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

Abstract

Cadmium (Cd) is a transition heavy metal that is able to accumulate in the central nervous system and may induce cell death through reactive oxygen species (ROS)-mediated mechanisms and inactivating the antioxidant processes, becoming an important risk factor for neurodegenerative diseases. The antioxidant effects of cannabinoid receptor modulation have been extensively described, and, in particular, β-Caryophyllene (BCP), a plant-derived cannabinoid 2 receptor (CB2R) agonist, not only showed significant antioxidant properties but also anti-inflammatory, analgesic, and neuroprotective effects. Therefore, the aim of the present study was to evaluate BCP effects in a model of Cd-induced toxicity in the neuroblastoma SH-SY5Y cell line used to reproduce Cd intoxication in humans. SH-SY5Y cells were pre-treated with BCP (25, 50, and 100 μM) for 24 h. The day after, cells were challenged with cadmium chloride (CdCl₂; 10 μM) for 24 h to induce neuronal toxicity. CdCl₂ increased ROS accumulation, and BCP treatment significantly reduced ROS production at concentrations of 50 and 100 μM. In addition, CdCl₂ significantly decreased the protein level of nuclear factor erythroid 2-related factor 2 (Nrf2) compared to unstimulated cells; the treatment with BCP at a concentration of 50 μM markedly increased Nrf2 expression, thus confirming the BCP anti-oxidant effect. Moreover, BCP treatment preserved cells from death, regulated the apoptosis pathway, and showed a significant anti-inflammatory effect, thus reducing the pro-inflammatory cytokines increased by the CdCl₂ challenge. The results indicated that BCP preserved neuronal damage induced by Cd and might represent a future candidate for protection in neurotoxic conditions.

Keywords: ROS; beta-caryophyllene; cadmium; inflammation; neurotoxicity.

Figure 1

Cytotoxicity assay evaluated in SH-SY5Y cell line treated with BCP at concentrations of 6.25, 12.5, 25, 50, 100, and 200 μM for 24 h. Values are expressed as the means ± SD of three experiments.

Figure 2

Representative morphological images of undifferentiated SH-SY5Y cells challenged with CdCl₂ and treated with different doses of β-caryophyllene (BCP). (A): Clusters of round control cells with euchromatic nuclei and short processes, particularly evident in the edges of the clusters. Occasional mitoses are present (arrowheads). (B): Cells challenged with CdCl₂ alone. Note the presence of heterochromatic or apoptotic (arrows) nuclei, the reduction in processes, and the absence of mitoses. (C): Cells challenged with CdCl₂ and treated with 25 μM of BCP. Euchromatic, heterochromatic, and apoptotic (arrow) nuclei, and occasional necrotic cells (*) are present. (D): Cells challenged with CdCl₂ and treated with 50 μM of BCP. Both mitotic (arrowhead) and apoptotic (arrow) cells are present. (E): Cells challenged with CdCl₂ and treated with 100 μM of BCP are arranged in clusters and show a morphological pattern similar to controls, with occasional mitoses (arrowheads). (Hematoxylin and Eosin stain. Scale bar = 50 μm; insets = 25 μm). (F): Mean number of undifferentiated SH-SY5Y cells/UA in the different groups examined. A = control; B = CdCl₂-challenged cells; C = CdCl₂-challenged cells treated with BCP 25 μM; D = CdCl₂-challenged cells treated with BCP 50 μM; E = CdCl₂-challenged cells treated with BCP 100 μM. Data are expressed as mean ± SD. * p ≤ 0.05 versus controls; § p ≤ 0.05 versus CdCl₂-challenged cells.

Figure 3

Representative morphological images of SH-SY5Y cells differentiated with RA, challenged with CdCl₂, and treated with different doses of BCP. (A): Cells differentiated with RA (controls) are isolated, with elongated or pyramidal bodies and evident processes (*). (B): RA-differentiated cells challenged with CdCl₂ show round shape, heterochromatic nuclei, and few processes. Some cells have apoptotic bodies (arrow). (C): In CdCl₂-challenged cells differentiated with RA and treated with BCP at 25 μM, either euchromatic or heterochromatic nuclei are present; occasional apoptotic bodies are evident (arrow). (D): CdCl₂-challenged cells differentiated with RA and treated with BCP at 50 μM show euchromatic nuclei and some short processes (*); some isolated apoptotic cells are present (arrow). (E): After treatment with BCP at the concentration of 100 μM, CdCl₂-challenged cells differentiated with RA show a morphological pattern similar to controls, with evident processes (*). (Hematoxylin and Eosin stain. Scale bar = 50 μm; insets = 25 μm). (F): Mean number of RA-differentiated SH-SY5Y cells/UA in the different groups examined. A = control; B = CdCl_2-challenged RA-differentiated cells; C = CdCl₂-challenged RA-differentiated cells treated with BCP 25 μM; D = CdCl₂-challenged RA-differentiated cells treated with BCP 50 μM; E = CdCl₂-challenged RA-differentiated cells treated with BCP 100 μM. Data are expressed as mean ± SD. * p ≤ 0.05 versus controls; § p ≤ 0.05 versus CdCl₂-challenged RA-differentiated cells.

Figure 4

The panel shows intracellular ROS accumulation evaluated by CM-H2DCFDA probe in undifferentiated SH-SY5Y cell line from ctrl (A), CdCl₂ (B), CdCl₂ + BCP 25 μM (C), CdCl₂ + BCP 50 μM (D), and CdCl₂ + BCP 100 μM (E). The graph (F) shows Nrf2 protein expression. The data are expressed as the means and SD of three experiments. * p < 0.05 vs. ctrl; # p < 0.05 vs. CdCl₂.

Figure 5

The panel shows intracellular ROS accumulation evaluated by CM-H2DCFDA probe in differentiated SH-SY5Y cell line from ctrl (A), CdCl₂ (B), CdCl₂ + BCP 25 μM (C), CdCl₂ + BCP 50 μM (D), and CdCl₂ + BCP 100 μM (E). The graph (F) shows Nrf2 protein expression. The data are expressed as the means and SD of three experiments. * p < 0.05 vs. ctrl; # p < 0.05 vs. CdCl₂.

Figure 6

The graphs represent Bcl-2 (A) and Bax (B) protein expression in undifferentiated SH-SY5Y cells and Bcl-2 (C) and Bax (D) in differentiated SH-SY5Y cells. The data are expressed as the means and SD of three experiments. * p < 0.05 vs. ctrl; # p < 0.05 vs. CdCl₂.

Figure 7

Assessment of apoptosis with TUNEL staining technique in undifferentiated SH-SY5Y cells challenged with CdCl₂ after treatment with different doses of BCP. (A): No TUNEL-positive cells are present in control cells. (B): Cells challenged with CdCl₂ alone. A large number of TUNEL-positive cells are present. (C): Cells challenged with CdCl₂ and treated with 25 μM of BCP. Many TUNEL-positive cells are observed. (D): Cells challenged with CdCl₂ and treated with 50 μM of BCP. An evident reduction in TUNEL-positive cells is present. (E): Cells challenged with CdCl₂ and treated with 100 μM of BCP. Only isolated TUNEL-positive cells are present. (F): Mean number of cells/UA in the different groups examined. Data are expressed as mean ± SD of three experiments. * p ≤ 0.05 vs. ctrl; # p < 0.05 vs. CdCl_2. Scale bar = 50 μm.

Figure 8

Assessment of apoptosis with TUNEL staining technique in RA-differentiated SH-SY5Y cells challenged with CdCl₂ and treated with different doses of BCP. (A): No positive cells are present in RA-differentiated control cells. (B): RA-differentiated cells challenged with CdCl₂ alone show a large number of positive cells. (C): In RA-differentiated cells challenged with CdCl₂ and treated with 25 μM of BCP many positive cells are observed. (D): RA-differentiated cells challenged with CdCl₂ and treated with 50 μM of BCP show an evident reduction in positive cells. (E): Only isolated positive cells are present in RA-differentiated cells challenged with CdCl₂ and treated with 100 μM of BCP. (F): Mean number of TUNEL-positive cells/UA in the different groups of RA-differentiated cells examined. Data are expressed as mean ± SD. * p ≤ 0.05 versus controls; # p ≤ 0.05 versus CdCl₂-challenged cells. Scale bar = 50 μm.

Figure 9

The graphs represent IL-1β (A), IL-6 (B), and TNF-α (C) mRNA expression assessed by RT-PCR in undifferentiated SH-SY5Y cells. The data are expressed as the means and SD of three experiments. * p < 0.05 vs. CTRL; # p< 0.05 vs. CdCl₂.

Figure 10

The graphs represent IL-1β (A), IL-6 (B), and TNF-α (C) mRNA expression assessed by RT-PCR in differentiated SH-SY5Y cells. The data are expressed as the means and SD of three experiments. * p < 0.05 vs. ctrl; # p< 0.05 vs. CdCl₂.