Curcumin structure-function, bioavailability, and efficacy in models of neuroinflammation and Alzheimer's disease

Abstract

Curcumin can reduce inflammation and neurodegeneration, but its chemical instability and metabolism raise concerns, including whether the more stable metabolite tetrahydrocurcumin (TC) may mediate efficacy. We examined the antioxidant, anti-inflammatory, or anti-amyloidogenic effects of dietary curcumin and TC, either administered chronically to aged Tg2576 APPsw mice or acutely to lipopolysaccharide (LPS)-injected wild-type mice. Despite dramatically higher drug plasma levels after TC compared with curcumin gavage, resulting brain levels of parent compounds were similar, correlating with reduction in LPS-stimulated inducible nitric-oxide synthase, nitrotyrosine, F2 isoprostanes, and carbonyls. In both the acute (LPS) and chronic inflammation (Tg2576), TC and curcumin similarly reduced interleukin-1beta. Despite these similarities, only curcumin was effective in reducing amyloid plaque burden, insoluble beta-amyloid peptide (Abeta), and carbonyls. TC had no impact on plaques or insoluble Abeta, but both reduced Tris-buffered saline-soluble Abeta and phospho-c-Jun NH(2)-terminal kinase (JNK). Curcumin but not TC prevented Abeta aggregation. The TC metabolite was detected in brain and plasma from mice chronically fed the parent compound. These data indicate that the dienone bridge present in curcumin, but not in TC, is necessary to reduce plaque deposition and protein oxidation in an Alzheimer's model. Nevertheless, TC did reduce neuroinflammation and soluble Abeta, effects that may be attributable to limiting JNK-mediated transcription. Because of its favorable safety profile and the involvement of misfolded proteins, oxidative damage, and inflammation in multiple chronic degenerative diseases, these data relating curcumin dosing to the blood and tissue levels required for efficacy should help translation efforts from multiple successful preclinical models.

Figures

Fig. 1

Schematic paradigms of curcumin and TC treatments. A, mice were treated with Curc or TC by gav, i.p., or i.m. using doses of 0.4, 0.4, or 0.2 µmol (148, 148, or 73.4 µg), respectively. B, aged Tg2576 APPsw transgenic mice were fed diets ad libitum with or without curcumin and TC for 4 months at 500 ppm (500 mg/kg chow).

Fig. 2

Detection of curcumin or TC in plasma and brain after acute injection or chronic feeding. Structural differences between curcumin (A) and TC (B) are indicated by circles, highlighting the presence (curcumin) or absence (TC) of diketone bridge. HPLC chromatograms with UV detection for plasma (C–D and G–J) and brain (E and F) are shown for curcumin (left) and TC (right). Control samples only showed the ISD peak, with no TC or curcumin peaks (C and D). At 262 nm, curcumin was detected at peak retention time 5.56 min, and ISD at 10.898 min (E, G, and I), whereas TC was detected at 10.6 min and ISD at 19.9 min (F, H, and J). mAU, milliabsorbance unit. I and J, chromatograms of plasma collected from mice fed for 4-month ad libitum feeding with curcumin in chow (2000 ppm; n = 5) revealed predominantly curcumin (I), but also significant bioconversion to TC (J). LC/MS/MS-MRM chromatograms of a brain extract from mice chronically fed the parent compound curcumin (K) show clear peaks for Curc (m/z 371→149 transition) and TMC (m/z 395.2→365.1 transition), and in addition a peak for TC (m/z 371.1→235 transition), which elutes as an earlier and broader peak on the chromatographic system used. ISD, internal standard.

Fig. 3

Curcumin and TC suppressed LPS-induced iNOS protein and mRNA. Mice injected with LPS or vehicle, were sacrificed 4 h after administration of curcumin (A) or TC (B), and the supernatant of TBS-extracted brains was electrophoresed on Western blot and immunostained with anti-iNOS and β-actin. Representative lanes and their densitometric quantitation are shown. C, RNA was extracted from brain and measured for iNOS mRNA using quantitative RT-PCR. D, percentage of iNOS inhibition was regressed on curcumin or TC concentrations. Closed circles, curcumin; open circles, TC. Values shown are the amount of iNOS mRNA as the mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent a significant difference compared with positive controls (LPS treatment alone; n = 4).

Fig. 4

Acute injection of TC or curcumin similarly attenuated LPS induced-IL-1β or F2 isoprostane. A, quantitation of IL-1β levels in mouse brain homogenates was determined by sandwich ELISA. LPS injection (i.p.) increased IL-β levels more than 6-fold, an effect that was partially (>50%) suppressed by acute administration of either curcumin or TC, regardless of route of administration. B, either curcumin or TC levels correlated positively with percentage of IL-1β inhibition. C, lipid extracts of brain were measured for 8-iso-PGF2α by ELISA. The 40% increase in 8-iso-PGF2α caused by LPS was partially reduced by i.p. injection and completely suppressed by i.m injection of either compound. D, brain curcumin or TC correlated positively with brain F2 isoprostane inhibition. Values shown are the mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent significant difference compared with positive controls (LPS treatment alone; n = 4).

Fig. 5

Acute curcumin or TC similarly suppresses LPS induction of brain NT, but curcumin is more effective at suppressing carbonyls. A, NT proteins increased more than 7-fold after LPS from mouse brain homogenates measured by Western blot with anti-nitrotyrosine antibody and normalized to β-actin. Injection (i.m.) of either compound suppressed NT induction. Injection (i.p.) of curcumin partially suppressed NT induction, whereas i.p. injection of TC did not affect NT induction. Oxidized protein levels of brain were determined in the lysis-extracted supernatant of the TBS-insoluble pellet using Oxyblot analysis with an anti-DNP antibody. Representative lanes are shown and quantified for curcumin- (B) or TC-(C) fed mice. Values shown are the mean ± S.D. *, p < 0.05 and **, p < 0.01 represent a significant difference compared with positive controls (LPS treated alone; n = 4).

Fig. 6

Brain levels and efficacy of curcumin and TC administered by gavage are increased by fasting. Four hundred and eighty micrograms of curcumin or TC was administered to mice by gavage with fasting (FAST) or nonfasting (nFAST). After 4 h, brains were removed, and then they were prepared for the analysis of curcumin and TC levels by HPLC (A) and iNOS protein (B) measured by Western blot of iNOS and normalized with β-actin (endogenous control). When administered with food, brain curcumin levels were not detectable, and TC levels were reduced 50% compared with if mice had fasted. Curcumin and TC reduced iNOS less if administered with food. Values shown are the mean ± S.D. *, p < 0.05 and **, p < 0.01 represent a significant difference compared with control (con, untreated; n = 4), curcumin, or TC by gav (n = 4, each treatment). ND, not detectable.

Fig. 7

Dietary curcumin seems more effective than TC in reducing plaque pathology in the Tg2576 APPsw mouse, but both reduce soluble Aβ levels. Curcumin or TC was administered to aged Tg2576 mice for 4 months (12–16 months old) in the chow (500 ppm) during accelerated plaque deposition. Representative micrographs stained with antibodies to Aβ (DAE; anti-Aβ1-13). Compared with Tg+ mice on control diet (A and B), mice on curcumin diet showed a noticeable reduction in plaque size and number (C and D). However, sections from mice on dietary TC (E and F) showed plaque distribution similar to those from mice on control diet. Although image analysis quantification confirmed that dietary curcumin could reduce plaque size (G) and plaque burden (H), dietary TC seemed to have no impact on plaque pathology. I, insoluble (guanidine-insoluble) and soluble Aβ (TBS-soluble) in cortical homogenates were also evaluated by Aβ sandwich ELISA. Curcumin, but not TC, reduced Aβ levels in detergent-insoluble fraction. J, in contrast, both drugs suppressed soluble Aβ levels (in TBS-extracted brain homogenates), with a trend for TC being more potent. Magnification bar, 75 µm. Statistical analysis of Western data were performed by one-way ANOVA and immunohistochemistry data by 2 × 2 ANOVA (treatment × brain region), and values shown are the mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent significant difference of control (n = 5) and standard diet (n = 5) compared with curcumin (n = 9) and TC (n = 7) treatment of Tg+ mice, respectively.

Fig. 8

Curcumin or TC diets ameliorated Tg2576-dependent glial activation. Micrographs demonstrated that compared with brains of Tg− mice (A and B), brains of Tg+ mice (C and D) showed increased staining for GFAP. Compared with Tg+ mice fed control diet, Tg+ mice fed either curcumin (E and F) or TC (G and H) in chow showed reduced GFAP (glial activation). Quantification of percentage GFAP staining demonstrated significant attenuation of transgene-dependent gliosis by both TC and curcumin (I). Values shown are the mean ± S.D.**, p < 0.01 represents significant difference of curcumin (n = 9) or TC (n = 7) treatment of mice compared with control diet (n = 5). Magnification bar, 75 µm.

Fig. 9

In APPsw Tg2576 mice, both dietary curcumin or TC similarly reduced IL-1β and pJNK, whereas curcumin, but not TC, reduced carbonyls. A, compared with Tg+ mice on control diet, mice fed curcumin or TC showed 25% reduction in IL-1β levels as measured by sandwich ELISA of TBS-extracted supernatant fraction of brain homogenate. B, compared with Tg+ mice on control diet brain curcumin reduced carbonyls measured on Western with anti-DNP antibody from the detergent buffer-extracts of brain homogenates. Although TC-fed mice showed a trend for reduction, it was not statistically significant. C, Western analysis of main pJNK bands of 46 and 56 kDa showed that compared with mice fed control diet, mice fed TC or curcumin showed reduced pJNK. D, densitometric quantification showed that compared with control fed Tg+ mice, mice fed curcumin or TC showed reductions in pJNK, with TC showing the greater reduction. E, pJNK was positively correlated with soluble Aβ. Values shown are the mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent significant difference of curcumin (n = 9) or TC (n = 7) treatment of Tg+ mice with control diet (n = 5).

Fig. 10

Differential impact of TC versus curcumin on iNOS, Aβ toxicity and Aβ aggregation in vitro. Primary cortical neuron cultures (A) or the microglial cell line BV-2 (data not shown) were treated with 1 µg/ml LPS, and iNOS protein levels in the detergent-soluble fraction were measured by Western blot with or without curcumin (2.5 µM), TC (2.5 µM), and curcumin + TC (1.25 µM each), using β-actin as an endogenous control to ensure equal protein loading. LPS induction of iNOS was attenuated by curcumin (50%), TC (80%), or combined curcumin + TC (95%). B, attenuation of Aβ42 oligomer (500 nM)-induced toxicity (% maximal LDH) in SH-SY5Y neuroblastoma by pretreatment with curcumin (40%), TC (20%), or curcumin + TC (70%). C, TC, but not curcumin minimized the reduction in viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium reduction] in MC65 neuroblastoma cells caused by intraneuronal expression of C99, 3 days after tetracycline withdrawal. D and E, Western blot for Aβ immunoreactivity bands (6E10) to assess drug effect on aggregation of specific molecular weight oligomers. Impact of cotreatment of TC and curcumin on aggregation of low-dose (5 µM) Aβ (D) or high-dose (67 µM) Aβ (E), initially monomerized with HFIP. F, both TC and curcumin reduced levels of oligomeric-specific antibody A11 during oligomerization of 11 µM Aβ, using starting curcuminoid/Aβ molar ratio of 1.45 to 1. Equal loading is demonstrated by a second dot blot with 6E10. Values shown are the mean ± S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 represent significant differences compared with positive control (n = 4) and treatments (n = 4).