Hesperetin Nanocrystals Improve Mitochondrial Function in a Cell Model of Early Alzheimer Disease

Abstract

Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP₆₉₅) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ_1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP₆₉₅ cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aβ_1-40 levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP₆₉₅ cells. Thus, it seems that the mechanisms involved may not be linked to altered Aβ production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD.

Keywords: Alzheimer disease; ROS; amyloid beta; hesperetin; mitochondria; mitochondria dysfunction; nanoparticles; peroxidase activity.

Figure 1

Respiration, ATP, Aβ_1-40 level, ROS level, and peroxidase activity of SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells. (A) Respiration of SH-SY5Y-MOCK and -APP cells adjusted to cell count. Respective mean values ± SD are shown. *p < 0.05, **p < 0.01, ***p < 0.001, and **** p < 0.0001. Significance was determined by Student’s unpaired t-test, N = 12. (B) SH-SY5Y-APP₆₉₅ cells exhibit reduced ATP levels compared to SH-SY5Y-MOCk control cells. ATP levels were determined after 24 h seeding by bioluminescence assay. Respective mean values ± SD are shown. Significance was determined by Student´s unpaired t-test (****p < 0.0001), N = 8. (C) Concentration of Aβ_1-40 levels in SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells. ** p < 0.01. Significance was determined by Student’s unpaired t-test. N = 6. (D) ROS levels in RUF/20⁴ cells in SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells. Respective mean values ± SD are shown. Significance was determined by Student´s unpaired t-test (*** p < 0.001), N = 8. (E) Peroxidase activity in SH-SY5Y-MOCK cells compared to -APP₆₉₆ cells. Respective mean values ± SD are shown. Significance was determined by Student´s unpaired t-test, N = 8. (F) Membrane integrity by LDH release level in RUF/20⁴ cells in SH-SY5Y-APP₆₉₅ cells compared to SH-SY5Y-MOCK cells. Respective mean values ± SD are shown, N = 4.

Figure 2

Effect of HstN and HstP on membrane integrity of SH-SY5Y-APP₆₉₅ cells. (A) Effect on membrane integrity after incubation with HstN. PC served as control. (B) Effect on membrane integrity after incubation with HstP. EtOH served as control. Respective mean values ± SD are shown, N = 4. Significance was determined by unpaired Student’s t-test. HstN, hesperetin nanocrystal; HstP, hesperetin in pure form; PC, PlantaCare; EtOH, ethanol.

Figure 3

Respiration, citrate synthase activity, and ATP level of SH-SY5Y cells after incubation with hesperitin nanocrystals (HstN). Cells were incubated for 24 h with 10 µM HstN or PlantaCare as control, N = 12. Respective mean values ± SD are shown. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Significance was determined by Student’s unpaired t-test. Respiration of (A) SH-SY5Y-MOCK and (B) SH-SY5Y-APP₆₉₅ cells adjusted to international units (IU) of citrate synthase activity. Citrate synthase activity of (C) SH-SY5Y-MOCK cells and (D) SH-SY5Y-APP₆₉₅ cells. Values are given as IU of citrate synthase activity. ATP level of (E) SH-SY5Y-MOCK cells and (F) SH-SY5Y-APP₆₉₅ cells after 24 h incubation with 0.01–10 µM HstN. Cells treated with cell culture medium served as control (100%).

Figure 4

Western blot analysis of mitochondrial respiratory chain complexes of five experiments. Protein levels of (A) complex I subunit NDUF88, (B) complex IV subunit COXII, and (C) complex V subunit ATP5D in % of control after incubation with 10 µM HstN or PC (control) in SH-SY5Y-APP₆₉₅ cells. (D) Lower part: representative Western blots of one experiment. GAPDH served as loading control. Respective mean values ± SD are shown. Significance was determined by Student’s unpaired t-test. HstN, hesperetin nanocrystal.

Figure 5

Effect of HstN and HstP on Aβ1-40 levels in SH-SY5Y-APP₆₉₅ cells. Concentration of Aβ_1-40 in SH-SY5Y-APP₆₉₅ cells: (A) incubated for 24 h with HstN, with PC as control, and (B) incubated for 24 h with HstP, with ethanol as control. Values are adjusted to protein concentrations. Respective mean values ± SD are shown, N = 6. Significance was determined with unpaired Student’s t-test. ns, not significant; HstN, hesperetin nanocrystal; HstP, hesperetin in pure form; PC, PlantaCare; EtOH, ethanol.

Figure 6

Effect of HstN and HstP on peroxidase activity in SH-SY5Y-APP₆₉₅ cells. Peroxidase activity in SH-SY5Y-APP₆₉₅ cells: (A) incubated for 24 h with HstN, with PC as control, and (B) incubated for 24 h with HstP, with ethanol as control. Respective mean values ± SD are shown, N = 10. Significance was determined by unpaired Student’s t-test. **** p < 0.0001. HstN, hesperetin nanocrystal; HstP, hesperetin in pure form; PC, PlantaCare; EtOH, ethanol.

Figure 7

Effect of Aβ on peroxidase activity in SH-SY5Y-MOCK cells incubated with different concentrations of Aβ for 24 h. Respective mean values ± SD are shown, N = 7. Significance was determined by one-way ANOVA. * p < 0.05. Aβ, amyloid beta.

Figure 8

Effects of HstN and HstP on peroxidase activity of cytochrome c: (A) incubated with HstN, with PC as control (N = 6), and (B) incubated with HstP, with ethanol as control (N = 5). Respective mean values ± SD are shown. Significance was determined with unpaired Student’s t-test. * p < 0.05. HstN, hesperetin nanocrystal; HstP, hesperetin in pure form; PC, PlantaCare; EtOH, ethanol.

Figure 9

Effect of HstN and HstP on expression of ROS production in SH-SY5Y-APP₆₉₅ cells: (A) incubated for 24 h with HstN, with PC as control, and (B) incubated for 24 h with HstP, with ethanol as control. Respective mean values ± SD are shown, N = 10. Significance was determined with unpaired Student’s t-test. * p < 0.05. HstN, hesperetin nanocrystal; HstP, hesperetin in pure form; PC, PlantaCare; EtOH, ethanol.