Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

(A-C). Effects of RBE on the phosphorylation of p38MAPK, ERK, and JNK in non-activated microglia. Cells were treated with RBE (50–300 μg/ml) for 24 h followed by cell lyses and protein estimation. During stimulation, one of the wells in 6-well plate was incubated with LPS (10 ng/ml) for 30 min to be used as positive control to validate the functionality of antibodies against activated state of kinases. Whole cell lysates were subjected to western blots analyses. Representative blots (upper panel) and densitometry analyses (lower panel) are shown: A) p38 MAPK, B) pERK, and C) pJNK. Statistical analyses were carried out by using one-way ANOVA with post hoc Student-Newman-Keuls test (multiple comparisons). Results are expressed as means ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 compared control cells. (TIF 963 kb

Impact of Silibinin A on bioenergetics in PC12APPsw cells and mitochondrial membrane properties in murine brain mitochondria

Age-related multifactorial diseases, such as the neurodegenerative Alzheimer’s disease (AD), still remain a challenge to today’s society. One mechanism associated with AD and aging in general is mitochondrial dysfunction (MD). Increasing MD is suggested to trigger other pathological processes commonly associated with neurodegenerative diseases. Silibinin A (SIL) is the main bioactive compound of the Silymarin extract from the Mediterranean plant Silybum marianum (L.) (GAERTN/Compositae). It is readily available as a herbal drug and well established in the treatment of liver diseases as a potent radical scavenger reducing lipid peroxidation and stabilize membrane properties. Recent data suggest that SIL might also act on neurological changes related to MD. PC12APPsw cells produce low levels of human Aβ and thus act as a cellular model of early AD showing changed mitochondrial function. We investigated whether SIL could affect mitochondrial function by measuring ATP, MMP, as well as respiration, mitochondrial mass, cellular ROS and lactate/pyruvate concentrations. Furthermore, we investigated its effects on the mitochondrial membrane parameters of swelling and fluidity in mitochondria isolated from the brains of mice. In PC12APPsw cells, SIL exhibits strong protective effects by rescuing MMP and ATP levels from SNP-induced mitochondrial damage and improving basal ATP levels. However, SIL did not affect mitochondrial respiration and mitochondrial content. SIL significantly reduced cellular ROS and pyruvate concentrations. Incubation of murine brain mitochondria with SIL significantly reduces Ca2+ induced swelling and improves membrane fluidity. Although OXPHOS activity was unaffected at this early stage of a developing mitochondrial dysfunction, SIL showed protective effects on MMP, ATP- after SNP-insult and ROS-levels in APPsw-transfected PC12 cells. Results from experiments with isolated mitochondria imply that positive effects possibly result from an interaction of SIL with mitochondrial membranes and/or its antioxidant activity. Thus, SIL might be a promising compound to improve cellular health when changes to mitochondrial function occur

Concentrations of total curcuminoids in plasma, but not liver and kidney, are higher in 18- than in 3-months old mice

Background: Curcuminoids (curcumin, demethoxycurcumin, bis-demethoxycurcumin) are lipophilic polyphenols thought to be effective in the prevention and treatment of neurodegenerative disorders, of which mitochondrial dysfunction is a prominent feature. In particular, older people may thus benefit from increasing their curcuminoid intake. However until now, it is not investigated if there exist age differences in the bioavailability of curcuminoids and therefore, it is unclear if curcumin doses have to be adjusted to age. Thus, we explored if the tissue concentrations and biological activities of curcuminoids are affected by age. Methods: We investigated age-differences in the bioavailability and tissue distribution of curcuminoids and mitochondrial function in 3- and 18-months old mice fed a control diet or identical diets fortified with 500 or 2000 mg curcuminoids/kg for 3 weeks. Therefore, we measured curcuminoid concentrations in plasma, liver, kidney, and brain, basal and stress-induced levels of adenosine triphosphate (ATP) and mitochondrial membrane potential (MMP) in dissociated brain cells and citrate synthase activity of isolated mitochondria. Results: Plasma but not liver and kidney curcuminoid concentrations were significantly higher in older mice. Age did not affect ATP concentrations and MMP in dissociated brain cells. After damaging cells with nitrosative stress, dissociated brain cells from old mice had a higher MMP than cells from young animals and were therefore more resistant. Furthermore, this effect was enhanced by curcumin. Conclusion: Our data suggest that age may affect plasma concentrations, but not the tissue distribution of curcuminoids in mice, but has little impact on mitochondrial function in brain cells

Beneficial Effects of Ethanolic and Hexanic Rice Bran Extract on Mitochondrial Function in PC12 Cells and the Search for Bioactive Components

Mitochondria are involved in the aging processes that ultimately lead to neurodegeneration and the development of Alzheimer’s disease (AD). A healthy lifestyle, including a diet rich in antioxidants and polyphenols, represents one strategy to protect the brain and to prevent neurodegeneration. We recently reported that a stabilized hexanic rice bran extract (RBE) rich in vitamin E and polyphenols (but unsuitable for human consumption) has beneficial effects on mitochondrial function in vitro and in vivo (doi:10.1016/j.phrs.2013.06.008, 10.3233/JAD-132084). To enable the use of RBE as food additive, a stabilized ethanolic extract has been produced. Here, we compare the vitamin E profiles of both extracts and their effects on mitochondrial function (ATP concentrations, mitochondrial membrane potential, mitochondrial respiration and mitochondrial biogenesis) in PC12 cells. We found that vitamin E contents and the effects of both RBE on mitochondrial function were similar. Furthermore, we aimed to identify components responsible for the mitochondria-protective effects of RBE, but could not achieve a conclusive result. α-Tocotrienol and possibly also γ-tocotrienol, α-tocopherol and δ-tocopherol might be involved, but hitherto unknown components of RBE or a synergistic effect of various components might also play a role in mediating RBE’s beneficial effects on mitochondrial function

Additional file 2: Figure S2. of Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

RBE did not significantly alter the protein levels of COX-2 in non-stimulated microglia. Cells were treated with RBE (50–300 μg/ml) for 24 h followed by lyses and protein estimation. During stimulation of microglia, one well of the 6-well plate was incubated with LPS (10 ng/ml) for 24 h to be used as positive control to validate the functionality of COX-2-specific antibody. Whole cell lysates were subjected to western blot for COX-2 and β-actin. Representative blots for COX-2 and β-actin are shown (upper panel) and densitometry analyses were performed (lower panel). To confirm equal sample loading, membranes were stripped and re-probed for β-actin and the data were used for normalization. Statistical analyses were carried out by using one-way ANOVA with post hoc Student-Newman-Keuls test (multiple comparisons). Results are expressed as means ± SEM of three independent experiments. *** p < 0.001 compared with control cells. (TIF 323 kb

Additional file 5: Figure S5. of Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

(A-D). Effects of α-tocopherol on the release of PGE2 and cytokines. Influence of α-tocopherol on the release of PGE2, TNF-α, IL-1β, and IL-6 in LPS-activated microglia was also examined. Cells were pre-treated with RBE (50–300 μg/ml); subsequently, LPS (10 ng/ml) was added for 24 h. Afterwards, release of A) PGE2, B) TNF-α, C) IL-1β, and D) IL-6, was analyzed by using immunoassays. Data are presented as percentage control of LPS. Results are expressed as means ± SEM of three to four independent experiments. Statistical analyses were carried out by using one-way ANOVA with post hoc Student-Newman-Keuls test (multiple comparisons). * p < 0.05; ** p < 0.01; *** p < 0.001 compared with LPS (10 ng/ml)-activated cells. (TIF 685 kb

Additional file 1: Figure S1. of Rice bran derivatives alleviate microglia activation: possible involvement of MAPK pathway

(A-K). Possible effects of rice bran extract (RBE) on the proliferation of microglia. Microglia were treated with either RBE (300 μg/ml) alone or RBE (50–300 μg/ml) in combination with LPS (10 ng/ml) for a total of 48 h. Thereafter, samples were stained with propidium iodide and processed for proliferation assay (for detailed protocol, see the “Methods” section) by using flow cytometer. Samples were acquired with the FL-2 fluorescence channel set to a linear scale, in order to amplify the diploid DNA peak. Graph A) represents the dot plot of cells acquired on the basis of side scattered light (SSC) and forward scattered light (FSC) and B) represents the pulse processing by using pulse area vs. pulse width. Graphs C-I) are representative histograms after each treatment. Markers represent the percentage of cells in the G0/G1, S, and G2/M phases from left to right, respectively. J) Represents histogram after 48 h treatment of M-CSF (50 ng/ml) used as positive control for proliferation. K) Showing quantification of microglial cells in each phase of cell cycle after respective treatments. Data are presented in percentage of cells in each phase. Results are expressed as means ± SEM of three independent experiments. Statistical analyses were carried out by using one-way ANOVA with post hoc Student-Newman-Keuls test (multiple comparisons). * p < 0.05; compared with percentage of control cells in each phase. (TIF 536 kb