Beta-Caryophyllene, a Plant-Derived CB2 Receptor Agonist, Protects SH-SY5Y Cells from Cadmium-Induced Toxicity

2.2. BCP Protects SH-SY5Y Cells from the Damage Induced by CdCl₂

In undifferentiated SH-SY5Y cells, control cells were numerous and arranged in clusters; they showed the typical flat morphology with short, stubby processes. and euchromatic nuclei with evident nucleoli; occasional mitoses were also observed (Figure 2A,F). A sharp reduction in the number of cells/unit area (UA) was observed in CdCl₂-challenged cells (Figure 2B,F). Cells showed heterochromatic nuclei and reduced or absent processes; no mitoses were detected, and some nuclei showed apoptotic bodies. A significant increase in the number of cells/UA was evident in CdCl₂ samples treated with BCP at a concentration of 25 μM (Figure 2C,F); cells showed either heterochromatic or euchromatic nuclei with evident nucleoli. Furthermore, occasional necrotic cells and apoptotic bodies were also present. BCP at a concentration of 50 μM increased the number of cells/UA (Figure 2D,F), as demonstrated by the augmented number of cells with euchromatic nuclei compared to those with heterochromatic nuclei. Moreover, both apoptotic and dividing cells were observed. A further increase in cells arranged in clusters was evident in CdCl₂-challenged cells treated with BCP at a concentration of 100 μM with a morphological pattern similar to controls (Figure 2E,F). Mitoses were present in many microscopic fields.

In SH-SY5Y cells differentiated with retinoic acid (RA), no clusters were present (Figure 3A). Cells were less numerous/UA when compared to control undifferentiated cells (Figure 3F) and generally isolated. Elongated or pyramidal bodies were detected, with evident processes and euchromatic nuclei (Figure 3A); no mitoses were observed. RA-differentiated cells challenged with CdCl₂ showed a significant increase in their number/UA (Figure 3F), a round shape with heterochromatic nuclei, and a reduction in their processes; in particular, some nuclei exhibited apoptotic bodies (Figure 3B). In CdCl₂-challenged samples differentiated with RA and treated with BCP at a concentration of 25 μM, a mild and not significant increase in the number of cells (Figure 3F) with either euchromatic or heterochromatic nuclei was observed; occasional apoptotic bodies were also detected (Figure 3C). BCP at the concentration of 50 μM induced a significant reduction in the number/UA (Figure 3F) of RA-differentiated cells challenged with CdCl₂, which showed euchromatic nuclei and some short processes (Figure 3D); additionally, some apoptotic cells were also present. CdCl₂-challenged cells differentiated with RA and treated with BCP at a concentration of 100 μM showed both cell number/UA and morphological pattern similar to controls, with evident processes (Figure 3E,F).

2.3. BCP Reduces Intracellular ROS Production

Intracellular ROS production was evaluated to investigate the possible antioxidant effects of BCP, both in undifferentiated and differentiated SH-SY5Y cells. CdCl₂ induced a marked increase in fluorescence signal in both cell lines, thus indicating an increased ROS accumulation in CdCl₂-challenged cells compared to controls (Figure 4B and Figure 5B). BCP significantly reduced ROS production at both concentrations of 50 and 100 μM (Figure 4D,E and Figure 5D,E), whereas a slight reduction in ROS levels was observed at the dose of 25 μM (Figure 4C and Figure 5C).

The BCP antioxidant effect was further confirmed by Western blot analysis by evaluating the nuclear factor erythroid 2–related factor 2 (Nrf2) expression. CdCl₂ significantly reduced the Nrf2 protein level in both undifferentiated and differentiated SH-SY5Y cells compared to unchallenged cells (p < 0.05 vs. ctrl; Figure 4F and Figure 5F). Nrf2 expression was significantly increased with BCP at the concentration of 50 μM in undifferentiated SH-SY5Y cells (p < 0.05 vs. CdCl₂; Figure 4F), whereas a significant increase in its expression was observed at the concentrations of 50 and 100 μM in differentiated SH-SY5Y cells (p < 0.05 vs. CdCl₂; Figure 5F), thus confirming the BCP anti-oxidant effect.

4.2. Cell Treatment

SH-SY5Y neuroblast-like cells were plated in a 6-well plate at a density of 1 × 10⁵ cells/well and incubated at 37 °C with a percentage of 5% CO₂ overnight. The day after, cells were differentiated in neuron-like cells. In particular, 10 µM of retinoic acid (RA) was added to each well for five days and the medium was changed every two days. At day 6, RA was removed, cells were washed three times with sterile phosphate-buffered saline (PBS) and brain-derived neurotrofic factor (BDNF; 50 ng/mL) was added for additional five days in order to improve RA effects on neuronal differentiation.

Undifferentiated SH-SY5Y cells and neuron cells were cultured in 6-well plates at a density of 2.5 × 10⁵ cells/well; 24 h after seeding, cells were pre-treated with β-caryophyllene (BCP) (Sigma-Aldrich, St. Louis, MO, USA) at the concentrations of 25, 50 and 100 μM for 24 h. The day after, the medium was replaced and the cells were stimulated with 10 μM of cadmium chloride (CdCl₂) (Sigma-Aldrich, St. Louis, MO, USA) for additional 24 h in order to reproduce an in vitro model of Cd-induced neuronal toxicity, as previously reported [45]. All treatments were performed in starvation conditions because essential elements of medium, such as zinc and calcium, could compromise Cd effects. At the end of the incubation period, cells were collected for molecular and morphological analyses.

4.3. MTT Assay

MTT assay was performed to evaluate BCP effects on cell viability. SH-SY5Y were seeded in a 96-well plate at a density of 1 × 10⁵ cells/well for 24 h. The day after, cells were treated with doubling concentrations of BCP (6.25, 12.5, 25, 50, 100, and 200 μM) for 24 h. Five hours before the end of the treatment, 20 μL of 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (Sigma-Aldrich, St. Louis, MO, USA) (0.5 mg/mL) was added into each well. 200 μL of dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) were added into each well to dissolve the insoluble formazan crystals. The reaction was read at 540 and 620 nm of absorbance by using VICTOR Multilabel Plate Reader (Perkin Elmer; Waltham, MA, USA), and the results were expressed as % of cell viability compared to untreated cells and reported as means and standard deviation (SD) [46].

4.4. Hematoxylin/Eosin Staining

The cells from both undifferentiated and RA-differentiated cells were fixed in 4% paraformaldehyde, washed in PBS, stained with hematoxylin and eosin (HE), and photographed with a Nikon Ci-L light microscope (Nikon Instruments, Tokyo, Japan). The images were obtained with a digital camera (Nikon DS-Ri2).

4.5. Evaluation of Apoptosis with Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay

An apoptosis detection kit (in situ Apoptosis Detection kit, Abcam, Cambridge, UK) was used for the TUNEL technique following the manufacturer’s instructions. In brief, cells from both undifferentiated and RA-differentiated cells were fixed in 4% paraformaldehyde, washed in PBS, and endogenous peroxidase activity was blocked with 3% H₂O₂ in methanol after permeabilization with proteinase K. Samples were incubated with terminal deoxynucleotidyl transferase, biotin-labeled deoxynucleotides, streptavidin-horseradish peroxidase conjugate, and lastly with the diaminobenzidine solution. No counterstaining was performed to better observe apoptotic cells. Cells were photographed with a Nikon Ci-L light microscope using a digital camera, the Nikon Ds-Ri2 [47].

4.6. Morphometric Evaluation

All images were saved as Tagged Image Format Files (TIFF), printed at the same final magnification, and blindly assessed by two trained observers. As to the specimens from both undifferentiated and RA-differentiated cells stained with HE, the mean number of cells per Unit Area (UA = 250 × 250 μm) was calculated from 20 UAs for each experimental group, obtained from micrographs taken with an objective of 20×. In order to evaluate apoptosis in both undifferentiated and RA-differentiated cells, the mean number of TUNEL-positive cells/UA was calculated from 20 UAs (UA = 250 × 250 μm) for each group, obtained from micrographs taken with an objective of 20×. In both procedures, cells overlapping the right and top boundaries were not included in the evaluation, whereas cells overlapping the left and bottom boundaries were counted.

4.7. Measurement of ROS Production

ROS production was studied by using the probe 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Thermofisher, Waltham, MA, USA). At the end of the treatment period, SH-SY5Y cells and neuron-like cells were incubated with 5 μM of CM-H2DCFDA for 1 h at 37 °C. Cells were then washed with PBS to remove the excess probe and observed with a fluorescent microscope at 10× magnification. The signal of fluorescence was quantified with ImageJ software for Windows (Softonic, Barcelona, Spain) [48].

4.8. RNA Isolation, cDNA Synthesis, and Real-Time Quantitative PCR Amplification

Total RNA was extracted from both cell lines using Trizol LS Reagent Kit (Life Technologies, Monza, Italy) and quantified with a spectrophotometer (NanoDrop Lite, Thermo Fisher, Waltham, CA, USA). 1.0 μg of total RNA was reverse transcribed in cDNA using the SuperScript IV Master Mix (Thermo Fisher, Carlsbad, CA, USA) and used for qPCR analysis to evaluate IL-6, IL-1β, and TNF-α gene expression using the BrightGreen 2X qPCR MasterMix-Rox (Applied Biological Materials, Vancouver, Canada) and the QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, CA, USA). β-Actin was used as a housekeeping gene, and the amplified PCR products were quantified by measuring the calculated cycle thresholds (CT) of target genes and β-actin mRNA. After normalization, the mean value of the normal control target levels was chosen as calibrator, and the results were expressed according to the 2^−ΔΔCt method as a fold change relative to normal controls. Primers used for targets and reference genes are listed in Table 1 [49].

4.9. Western Blot Analysis

At the end of the experimental procedure, SH-SY5Y cells and neuron-like cells were collected and homogenized in RIPA buffer (25 mM Tris/HCl, pH 7.4; 1.0 mM EGTA (Ethylene glycol bis(2-aminoethyl ether)-N,N,N’,N’-tetraacetic acid); 1.0 mM EDTA(Ethylenediaminetetraacetic acid dipotassium salt dihydrate) with NP40 (1%), phenyl methylsulfonyl fluoride (PMSF, 0.5%), aprotinin, leupeptin, and pepstatin (10 μM each)) and centrifuged at 15,000× g for 15 min at 4 °C; the supernatant obtained from each sample was used to measure the total protein content using the Bio-Rad protein-assay kit (BioRad, Hercules, CA, USA). 30 μg of proteins were run in a 10% sodium dodecyl sulphate (SDS) polyacrylamide gel and then transferred to PVDF membranes using a specific transfer buffer (39 mmol/L glycine, 48 mmol/L Tris, pH 8.3, and 20% methanol) at 300 mA for 2 h. The obtained membranes were blocked with 5% non-fat dry milk in Tris buffered saline (TBS) 0.1% for 1 h at room temperature and incubated with primary antibodies for Bax and Nrf2 (Cell Signaling, Danvers, MA, USA) and Bcl-2 (Abcam, Cambridge, UK) diluted in TBS-0.1% Tween, overnight, at 4 °C. The day after, membranes were washed with TBS-0.1% Tween and incubated with a secondary peroxidase-conjugated goat anti-rabbit antibody (KPL, Gaithersburg, MD, USA) for 1 h at room temperature. After washing with TBS-0.15% Tween buffer, membranes were analyzed by the enhanced chemiluminescence system (LumiGlo reserve; Seracare, Milford, MA, USA). The protein signal was detected and quantified by scanning densitometry using a bio-image analysis system (C-DiGit, Li-cor, Lincoln, NE, USA). Results were expressed as relative integrated intensity, and β-actin (Cell Signaling, Danvers, MA, USA) was used to confirm equal protein loading in all samples [27].