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Introduction
Neuroprotection is the prevention of neuronal cell death by intervening and inhibiting the pathogenetic process that causes neuronal dysfunction and death1. Compounds with this activity have the potential to treat neurodegenerative diseases. Although some therapies, such as pharmaceutical, herbal agents, surgical methods, exercise, ultrasound, and photobiomodulation2, have been approved for managing and caring for these diseases, until now there is no cure.
Compounds like bilobalide and the current medicinal drugs valproic acid, furosemide, and metformin have shown neuroprotective effects in some studies, so they have the potential to be pharmacologically repositioned for other therapeutic use. On the other hand, cannabidiol (CBD), the main non-psychoactive phytocannabinoid of marijuana, has been proposed as an alternative for the treatment of several neuropsychiatric and neurological disorders3. This molecule has shown a neuroprotective effect through several mechanisms such as anti-inflammatory, immunomodulatory, decreasing inflammatory cytokines.4
The search for neuroprotective compounds is needed considering that there is a lack of medicinal drugs that limit neuronal damage; the potential neuroprotection of CBD; and the increased use of non-prescribed CBD products for treating different conditions. The objective of this study was to evaluate the neuroprotective activity of CBD individually and mixed with valproate, furosemide, bilobalide, and metformin in an in vitro model of neurotoxicity.
Materials and methods
CBD was purchased from Spex CertiPrep (Metuchen, NJ, USA), valproic acid, bilobalide, metformin, bilobalide, Trolox, glutamate, DCFDA, xanthine oxidase, 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), dimethyl sulfoxide, MTT, H2O2 from Sigma-Aldrich, (St Louis, MO, USA). Furosemide from PiSA Laboratories (Santa Catarina, Mexico), RPMI-1640 medium, and kit EnzChek® Caspase-3 assay Kit #2 from Thermo Fisher Scientific. The PC12 cell line is part of the UANL molecular pharmacology and biological models laboratory catalog.
Cell culture
The PC12 cell was grown in RPMI-1640 medium, 20% fetal bovine serum, and 1% antibiotics (5 mg/mL penicillin, 5 mg/mL streptomycin). They were cultured in 25 cm2 tissue-culture flasks at 37°C, 5% CO2, and humidified atmospheric conditions. The medium was replaced every 2 days. Confluent cultures were washed with phosphate-buffered saline and detached with trypsin/ethylenediaminetetraacetic acid solution. Third passage cells with 80% confluence were used for experiments.
The concentrations used for each compound were as follows: CBD 5 μM5, valproate 433.4 μM (therapeutic dose for seizure in patients with epilepsy and mood disorders)6, bilobalide 15.3 μM (is the IC50 value)7, furosemide 604.7 μM (is the ED50)8, and metformin 1548.4 μM (neuroprotective against H2O2)9. Each compound was dissolved in an FBS-free medium.
Neurotoxicity assay
Ten thousand cells were placed in 96-well plates and incubated for 24 h. Compounds were added alone and in a binary combination with CBD at the concentrations previously mentioned and incubated for 24 h. The medium was replaced with [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) solution (0.5 mg/mL) and incubated for 3 h. Formazan crystals were solved with a solution of isopropyl alcohol/HCl (10%), and after 30 min, absorbance was measured at 550 nm in a microplate enzyme-linked immunosorbent assay reader ELx800 (Biotek instruments). Results were expressed as a percentage of cell viability10. Glutamate (25 mM) was the positive control for neurotoxicity, and glutamate-free cells as a control of 100% cell viability.
Neuroprotection assay
Ten thousand cells were placed in 96-well plates and incubated for 24 h. Then, a 2 h pre-treatment11 with the compounds alone and mixed with CBD was added. Thereafter, 0.1 mL of glutamate 25 mM12 was added, and incubated for 22 h. The medium was removed and replaced with MTT solution (0.5 mg/mL). The same protocol for MTT assay was performed. Results were expressed as a percentage of cell viability. Trolox® 50 μM was the positive neuroprotection control13.
Two different concentrations of each drug, including CBD, were used for the preliminary evaluation of the combinations of CBD with the other studied drugs. The first concentration was the same used for the neuroprotective and neurotoxic assays; the second concentration was ½ of the concentrations previously mentioned. The increase or decrease in cell viability was compared between the CBD combinations and the individual drugs.
Antioxidant activity
0.1 mL of each drug, alone and mixed with CBD 5 μM was placed in a 96-well plate, then 0.1 mL of 100 μM DPPH14 solution was added. The microplate was incubated in darkness at room temperature for 15 min, then absorbance was measured at 517 nm. The negative control was wells containing DPPH solution. Results were expressed as a percentage of DPPH inhibition.
Inhibition of reactive oxygen species (ROS) production
Ten thousand cells/well were pretreated with CBD alone and mixed with the other molecules. Then, H2O2 (0.1 mM) was added, and incubated for 1 h. Then, 2’,7’-dichlorofluorescein diacetate (H₂DCFDA)15 solution (0.05 mg/mL) was added and incubated for 20 min. Fluorescence was measured at 485 nm of excitation and 530 nm of emission. Negative control was cells treated with H2O2 0.1 mM.
Caspase-3 activity
The EnzChek® Caspase-3 Assay Kit#2 was used according to the manufacturer’s instructions. The assay was performed only for bilobalide and metformin alone and mixed with CBD. First, 1,000,000 cells were placed in a 35-mm petri dish and exposed to bilobalide and metformin alone and mixed with CBD. Glutamate 25 mM was added and incubated for 2 h. The medium was removed, cells harvested, washed with PBS, lysed, and centrifuged. To 0.05 mL of supernatant, 0.05 mL of the caspase-3 substrate Z-DEVD-R110 was added and set in darkness for 30 min at room temperature. Fluorescence was measured at 496 nm/520 nm excitation and emission in Fluoroskan Ascent (Thermo Fisher). Trolox® 50 μM was the positive control of inhibition of caspase-3, and glutamate 25 mM was the negative control.
Statistical analysis
Data are expressed as mean ± standard error of the mean of three independent experiments performed in triplicate (n = 9). Statistical comparisons were performed using one-way analysis of variance with Tukey’s multiple comparisons test. The statistical software GraphPad Prism 5 was used for the analysis. A p < 0.05 was considered significant.
Results
Neurotoxicity assay
Individually, CBD and other drugs did not show neurotoxic activity, their cell viability ranging from 77.3 to 106.5%. Furosemide had significantly lower cell viability than CBD (77.3 vs. 106.5%; p < 0.0001). Combinations of CBD with other drugs did not show neurotoxicity, and the cell viability was above 80%. All drugs, both individually and combined with CBD, significantly increased cell viability compared to the neurotoxic control, glutamate 25 mM (cell viability 47%; p < 0.05); however, the cell viability of the combination of CBD with the other compounds was not better than individual drugs (Fig. 1).
Neuroprotection activity of individual molecules
The pre-treatment of PC12 cells with CBD, and subsequent exposition to glutamate, 25 mM, significantly increases 22.6% cell viability than non-pretreated cells (cell viability = 47%; p < 0.05). This neuroprotective activity, however, was lesser than the positive control of neuroprotection Trolox 50 μM (cell viability = 103.4%). Bilobalide (15.3 μM) showed neuroprotection (cell viability = 97.6%) similar to Trolox. The other studied compounds did not show neuroprotective activity (Fig. 2).
Neuroprotection activity of combined molecules
Only the pre-treatment of CBD combined with bilobalide showed neuroprotective activity since significantly increased the viability (108.3%) of glutamate-exposed cells (p < 0.05). However, this neuroprotection was not different than neuroprotection of individual compounds (Fig. 2).
Antioxidant activity
All drugs, individually and combined with CBD, showed lower antioxidant activity than Trolox 50 μM (71.35%) (p < 0.0001). The values of antioxidant activity ranged from 3.87% to 9.3% with no significant difference among them.
Inhibitory activity of ROS production
Only CBD, bilobalide, and metformin decreased the production of ROS (38.2%, 50.1%, and 39.5%, respectively) compared with H2O2 0.1 mM. However, these values were minor than Trolox 50 μM (78%) (p < 0.0001). The capacity of CBD to inhibit ROS production did not improve when it was mixed with the other molecules (Fig. 3).
Caspase-3 activity
Only CBD, alone and mixed with bilobalide, significantly decreased caspase-3 activity (40.9 and 38.5%, respectively). Although the combination of bilobalide plus CBD significantly decreased caspase 3 activity, this activity was not better than the activity of individual drugs (Fig. 4). There was no difference between the control (Trolox), CBD, and the combination CBD plus bilobalide.
Discussion
Individually, CBD and the other molecules studied did not showed neurotoxicity in PC12 cells. Previous reports indicate that CBD (10 μM) and valproate (1-5 mM) are neurotoxic in higher doses than used in the present study16,17. Bilobalide and metformin (2 mM) do not have neurotoxic activity in the PC12 cells18,19 and SH-SY5Y cells,20 respectively, which is similar to the present study. In the case of furosemide, there are no previous studies.
The pre-treatment of CBD showed neuroprotection that was similar to previous studies that use different cells and neurotoxic agent5. The mechanisms reported include decreased ROS accumulation, lipid peroxidation, caspase-3, DNA fragmentation, attenuation of intracellular calcium, and inhibition of iNOS and NO production21,22. The inhibition of ROS production, and decrease in caspase-3 activity (40.9%) observed in the present study also has been reported in SH-SY5Y and PC12 cells23,24.
Valproate does not show neuroprotective activity, which is contrary to previously reported, this could be due to the different dose and neurotoxic agents used. In a study, valproate (50-400 μM) increases cell viability (> 75%) of PC12 cells exposed to aluminum maltolate (1000 μM), the mechanism was by minor apoptosis, decreased ROS, catalase activity, and reduced mitochondrial membrane potential25. In another study with motor neurons treated with glutamate 100 μM, valproate increased cell viability through the inhibition of histone deacetylase26 and acetylation of factor transcription SP127,28.
In the present work, furosemide was studied for the 1st time in PC12 cells and did not show neuroprotective activity. This is contrary to reported previously in microglial cell line SIM-A9, where it decreased the proinflammatory microglia phenotype M1 and favored the anti-inflammatory phenotype M2 on cells exposed to lipopolysaccharides (5 ng/mL)29. The antioxidant activity was similar to that previously reported, but the inhibition of ROS production was different than reported in SIM-A9 cells29,30.
As has been previously reported in PC12 cells (25-100 μM), and primary culture of astrocytes (100 μM), bilobalide has neuroprotective activity. Mechanisms include inhibition of ROS production by glucose and oxygen deprivation31, decreased apoptosis by BAX, and caspase-3 activation induced by ROS19. The inhibition of ROS production is similar to that obtained in the present study.
Metformin (1.54 mM) did not show neuroprotective activity which is contrary to other studies and is explained by the different doses used. Higher doses (2 mM) demonstrated neuroprotective activity in PC12 cells and primary culture of hippocampal neurons exposed to H2O2 100 μM. The mechanism includes decreased apoptosis and necrosis, less intracellular ROS production, and protected mitochondrial membrane potential by AMPK activation9. Antioxidant activity (DPPH inhibition) was lower (5.14% at 1.54 mM) than previously reported, and it is explained by the higher doses used (31% at 20 mM)32. The decreased ROS production by metformin (39.4%) is similar to previously reported9. Contrary to other studies metformin does not significantly decrease caspase-3 activity and is explained by the different cells and concentrations used33.
Results showed that CB combined with the studied drugs neither improve nor worsen the neuroprotection of each individual drug. Although with limitations, these results imply that the combinations studied do not produce a beneficial synergic effect in PC12 cells. It is necessary to confirm this with an isobolographic analysis.
There are no studies about the neuroprotective activity of CBD combined with these drugs in vitro or in vivo models; however, there are some anecdotal findings. In the case of valproate, a clinical case reports that CBD does not modify the antiepileptic effect of valproate34. In a clinical trial, CBD plus valproate increased hepatic aminotransferase enzymes35. Although the coadministration of CBD and furosemide has not been evaluated, some neuroprotective activity is possible since furosemide has a neuroprotective activity similar to CBD in an in vivo binge model induced by ethanol in rats. The combination of CBD plus bilobalide could have pharmacokinetic interaction since both molecules interact with the CYP1A2 isoform. The same could be for the combination of CBD plus metformin since both decrease CYP3A4 expression36.
Limitations
Only two concentrations of each combination were used in this study. This approach does not permit concluding the type of interactions, such as synergism, summation, potentiation, or antagonism. However, our study permits discarding, in a preliminary way, a beneficial synergic effect of the CBD combination with the study drugs in vitro.
Conclusion
CBD and bilobalide present neuroprotective activity in PC12 cells exposed to glutamate 25 mM. The neuroprotective activity of CBD did not improve when combined with furosemide, valproate, and metformin. Bilobalide, CBD, and metformin decrease ROS production. Only CBD decreases caspase-3 activity.
