaluate the anti-inflammatory propriety of CBD on LPS-stimulated primary astrocytes and to determine if the anti-inflammatory effect is associated with CB1 receptor activation, concentration levels of TNFα and IL-6 were measured in the control group after LPS (0.2 µM), CBD, SR141716A, CBD + LPS, SR + LPS, and SR + CBD + LPS treatments (Fig. 4).Two-way ANOVA analysis demonstrated a significant effect of CBD (F (3, 24) = 15.71,p < 0.0001) on LPS-induced increase in the release of TNFα and IL-6 by astrocytes without significant interaction (F (3, 24) = 1.146, p = 0.3506).Bonferroni's post-hoc analysis revealed that 0.2 µM of LPS significantly increased the concentration of TNFα and IL-6 when compared to the control group (p = 0.0015 and p = 0.0147, respectively), and the CBD treatment of LPSstimulated astrocytes significantly decreased TNFα and IL-6 concentrations compared to RS + LPS group (p = 0.0059 and p = 0.0015, respectively).CBD significantly reduced IL-6 compared to LPS-stimulated astrocytes (p = 0.0028) (Fig. 4).These results suggest that CB1 receptors might be implicated in cytokine regulation as CB1-receptor blocked with SR141716A significantly increased TNFα and IL-6 production.Moreover, our results suggest that the CBD effect on LPS-induced overproduction of cytokines may not be mediated directly by astroglial CB1 receptors activation, as there was no significant difference between CBD: LPS group Fig. 2 CBD's effect against LPS-induced impairments on astrocytic mitochondrial respiratory parameters depends on the inhibition of CB1 receptors by SR141716A.The oxygen consumption rate (OCR) parameters were measured by using selective inhibitors, which were injected during the assay, oligomycin (0.5 µM), carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone (FCCP, 1 µM), rotenone (0.5 µM), and antimycin A (0.5 µM) as depicted in the representative oxygen consumption rate (OCR) tracing (A).The different OCR parameters are non-mitochondrial oxygen consumption (B), basal respiration (C), maximal respiration (D), ATP-linked respiration (E), proton leak-linked respiration (F), spare respiratory capacity/reserve capacity (G), and coupling efficiency (H).Data are presented as the mean of 5 independent biological experiments with three technical replicates and expressed as means ± SD and median ± interquartile for proton leak-linked respiration, spare respiratory capacity, and coupling efficiency parameters.Significance was assessed by Bonferroni post-hoc test when there were significant interactions between groups; *p < 0.05 compared to the control group (Vehicle: Saline) and from # p < 0.05 compared to LPS:SR + CBD group and CBD: LPS incubated with SR141716A (p = 0.5314 and p = 0.9815, respectively for TNFα and IL-6 concentrations).

CBD Reduces LPS-Induced Oxidative Stress in Cultured Primary Astrocytes
CBD's effect on LPS-induced oxidative stress has been explored by measuring the levels of total intracellular superoxide and hydroxyl radical in astrocytes (Fig. 5).Twoway ANOVA analysis demonstrated a significant interaction between CBD and LPS treatments (F (3,24) = 4.179; p = 0.0163).Dunn's multiple comparisons test revealed that LPS-stimulated astrocytes have significantly increased the total intracellular ROS concentration when compared to the control group (p = 0.0003), CBD-treated astrocytes (p < 0.0006), and SR + CBD treated group (p < 0.0024).But the treatment with CBD significantly reduced ROS concentration (p = 0.0011).However, the effect of CBD on ROS levels in LPS-stimulated astrocytes did not change following co-treatment with SR141716A (p = 0.0143), (Fig. 5).This result suggests that the CBD effect is not mediated directly by astroglial CB1 receptors.

Discussion
This study aimed to determine (1) how LPS affects the mitochondrial metabolism of astrocytes and (2) CBD's effect on metabolic activity and pro-inflammatory cytokine release in LPS-treated astrocytes.Our findings show that CBD can modulate the bioenergetic profile of primary astrocytes previously stimulated by LPS, which is an inflammatory-inducing bacterial membrane protein.The CBD-induced bioenergetic modulation consisted of modifying both; oxidative phosphorylation parameters, such as mitochondrial membrane proton leak and mitochondrial spare respiratory capacity and glycolytic parameters, such as glycolytic capacity and astrocytic glycolytic reserve.The metabolic change induced by CBD was correlated to changes in pro-inflammatory cytokines TNFα and IL-6 concentrations.CB1 receptor blocker SR141716A did not impair the anti-inflammatory effect of CBD suggesting that the CBD anti-inflammatory effects may not be mediated by CB1 receptors.Our findings have also shown that CBD reduces ROS production in astrocytes stimulated by LPS.Moreover, SR141716A did not attenuate CBD's effect on ROS production, suggesting that

D) E) F)
Fig. 3 CBD effect on astrocytes glycolytic parameters depends on inhibition of CB1 receptors by SR141716A.The different ECAR parameters were measured by using selective inhibitors, which were injected during the assay, glucose (10 mM), oligomycin (1 µM), and 2-deoxy-glucose (50 mM) as depicted in the representative extracellular acidification rate (ECAR) tracing (A).Glycolysis (B), glycolytic capacity (C), glycolytic reserve (D), glycolytic reserve as % (E), non-glycolytic acidification rate (F) measured using Seahorse Glycolysis Stress Test kit.Data are presented as the mean of 4 independent biological experiments with three replicates and expressed as means ± SD and median ± interquartile for glycolytic reserve as % and non-glycolytic acidification rate parameters.Significance was assessed by Bonferroni post-hoc test when there were significant interactions between groups; from *p < 0.05 compared to all groups, $ p < 0.05 compared to Vehicle:CBD group, and # p < 0.05 compared to Vehicle:RS + CBD group CBD-induced ROS reduction in LPS-stimulated astrocytes may not be directly mediated by CB1 receptors.Furthermore, our results suggest that CB1 receptors are implicated in the anti-inflammatory response and the regulation of ROS production by LPS-stimulated astrocytes; as CB1 blockade by SR141716A exacerbated the inflammatory response in LPS-stimulated astrocytes by increasing TNFα and IL-6 levels, as well as ROS production.LPS-stimulated astrocytes demonstrated a significant decrease in basal mitochondrial respiration that reflects the proportion of OCR used by cells to meet their endogenous ATP demand (Divakaruni et al. 2014).This decrease could be related to a change in the electron chain transport (ECT) activity, in particular, in ECT complex I, the site of NADH dehydrogenase and source of reactive nitrogen (RNS) and reactive oxygen species (ROS) release.These oxidative forms are produced after TLR4 pattern recognition receptor activation by LPS (Lopez-Fabuel et al. 2016) and the activation of their underlying pathways such as mitogen-activated protein kinase (MAPK), myeloid differentiation protein 88-nuclear factor-κB (Myd88-NFκB), and Janus kinases/ signal transducers and activators of transcription (JAK1/ STAT1) (Gorina et al. 2011).Consequently, triggering the generation of ROS and RNS from NADPH oxidase (NOX) Fig. 4 CBD reduces cytokine release on primary astrocytes stimulated with LPS.Concentration levels of pro-inflammatory cytokines, TNFα, and IL-6 were measured in pg/ml by ELISA.Data represented as means ± SD were derived from four separate biological experiments.Data are analyzed via Bonferroni post-hoc, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared to all LPS: saline group; @ p < 0.05, @@ p < 0.01, @@@ p < 0.001, and @@@@ p < 0.0001 compared to RS:LPS group; $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, and $$$$ p < 0.0001 compared to RS + CBD:LPS group; and ## p < 0.01 vs CBD:LPS group @@@@ *** * ** @@@@ @@@ @@@ @@@@ @ Fig. 5 CBD inhibited over production of the total intracellular superoxide and hydroxyl radical (ROS) by LPS-stimulated astrocytes.ROS generation is represented as relative concentration to the control group (Vehicle:Saline).Data expressed as median ± interquartile were derived from four separate biological experiments.Data are analyzed via Dunn's multiple comparisons test; *p < 0.05, **p < 0.01, and ***p < 0.001 vs Saline:LPS group; @@ p < 0.01, @@@ p < 0.001, and @@@@ p < 0.0001 compared to RS:LPS group and nitric oxide synthase (iNOS), respectively (Takahashi 2021), both ROS and RNS inhibit ECT and decrease mitochondrial membrane potential (O-Uchi et al. 2014).Recently, it was reported that LPS-stimulated astrocytes activate TLR4 and induce significant production of ROS without RNS production.In astrocytes, the stimulation of TLR4 by LPS induces the activation of the MAPK phosphorylating pathway, underlying ROS release, and subsequent enhancement of PPP flux, as has been suggested by Liyzumi et al. and Takahashi (Iizumi et al. 2016;Takahashi 2021).
On another hand, we suggest that CBD metabolic effects seem to depend on the inhibition of CB1 receptors by SR141716A, where it significantly decreases proton leak, increases spare respiratory capacity, and enhances coupling efficiency in LPS-stimulated astrocytes.In this respect, it was reported that SR141716A significantly decreases CB1 receptor expression and promotes peroxisome proliferatoractivated receptor-γ (PPARγ) activation which has a key role in astrocytic metabolism by upregulating glucose, glutamate uptake, and lactate production and release (Wang et al. 2015;Iglesias et al. 2017).These findings support the role of PPARγ activation through CB1 receptors inhibition in CBD anti-inflammatory effects.Moreover, our data indicate that CBD at 1 µM prevents LPS-induced mitochondrial damage only when administered in combination with SR141716A.Knowing that SR141716A alone does not affect astrocytic metabolism, it suggests that CBD-induced metabolic effect could be potentiated by negative allosteric modulation of the CB1 receptors as has been suggested by Laprairie et al. (2015).In line with our findings, Sun et al. (2017) confirmed CBD's effect on mitochondrial bioenergetics in HT22 cells (neuronal cell line) demonstrating that CBD at a dose of 5 µM enhances basal respiration, ATP production-linked oxygen consumption, maximal respiration, and the spare respiratory capacity (Sun et al. 2017).
Lastly, we investigated CB1 receptor inhibition on astrocytic glucose metabolism by measuring the glycolytic flux.Similar to respiratory mitochondrial data, the glycolytic analysis revealed that the glycolytic capacity and glycolytic reserve were increased only following co-administration of CBD with SR141716A while CBD alone had no effect.
Taken together, our data suggest that astrocyte co-treatment with CBD and RS may provide a significant enhancement to both glycolytic capacity and glycolytic reserve under inflammatory conditions induced by LPS-stimulation.It has been shown that CBD effect on astroglial CB1 receptor located on plasma membrane may prevent mitochondrial CB1 receptors (mtCB1R) activation needed for controlling lactate release and its subsequent related neuronal bioenergetic stress (Jimenez-Blasco et al. 2020).It is well-known that glucose conversion to lactate is critical in the maintenance of neuronal homeostasis (Staricha et al. 2020).Thus, under noninflammatory conditions, astrocytes rely more on glycolysis than oxidative phosphorylation to generate energy and to produce lactate needed by neurons through a mechanism that implicates the endocannabinoid system.Several studies show that CBD has a potent role in mitochondrial chain respiratory regulation and glucose metabolism by enhancing glucose consumption and lactate release (Singh et al. 2015;Sun et al. 2017).However, our investigation outcomes suggest that CBD markedly enhances mitochondrial bioenergetics and promotes protectiveness against LPS-induced inflammation only when applied in combination with CB1 receptor inhibitors.
Besides astrocytes major role in maintaining neuronal energy balance, astrocytes are involved in sustained inflammation in response to infections or diseases (Khandelwal et al. 2011;Klegeris 2021).It has been shown that astrocytic stimulation by LPS induces a significant increase in proinflammatory markers such as TNFα and IL-6 and increases cells' oxidative phosphorylation without affecting their glycolytic metabolism (Krasowska-Zoladek et al. 2007, Jiang et al. 2009;Zamanian et al. 2012).Therefore, we investigated if CBD treatment may prevent the release of these cytokines TNFα and IL-6.Our obtained results showed that CBD at a dose of 1 µM significantly decreases TNFα and IL-6 release following astrocytic LPS-stimulation (0.2 µM).This result partially corroborates the findings of a recent study showing that CBD indeed attenuates IL-6 release in astrocytes without affecting TNFα release (Wu et al. 2021).This discrepancy might be explained, first, by differences in the experimental approaches, as in our study, astrocytes were incubated with LPS (0.2 µg/ml) overnight however in their protocol, astrocytes were stimulated with LPS (0.1 µg/ml) only for 2 h.Secondly, CBD was applied at a dose of 1 µM, however, in Wu et al.'s study, CBD was applied at a dose of 5 µM.Knowing that CBD has biphasic proprieties may also explain its lack of effect on TNFα release (Wu et al. 2021).Moreover, this difference could be related to the developmental state of the cells (Mecha et al. 2012).CBD's effect on LPS-induced TNFα and IL-6 release were not conditioned by CB1 receptor inhibition and by virtue of its effect on metabolism.Thus, CBD's effect on IL-6 and TNFα release following LPS stimulation could be mediated by other cannabinoidergic pathways.This hypothesis is supported by other studies showing that CBD's anti-inflammatory effect may involve neither CB1 nor CB2 receptors.In contrast, CBD may act through other pathways such as Wnt3 and NF-κB to inhibit astrocytic cytokine release (Kozela et al. 2010;Esposito et al. 2011;Vallée et al. 2017;Cardinal von Widdern et al. 2020;Wu et al. 2021).However, more studies are needed to confirm these hypotheses.On another hand, regarding CBD well-known neuroprotective properties associated with its potent antioxidant capacity (Kim et al. 2021b), our results showed that CBD has the potency to reduce LPS-induced over-production of intracellular ROS in primary astrocytes.This effect is not mediated directly by CB1 receptors activation, as CB1 blockade by SR141716A not counteracted CBD's effect on ROS release.These results were in line with previous studies demonstrating a similar effect of CBD in LPS-induced oxidative stress in glial cell models.Sonego et al. (2018) observed that CBD (10 µM) attenuates intracellular ROS release in primary murine microglia activated by LPS (10 ng/mL).These studies also suggested that CBD-induced attenuation of ROS release might be mediated by its inhibitory effect on the NF-κB signaling pathway.It attenuates NF-κB phosphorylating transcription factor subunit p65 in LPS-stimulated glial cells (Cardinal von Widdern et al. 2020).Also, CBD may inhibit ROS release in LPS-stimulated cells by limiting NADPH production and glucose uptake regarding the fact that glucose is an essential element in the regeneration of NADPH used by NADPH oxidase (NOX) to catalyze ROS production (Kim et al. 2021a, b).Yet, the exact mechanisms by which CBD inhibits the over-production of intercellular ROS induced by LPS remain unresolved.
Furthermore, our study revealed that the application of SR141716A exacerbates TNFα and IL-6 release and ROS production by LPS-stimulated astrocytes.This result was in line with findings from a recent study investigating the role of CB1 receptors on the regulation of microglia/macrophage polarization (Lou et al. 2018).This study revealed that the inhibition of the CB1 receptors by SR141716A up-regulates pro-inflammatory cytokines, including TNFα and IL-6, and increases nitric oxide (NO) release by microglia/macrophages.Also, this study suggested that the SR141716A effect is related to the TLR-4 and NF-κB/p65 overexpression on microglia/macrophages (Lou et al. 2018).Thus, a next study on the evaluation of NFκB expression in astrocytes could indicate a possible mechanism for CB1.
Our study has provided a new finding alongside the growing evidence that confirms the role of the cannabinoidergic system in the regulation of astrocytes-mediated neuronal circuits homeostasis maintaining and metabolic support (Covelo et al. 2021;Iannotti et al. 2016;Navarrete and Araque 2010).Two lines of data supporting this, first, CB1 receptors expressed in the astrocytic membrane are potential modulators of neurotransmitter reuptake mechanisms by affecting both neurotransmitters and gliotransmitters release (Djeungoue-Petga and Hebert-Chatelain 2017; Kovács et al. 2017;Martinez Ramirez et al. 2022).Secondly, CB1 receptors expressed in the mitochondrial membrane of astrocytes have a noticeable effect on mitochondrial performance by decreasing ROS release (Urcaregui 2021;Robledo-Menendez et al. 2022).Hence, that opens up novel strategies for exploring interventional therapy for neurodegenerative disorders through modulating astrocytes functions.
Among limitations of our study is that we don not explore yet the underlining mechanism.Thus, a study on CB1 knockout mice by using a transgenic model is needed to confirm our hypotheses and greatly extend our knowledge of the molecular mechanisms underlying CBD effects much beyond what has been determined from the CB1 receptor-blocked model by SR141716A.In the current study, CBD effects were evaluated on metabolic and inflammatory parameters in LPS-stimulated primary astrocytes.Therefore, our findings highlight that LPSinduced pro-inflammatory cytokines release is mediated by CB1, and this is the crucial point of our study.In this respect, we will conduct a future study to perform more specific experiments related to astrocytic inflammatory response to show how endocannabinoid system could regulate this response.

Conclusion and Perspectives
In summary, our results suggest that CBD exerts a potent neuroprotective effect against LPS-stimulation in primary astrocytes by enhancing mitochondrial bioenergetic profile, attenuating pro-inflammatory cytokines release, and inhibiting LPS-induced ROS release.We suggest that these beneficial effects of CBD in LPS-induced toxicity in astrocytic cells are not mediated directly by CB1 receptors, while these receptors seem to have a key role in the anti-inflammatory response of the endocannabinoid system on astrocytes, as their specific inhibition by SR141716A led to increased proinflammatory cytokines release and ROS production.The current study investigated specific metabolic and inflammation parameters, therefore, more studies using transgenic models of CB1 receptors are needed to confirm our hypotheses on the molecular pathways.We suggest that CBDinduced bioenergetic regulation and anti-inflammation could be related to its activation of the pentose-phosphate pathway (PPP), a major branch of glycolysis that maintains redox balance and energy conservation under oxidative stress conditions.However, more in-depth works are required to investigate the potential therapeutic effect of CBD and the cell signaling pathways underlying its effect.