Nigella and Milk Thistle Seed Oils: Potential Cytoprotective Effects against 7β-Hydroxycholesterol-Induced Toxicity on SH-SY5Y Cells

Abstract

Oxysterols are assumed to be the driving force behind numerous neurodegenerative diseases. In this work, we aimed to study the ability of 7β-hydroxycholesterol (7β-OHC) to trigger oxidative stress and cell death in human neuroblastoma cells (SH-SY5Y) then the capacity of Nigella sativa and Milk thistle seed oils (NSO and MTSO, respectively) to oppose 7β-OHC-induced side effects. The impact of 7β-OHC, associated or not with NSO or MTSO, was studied on different criteria: cell viability; redox status, and apoptosis. Oxidative stress was assessed through the intracellular reactive oxygen species (ROS) production, levels of enzymatic and non-enzymatic antioxidants, lipid, and protein oxidation products. Our results indicate that 7β-OHC (40 µg/mL) exhibit pr-oxidative and pro-apoptotic activities shown by a decrease of the antioxidant enzymatic activities and an increase of ROS production, lipid, and protein oxidation end products as well as nitrotyrosine formation and caspase 3 activation. However, under the pre-treatment with NSO, and especially with MTSO (100 µg/mL), a marked attenuation of oxidative damages was observed. Our study suggests harmful effects of 7β-OHC consisting of pro-oxidative, anti-proliferative, and pro-apoptotic activities that may contribute to neurodegeneration. NSO and especially MTSO showed potential cytoprotection against the cytotoxicity of 7β-OHC.

Keywords: 7β-hydroxycholesterol; antioxidants; antioxidants enzymes; cellular oxidative stress; neuroblastoma cells; neurodegenertion.

Figure 1

Antioxidant activities of NSO and MTSO. (A) DPPH assay. (B) FRAP assay. (C) Iron chelating Assay. NSO: Nigella seed oils; MTSO: Milk Thistle seed oils; EDTA: Ethylenediaminetetraacetic acid; AA: Ascorbic acid; FRAP: Ferric Reducing Antioxidant Power; DPPH 2,2-diphenyl-1-picrylhydrazyl.

Figure 2

Effect of 7β-OHC on cell viability and proliferation. (A) MTT assay: Cells were incubated with different concentrations of 7β-OHC (10–60 µg/mL) for 24 h. (B) MTT assay: Cells were incubated with different concentrations of H₂O₂ (50–150 µM) for 24 h. Data are represented as mean ± SD. 7β-OHC: 7β-hydroxycholesterol; H₂O₂: Hydrogen peroxide. Statistical analyses were performed using the Mann–Whitney test or student t test. * Statistical differences were significant between the control and 7β-OHC or H₂O₂-treated cells (p < 0.05).

Figure 3

Effect of 7β-OHC on Intracellular ROS production by H₂DCFDA fluorescence assay. Data are represented as mean ± SD. ROS: reactive oxygen species; H₂DCFDA: 2′7′-dichlorofluorescein diacetate; DCF: 2′7′-dichlorofluorescein; 7β-OHC: 7β-hydroxycholesterol; H₂O₂: Hydrogen peroxide. Statistical analyses were performed using the Mann–Whitney test or student t test. * Statistical differences were significant between the control and 7β-OHC or H₂O₂-treated cells (p < 0.05).

Figure 4

Effect of NSO and MTSO associated with 7β-OHC on cell viability and proliferation. (A) effect of NSO (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the MTT assay. (B) effect of MTSO (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the MTT assay. (C) effect of trolox (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the MTT assay. (D) effect of NSO (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the trypan blue exclusion assay. (E) effect of MTSO (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the trypan blue exclusion assay. (F) effect of trolox (50–150 µg/mL) associated with 7β-OHC (40 µg/mL) on cell viability and proliferation by the trypan blue exclusion assay. Data are represented as mean ± SD. 7β-OHC: 7β-hydroxycholesterol; NSO: Nigella sativa seed oil; MTSO: Milk Thistle seed oil; MTT: Methyl thiazolyldiphenyl-Tetrazolium Bromide. Statistical analyses were performed using the Mann–Whitney test or student t test. * Statistical differences were significant between the control and or 7β-OHC-treated cells (p < 0.05). ^# Statistical differences were significant between 7β-OHC-treated cells and 7β-OHC + (NSO or MTSO) -treated cells (p < 0.05).

Figure 5

The cytoprotective effects of NSO and MTSO to proteins. (A) Confocal immunofluorescence microscopy observation of Nitrotyrosine (green fluorescence) with DAPI-stained nuclei (blue fluorescence) in SHSY5Y cells. (B) Nitrotyrosine quantification. Data are shown as mean ± SD. A.U: arbitrary units; 7β-OHC: 7β-hydroxycholesterol; NSO: Nigella sativa seed oil; MTSO: Milk Thistle seed oil. Statistical analyses were performed using the Mann–Whitney test or student t test. * Statistical differences were significant between the control and 7β-OHC-treated cells (p < 0.05). ^# Statistical differences were significant between the 7β-OHC-treated cells and 7β-OHC + (NSO or MTSO)-treated cells (p < 0.05).

Figure 6

The cytoprotective effects of NSO and MTSO against cell death (A) Confocal immunofluorescence microscopy observation of cleaved caspase 3 (green fluorescence) with DAPI-stained nuclei (blue fluorescence) in SHSY5Y cells. (B) Cleaved caspase 3 quantification. Data are shown as mean ± SD. A.U: arbitrary units; 7β-OHC: 7β-hydroxycholesterol; NSO: Nigella sativa seed oil; MTSO: Milk Thistle seed oil. Statistical analyses were performed using the Mann–Whitney test or student t test. * Statistical differences were significant between the control and 7β-OHC-treated cells (p < 0.05). ^# Statistical differences were significant between the 7β-OHC-treated cells and 7β-OHC + (NSO or MTSO)-treated cells (p < 0.05).