Neuroprotective Effects of Açaí ( Euterpe oleracea Mart.) against Rotenone In Vitro Exposure

Abstract

Neuropsychiatric diseases, such as bipolar disorder (BD) and schizophrenia (SCZ), have a very complex pathophysiology. Several current studies describe an association between psychiatric illness and mitochondrial dysfunction and consequent cellular modifications, including lipid, protein, and DNA damage, caused by cellular oxidative stress. Euterpe oleracea (açaí) is a powerful antioxidant fruit. Açaí is an Amazonian palm fruit primarily found in the lowlands of the Amazonian rainforest, particularly in the floodplains of the Amazon River. Given this proposed association, this study analyzed the potential in vitro neuropharmacological effect of Euterpe oleracea (açaí) extract in the modulation of mitochondrial function and oxidative metabolism. SH-SY5Y cells were treated with rotenone to induce mitochondrial complex I dysfunction and before and after we exposed the cells to açaí extract at 5 μg/mL. Treated and untreated cells were then analyzed by spectrophotometric, fluorescent, immunological, and molecular assays. The results showed that açaí extract can potentially increase protein amount and enzyme activity of mitochondrial complex I, mainly through NDUFS7 and NDUFS8 overexpression. Açaí extract was also able to decrease cell reactive oxygen species levels and lipid peroxidation. We thus suggest açaí as a potential candidate for drug development and a possible alternative BD therapy.

Figures

Figure 1

Representative high performance liquid chromatography profile of Euterpe oleracea freeze-dried hydroalcoholic extract. Gallic acid (peak 1), catechin (peak 2), chlorogenic acid (peak 3), caffeic acid (peak 4), p-coumaric acid (peak 5), epicatechin (peak 6), orientin (peak 7), vitexin (peak 8), cyanidin-3-0-glucoside (peak 9), luteolin (peak 10), apigenin (peak 11), and chrysin (peak 12).

Figure 2

Cell viability measurements. (a) SH-SY5Y cells exposure to 5, 15, and 30 nM of rotenone during 24 h showing significant cell mortality for all concentrations; (b, c, and d) SH-SY5Y cells exposure to different concentrations of açaí freeze-dried hydroalcoholic extract during 24, 48, and 72 h showing a hormetic cell response; (e–p) microscopic scanning of SH-SY5Y cells exposure to rotenone and/or açaí freeze-dried hydroalcoholic extract (5 μg/mL) showing the cytotoxic effect of rotenone and the protective action of açaí freeze-dried hydroalcoholic extract. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 3

Cellular oxidative phosphorylation pathway measurements. (a–j) Rotenone exposition decreased mitochondrial complex I and increased mitochondrial complexes II and III, and açaí freeze-dried hydroalcoholic extract normalized the protein expressions. (k and l) Rotenone decreased mitochondrial complex I enzyme activity and açaí freeze-dried hydroalcoholic extract was able to increase enzyme activity in both designs of experiment. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 4

Mitochondrial complex I subunits analysis by western blot and qRT-PCR assays. The order of treatment for all the blot images: 0, Rot. 5, Rot. 15, Rot. 30, A+R5, A+R15, A+R30, R5+A, R15+A, and R30+A; (a and b) NDUFS7 protein expression analysis; (c and d) NDUFS8 protein expression analysis; (e and f) NDUFV1 protein expression analysis; (g and h) NDUFV2 protein expression analysis. Gene expression analysis follows the same graph order where gray means normal gene expression, green means downregulation gene expression, and red means upregulation gene expression. (i and j) NDUFS7 gene expression analysis; (k and l) NDUFS8 gene expression analysis; (m and n) NDUFV1 gene expression analysis; (o and p) NDUFV2 gene expression analysis. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 5

Cell oxidative metabolism biomarker measurements. (a and b) Total levels of ROs measured by DCFH-DA assay; (c and d) lipid peroxidation measured by TBARS assay. While rotenone increased ROs levels in a dose-dependent way and also lipid peroxidation, açaí freeze-dried hydroalcoholic extract was able to decrease both biomarkers compared to the negative control. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Figure 6

Protective effects of açaí supplementation in neuron-like cells SH-SY5Y exposed to rotenone. ① Rotenone causing mitochondrial complex I dysfunction, increasing superoxide production, and decreasing ATP synthesis; ② açaí freeze-dried hydroalcoholic extract supplementation acting at cell nucleus; ③ açaí freeze-dried hydroalcoholic extract increasing NDUFS7 and NDUFS8 gene expression in particular; ④ NDUFS7 and NDUFS8 protein production in response to the gene overexpression; ⑤ mitochondrial complex I assembly by new protein subunits; ⑥ renormalization of the mitochondrial electron transport chain, decreasing the oxidative stress, and normalizing ATP synthesis. ⑦ Decreased lipid peroxidation in consequence of the oxidative metabolism balance recovery.