Isobolographic Analysis of the Cytoprotective Effect of Dapsone and Cannabidiol Alone or Combination upon Oxygen-Glucose Deprivation/Reoxygenation Model in SH-SY5Y Cells

2.2. Cell Culture

The human neuroblastoma SH-SY5Y cell line was purchased from ATCC, Manassas, VA, USA. Cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DMEM/F12) (Gibco-Thermoscientific, Grand Island, NY, USA) supplemented with 100 U/mL penicillin/streptomycin, 10% fetal bovine serum (FBS) certified (16000044) (Gibco-Thermoscientific, Grand Island, NY, USA) and 0.2% sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA). The cells were maintained in an incubator at 37 °C in a saturated humidity atmosphere containing 95% air and 5% CO₂; these conditions were used in all experiments unless otherwise stated. The culture medium was replaced every three days, and the cells were detached and subcultured at a ratio of 1:4 in standard culture medium and incubation conditions once they had achieved a confluence of no more than 90% through trypsin treatment. Before conducting any assays, the SH-5HSY cells were differentiated according to the protocol described by Teppola [17]. In summary, cells in suspension were seeded in microplates (dimension according to each experiment) at a concentration of 40,000 cells/well and standard culture medium supplemented with 10 µM all-trans retinoic acid (RA) (Sigma Aldrich, Milan, Italy) incubated in conditions described before for six days to induce differentiation. All experiments were performed in nine replicates per group.

2.3. MTT Cytotoxicity Assay

Cell viability was measured at the end of the exposure period using the 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay described by Martinez et al. [18], which consisted of replacing the culture medium with the treatments, with 200 μL of MTT dissolved in standard culture medium at a concentration of 1 mg/mL and incubating the microwell for 4 h in standard conditions. Finally, the medium containing MTT was withdrawn, leaving the well dry, and 200 μL of DMSO was added to each well to dissolve the purple formazan crystals. After 10 min, the absorbance of each well was measured using a microplate spectrophotometer (BioTek Eon) set to 490 nm. The following formulas were used to calculate cell viability: Cell viability (%) = cells in the experimental group divided by cells in the control group = 100% and % of Lethality = 100 − cell viability (%).

2.4. Mean Cytotoxic Concentration (LC₅₀) Assay

The median lethal concentrations (LC₅₀) of DDS and CBD were evaluated by subjecting SH-5HSY differentiated cells, which were cultivated in a 96-well microplate, to various doses ranging from 2.5 to 604 µM and 0.05 to 477 µM, respectively, for 24 h. The compounds were diluted in dimethyl sulfoxide (DMSO) to achieve a final concentration of 0.01% in the culture medium. DDS, CBD, and their combinations were diluted in all subsequent tests using the above method. After 24 h, the MTT method was employed to assess cell viability, which was subsequently quantified as a percentage relative to the viability of unexposed cells.

2.5. Oxygen–Glucose Deprivation/Reoxygenation (OGD/R) Model and Treatment

Culture under OGD/R conditions mimics the cellular conditions of cerebral ischemia and was used to assess DDS and CBD’s cytoprotective capacity. According to the protocol above, differentiated SH-SY5Y cells in 96-well microplates had the culture medium removed. After three washes with Hank’s buffered saline solution (HBSS), the HBSS was replaced with standard culture medium without glucose and incubated in a humidity-saturated anaerobic chamber with an atmosphere consisting of 5% CO₂ balanced with N₂ and tempered at 37 °C for 6 h. Under these conditions, cell viability decreased by 50%. Then, the glucose-free medium was replaced with a regular culture medium enriched with the DDS and CBD treatments, respectively. The working concentration ranges for DDS (6.04–30.6 µM) and CBD (0.047–0.24 µM) were determined following the cytotoxicity study detailed in the preceding paragraphs. The greatest concentration of each agent found to maintain 90% cell viability was used to determine these ranges. The plates supplemented with the treatments were incubated for 24 h in normoxic and glucose-containing culture media. After this period, the MTT method was used to reveal cell viability and expressed as a percentage of untreated cell viability. The cytoprotection index was computed using the viability percentage values, employing the following formula:

The effective cytoprotective concentration of 50 (EC₅₀) of each component, EC₅₀-DDS and EC₅₀-CBD, was computed using concentration–effect relationship curves and logit–probit analysis [19].

2.6. Assessment of the Effect of DDS–CBD Combination Using Isobolographic Analysis

An isobolographic analysis [20] was performed to investigate the pharmacological interactions between DDS and CBD. According to Miranda et al. [21], combinations of fixed proportions were made with the EC₅₀-DDS and EC₅₀-CBD values, and the culture medium was supplemented with the following mixture of DDS–CBD at proportions: 1:1, 1:4, and 4:1, respectively. Mixture concentrations were determined using the following formulas for each drug: 1:1, EC₅₀ = 0.5 (EC₅₀ DDS) + (1 − 0.5) (EC₅₀ CBD); 1:4, EC₅₀ = 0.25 (CE₅₀ DDS) + (1 − 0.25) (EC₅₀ CBD); 4:1, CE₅₀ = 0.75 (EC₅₀ DDS) + (1 − 0.75) (EC₅₀ CBD). All treatments were dissolved in 0.01% DMSO mixes. According to the methodology stated at the beginning of the section, differentiated SH-SH5Y cells were exposed to OGD/R culture conditions for 6 h before being replaced with glucose-rich culture media containing the appropriate DDS–CBD treatment mixes and controls, respectively. Under standard conditions, the plate was incubated for 24 h. Finally, using the MTT method, the percentage of cell viability was calculated by contrasting cells subjected to culture standard conditions. Using the cell viability data, under OGD/R culture conditions, the mixing ratio that was significantly more protective was chosen.

In order to calculate the mean experimental effective concentration (EC₅₀E) that protects SH-5HSY cell viability from OGD/R conditions, the range of concentrations required a second OGD/R experiment that was designed around the DDS/CBD mixture ratio that resulted in the highest percentage of cell viability. To determine the concentrations of the working solutions, values were established upstream and downstream of the most effective mixing concentration, excluding this same value from the assay. All treatment solutions were prepared with a culture medium enriched with the compounds of each concentration previously dissolved in 0.01% DMSO. As previously mentioned, the MTT technique was employed to assess cell viability. Subsequently, the percentage viability was determined by comparing the results to a control group of cells from the identical subculture subjected to standard conditions for 30 h.

Based on the assumption that the interaction between the two substances was additive, a simple graph was created depicting the ratio of DDS to CBD equieffective concentration ratios [22]. This was performed using Cartesian coordinates to place EC₅₀-DDS (value ± SEM) on the y-axis and EC₅₀-CBD (value ± SEM) on the x-axis. An isobola (straight line) was created from the two concentrations. The DDS-to-CBD-concentration ratios given below are those that should result in the average cytoprotective effect if the additive-type interaction between DDS and CBD occurs.

The method below can be used to calculate the additivity index (i) for any given point in this interaction, as well as the precise values of its DDS and CBD components [23]:

Which represents the concentrations of DDS and CBD that produce the desired effect when taken together; in this case, they are represented by C1-DDS and C2-CBD, respectively, while EC₅₀-DDS and EC₅₀-CBD represent the corresponding values of the mean effective individual cytoprotective concentrations of the DDS and CBD, respectively. To find the relevant concentrations, the following formula was used to compute the corresponding EC₅₀-DDS and EC₅₀-CBD fractions:

where f-DDS and f-CBD are the fractions of DDS and CBD, respectively. Finally, the interaction index was calculated using the formula

Theoretical effective concentration (EC₅₀T) is derived from the above equations with the experimental values of the EC₅₀ of individual effects of DDS and CBD, respectively, and EC₅₀E is obtained directly from the concentration–effect curve of the most effective mixture. The interaction indices of the experimental combination (EC₅₀E) and the theoretical (EC₅₀T) were then compared using Student’s t-test, with p < 0.05 indicating a significant difference. Tallarida [24] defines a synergistic (sub-additive) interaction as I < 1.0, additive (I = 1.0), or antagonistic (supra-additive) interaction as I > 1.0.

Finally, to determine the parameters for evaluating the pharmacological and biochemical activities of the DDS–CBD mixture, the EC₉₀E obtained from the concentration-effect curve mentioned above was used.

2.7. Lactate Dehydrogenase (LDH) Assay

Cytotoxicity was assessed through the release of LDH [25]. SH-SY5Y cells from different groups were cultured in 96-well plates (4 × 10³ cells/well), subjected to oxygen–glucose deprivation for 6 h, and reoxygenation (OGD/R) for 24 h with pharmacological treatment. LDH released from cells was detected using the LDH assay kit (11644793001, Roche). The supernatant (100 μL) and reaction mix (100 μL) were mixed in 96-well plates and incubated for 1 h at 37 °C. The absorbance was measured at 490 nm using a microplate spectrophotometer (BioTek Eon™). Results were expressed as a percentage of control group cells. All experiments were performed in nine replicates per group. The LDH release rate was calculated with the following formula:

2.8. Measurement of Intracellular ROS

5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) is an indicator of general oxidative stress used to measure the level of intracellular ROS production. Seeding 96-well plates with 1 × 10³ cells/well for 24 h promotes cell adhesion. After being washed twice in Hank’s medium, the cells were exposed to OGD for 6 h before being reoxygenated with 100 mL of the alternative test treatments for 24 h. Following exposure, cells were washed twice with Hank’s solution and incubated for 1 h in the dark in an incomplete culture medium (0.02 M CM-H2DCFDA) (medium without FBS supplement). A Varioskan lux multi-plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used at 485 nm for excitation and 528 nm for emission to measure the fluorescence intensity.

2.9. Measurement of Reduced Glutathione (GSH)

After seeding SH-SY5Y cells in 6-well plates and treating them with OGD/R conditions, the cells were harvested and sonicated in SSF at cold temperatures, 25% metaphosphoric acid was added to the sample in a 1:1 ratio, and the mixture was centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was then taken, and EDTA buffer and the O-phthalaldehyde indicator were added. It was incubated at room temperature for 15 min with a 1 mg/mL concentration. The fluorescence intensity was measured using a Varioskan lux multi-plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with an excitation wave of 350 nm and an emission wave of 420 nm. GSH (5 mg/mL) generated a standard curve.

2.10. Caspase-3 Activity Assay

Cellular apoptosis was assessed with a caspase-3 colorimetric kit (APT165, Merck Millipore) that identifies the DEVD amino acid sequence. A concentration of 1 × 10⁶ cells/mL was placed in a 6-well plate, and they were subjected to oxygen–glucose deprivation for 6 h and reperfusion (OGD/R) for 24 h with drug treatment divided into different groups. The cells were recovered by scraping and centrifuged at 1500 rpm for 10 min; the supernatant was removed, and the button was reconstituted with 1× buffer for 5 min. The supernatant was taken and placed in a 96-well plate. Finally, 5× assay buffer and caspase-3 substrate were added; they were incubated at 37 °C for 1 h. A standard curve of p-nitroanilide (pNA) in a concentration range of 10 μM to 1 mM was generated by diluting the pNA stock solution in 1× assay buffer. Then, 100 μL of each dilution was placed in the ELISA plate, and they were read together with the samples in a fluorescence spectrophotometer at 405 nm using a microplate spectrophotometer (BioTek Eon™, Winooski, VT, USA). The results were expressed as a percentage of control group cells. All experiments were performed in nine replicates per group. Caspase-3 activity was expressed as a percentage compared to the control group.

3.2. Cytoprotective Effect of Dapsone and CBD in a Model Oxygen Glucose Deprivation/Reoxygenation in SH-SY5Y Cells

In this section, the cytotoxic threshold values from the previous experiment were utilized to determine the concentrations of DDS (6.04, 7.65, 9.66, 12.08, 15.30, 19.33, 24.16, and 30.6 µM) and CBD (0,04, 0.06, 0.07, 0.09, 0.11, 0.15, 0.18, and 0.24 µM) to assess the cytoprotective effect separately of both in the OGD/R model. The viability of the cells was significantly higher in the group pretreated with 30.6 µM DDS, showing an 88.18 ± 1.6% response compared to the OGD/R without treatment, as shown in Figure 2 panel A, while CBD had its most significant effect at 0.15 µM concentration, which shows 76.35 ± 2.2% cytoprotection when compared to the OGD/R group without treatment (Figure 2, panel B). The effective cytoprotective concentration 50 (EC₅₀) of DDS and CBD obtained were 10.06 ± 0.22 µM and 0.07 ± 0.004 µM, respectively.

3.3. Effect of DDS–CBD Combination by the Isobologram Method

Table 1 provides a summary of the calculated concentrations of the components of the DDS–CBD mixture in each of the evaluated proportions, as determined by the EC₅₀ value of each agent [26]. The exposure to all combinations significantly increased cell viability in SH-SY5Y cells under oxygen–glucose deprivation and reoxygenation (OGD/R). However, the combination with a 1:4 mix ratio showed the maximum effect on the other combinations, where an effect of 91.62% cell viability recovery response was observed (Figure 3, panel A).

The range of dilutions for the 1:4 drug mixture was determined by the cytoprotective activity of 91.62% of the 1:4 ratio, which had a final concentration of 2.55 µM (Table 1). An independent assay was conducted to determine the experimental 50% effective concentration (EC₅₀E) of the 1:4 ratio of DDS–CBD, resulting in 0.26 µM, as shown in Figure 3 panel B.

The value of the theoretical 50% effective concentration of the 1:4 ratio mixture, as determined by the formula provided in Table 1, was 2.55 µM, shown by the isobologram in panel C of Figure 3. As stated in Table 2, a comparison of the EC₅₀T and EC₅₀E values revealed a significant difference of p < 0.05 and an interaction index of 0.10.

3.4. DDS–CBD Treatment Improves Cell Viability and Attenuates Oxidative Damage

Figure 4, Panel A shows the viability of SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD/R), where it is decreased in those cells that did not receive treatment (47.19 ± 1.58). The groups treated with DDS (51.69 ± 2.21) and CBD (50.38 ± 2.01) do not show a statistically significant difference compared to the group subjected to conditions of OGD/R. In contrast, the group treated with the DDS–CBD (89.37 ± 1.44) mixture presents a statistically significant difference vs. group OGD/R.

Similarly, the measurement of cellular LDH release revealed the conditions of cells’ cytotoxicity. The result demonstrated that the DDS–CBD combination is an efficient treatment against cellular oxidative damage because the group treated with this mixture at a total concentration of 7.86 µM (7.55–0.311 µM) showed a significant decrease in the release of LDH (17.86% ± 3.31) compared to the group subjected to conditions of oxygen–glucose deprivation without treatment (OGD/R) where an increase of 61.22% ± 5.9 vs. control group is observed, while the OGD/R-DDS and OGD/R-CBD groups do not show a statistically significant difference (p < 0.05) compared to the OGD/R group as shown in Figure 4, panel B.

3.5. DDS–CBD Combination Decreased Oxidative Stress Induced by OGD/R

The effect of the DDS–CBD combination on the oxidative stress induced by OGD/R was evaluated by detecting levels of ROS and GSH. Figure 5 (Panel A) shows a considerable rise in ROS levels in the OGD/R group (73.7%) vs. the control group (173.7 ± 5.8). In comparison, the group treated with the DDS–CBD combination increased only by 19% vs. the control group (119.01 ± 6.72), indicating a considerable reduction in ROS levels. The damage caused by OGD/R significantly decreased GSH levels (Figure 5 Panel B) (116.45 ± 7.82). On the contrary, when the cells were treated with DDS–CBD (164.21 ± 13.6), the GSH levels were increased compared to the OGD/R group, which showed a statistically significant difference, while the drugs separately showed no difference with the OGD/R group.