Nutr Res Pract.  2020 Oct;14(5):438-452. 10.4162/nrp.2020.14.5.438.

Curcumin and hesperetin attenuate D-galactose-induced brain senescence in vitro and in vivo

• Lee J
• Kim YS
• Kim E
• Kim Y
• Kim Y

• KMID: 2506882
• DOI: http://doi.org/10.4162/nrp.2020.14.5.438

Abstract

BACKGROUND/OBJECTIVES
Brain senescence causes cognitive impairment and neurodegeneration. It has also been demonstrated that curcumin (Cur) and hesperetin (Hes), both antioxidant polyphenolic compounds, mediate anti-aging and neuroprotective effects. Therefore, the objective of this study was to investigate whether Cur, Hes, and/or their combination exert anti-aging effects in D-galactose (Dg)-induced aged neuronal cells and rats.
MATERIALS/METHODS
SH-SY5Y cells differentiated in response to retinoic acid were treated with Cur (1 μM), Hes (1 μM), or a combination of both, followed by 300 mM Dg. Neuronal loss was subsequently evaluated by measuring average neurite length and analyzing expression of β-tubulin III, phosphorylated extracellular signal-regulated kinases, and neurofilament heavy polypeptide. Cellular senescence and related proteins, p16 and p21, were also investigated, including their regulation of antioxidant enzymes. In vivo, brain aging was induced by injecting 250 mg/kg body weight (b.w.) Dg. The effects of supplementing this model with 50 mg/kg b.w. Cur, 50 mg/kg b.w. Hes, or a combination of both for 3 months were subsequently evaluated. Brain aging was examined with a step-through passive avoidance test and apoptosis markers were analyzed in brain cortex tissues.
RESULTS
Cur, Hes, and their combination improved neuron length and cellular senescence by decreasing the number of β-gal stained cells, down-regulated expression of p16 and p21, and up-regulated expression of antioxidant enzymes, including superoxide dismutase 1, glutathione peroxidase 1, and catalase. Administration of Cur, Hes, or their combination also tended to ameliorate cognitive impairment and suppress apoptosis in the cerebral cortex by downregulating Bax and poly (ADP-ribose) polymerase expression and increasing Bcl-2 expression.
CONCLUSIONS
Cur and Hes appear to attenuate Dg-induced brain aging via regulation of antioxidant enzymes and apoptosis. These results suggest that Cur and Hes may mediate neuroprotective effects in the aging process, and further study of these antioxidant polyphenolic compounds is warranted.

Keyword

Curcumin; hesperetin; brain; aging; D-galactose

Figure

• 

  Fig. 1 Effects of Cur, Hes, and their combination on cell viability and proliferation. MTT assays were performed to evaluate cell viability and proliferation. (A) SY5Y cells were treated with Dg (300 mM or 400 mM) for 48 h. (B, C) SY5Y cells were pretreated with Cur or Hes (0.3, 1, or 3 μM) for 24 h, and then were treated with 300 mM Dg for 72 h. Data are represented as the mean ± SEM. Letters label the bars which represent significant differences (P < 0.05).Ctrl, control; Dg, D-galactose; Cur, curcumin; Hes, hesperetin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; SEM, standard error of the mean.

• 

  Fig. 2 Effect of Cur (1 μM) and Hes (1 μM) on synaptic loss and neuronal degeneration in Dg-induced aged SH-SY5Y cells. (A) Representative images (100× magnification) are shown. Neurite length was measured by Image J software. (a) Ctrl; (b) D-gal; (c) Cur; (d) Hes; (e) Combi; (f) Quantified average neurite length from each group. (B, C) The protein expressions of neuronal markers, β-tubulin III, p-ERK, and NEFH were analyzed by Western blot assay. α-tubulin was used as a loading control. (B) Representative blots, (C) quantified proteins levels. Data are represented as the mean ± SEM. Letters label the bars which represent significant differences (P < 0.05).Ctrl, control; Dg, D-galactose; Cur, curcumin; Hes, hesperetin; Combi, curcumin + hesperetin; ERK, extracellular signal-regulated kinases; NEFH, neurofilament heavy polypeptide; SEM, standard error of the mean.

• 

  Fig. 3 Effect of Cur (1 μM) and Hes (1 μM) on cellular senescence in Dg-induced aged SH-SY5Y cells. (A) Representative images (100× magnification) are shown. Cellular senescence was examined by SA-β-gal staining. (a) Ctrl; (b) D-gal; (c) Cur; (d) Hes; (e) Combi; (f) Quantification of β-galactosidase stained cells. (B, C) The protein expressions of cellular senescence markers, p16 and p21 were analyzed by Western blot assay. α-tubulin was used as a loading control. (B) Representative blots, (C) quantified proteins level data are represented as the mean ± SEM. Letters label the bars which represent significant differences (P < 0.05).Ctrl, control; Dg, D-galactose; Cur, curcumin; Hes, hesperetin; Combi, curcumin + hesperetin; SEM, standard error of the mean.

• 

  Fig. 4 Effect of Cur (1 μM) and Hes (1 μM) on the expression of anti-oxidant enzymes in Dg-induced aged SH-SY5Y cells. The protein expressions of anti-oxidant enzymes, SOD1, Gpx1, and catalase were analyzed by Western blot assay. α-tubulin was used as a loading control. (A) Representative blots, (B) quantified proteins level. Data are represented as the mean ± SEM. Letters label the bars which represent significant differences (P < 0.05).Ctrl, control; Dg, D-galactose; Cur, curcumin; Hes, hesperetin; Combi, curcumin + hesperetin; SOD1, superoxide dismutase 1; Gpx1, glutathione peroxidase 1; SEM, standard error of the mean.

• 

  Fig. 5 Effect of Cur and Hes on cognitive impairment and neuronal apoptosis in Dg-induced aged rats. (A) Memory retention was investigated by passive avoidance test in Dg-induced aged rats. Training latency was measured on acquisition day. After 24 h of electric shock, memory retention was recorded to a maximum of 180 sec. (B, C) The apoptosis-associated proteins expressions were analyzed in cerebral cortex of Dg-induced aged rats. β-actin was used as a loading control. (B) Representative blots, (C) quantified protein levels. Data are represented as the mean ± SEM. Letters label the bars which represent significant differences (P < 0.05).Ctrl, control (n = 10); Dg, 250 mg/kg b.w D-galactose (n=10); Cur, 250 mg D-galactose + 50 mg/kg b.w. curcumin (n=10); Hes, 250 mg/kg b.w. D-galactose + 50 mg hesperetin (n = 9); Combi, 250 mg/kg b.w. D-galactose + 50 mg/kg b.w. curcumin + 50 mg/kg. b.w. hesperetin (n=10); SEM, standard error of the mean.

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