Previous measurements of curcumin or TC using our published HPLC methods showed detection of TC or curcumin in tissue samples spiked with TC or curcumin. However, it was not known whether these methods were sufficiently sensitive or specific to detect unconjugated TC or curcumin in animals treated with curcumin or TC before sacrifice. We delivered curcumin or TC using three routes of administration to investigate the impact of GI tract on tissue levels and drug efficacy. The structures of curcumin and TC are shown in , respectively, illustrating that the double bonds if curcumin flanking the β-diketone bridge are lost upon reduction to TC. Data demonstrated successful detection of these compounds in treated mice (; ). Representative chromatograms from brains or plasma of vehicle-treated mice showed the internal standard peak, without signal for curcumin or TC, respectively (). However, when mice were injected i.m. with either drug, their respective peaks were present in both brain () and plasma fractions ().
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 (more ...)
Detection of curcumin and TC in plasma and brain after multiple dosing routes
We also examined metabolism of curcumin. Although in the acute study we did not detect metabolites of curcumin in the brain or plasma, measurable levels were obtained in the chronic study. HPLC analysis revealed that plasma containing the parent compound curcumin () also contained the metabolite TC (; ). The plasma values for the metabolite of mice fed 500 ppm curcumin were close to the lower limit of detection, but they were higher in animals fed a higher dose (2000 ppm). Likewise, the metabolite was also detected by LC/MS/MS in the brains of mice chronically fed the parent compound curcumin, using LC/MS/MS (; ). Brain levels of the metabolite were approximately 6-fold less than that of the parent compound ().
Parent and metabolite compounds in plasma and brain after chronic dietary administration
(acute study) shows that plasma levels of drug are higher with TC than with curcumin, regardless of route of administration. Similar results are observed in chronic study ().
Brain-to-plasma ratios of parent compounds were calculated. In the acute gavage study (), curcumin ratios were approximately 5-fold higher in brain than plasma), whereas TC brain-to-plasma ratios were much lower (), particularly in chronically fed mice (where brain TC levels were ~0.5-fold that of plasma; ).
Brain levels of the curcuminoids ranged from 0.7 to 6 µM for TC and from 1.4 to 3 µM for curcumin, depending on route of administration. We then assessed the association of these brain levels with anti-inflammatory effects, using iNOS as an index of inflammation. We induced inflammation by injecting LPS (i.p.), and then we analyzed brains of TC- or curcumin-treated mice for iNOS protein () and mRNA (). LPS caused robust increases in iNOS protein and mRNA. Although i.m. injection, which produced the highest brain drug levels, tended to be slightly more effective in attenuating these LPS responses, all routes of administering curcumin and TC caused large decreases in iNOS protein and mRNA. Regression analysis showed a close correlation between inhibition of iNOS mRNA and increasing concentrations of the administered curcuminoids (). The EC50 value for iNOS mRNA inhibition in vivo was 1.186 and 0.701 µM for curcumin and TC, respectively.
Because LPS induces CNS expression of IL-1β (Hsu and Wen, 2002
), an inflammatory cytokine known to be elevated in neuroinflammatory diseases, including AD (Akiyama et al., 2000
), we evaluated the relative impact of curcumin or TC on IL-1 β induction. Although LPS effectively induced robust elevations in IL-1 β brain levels compared with vehicle, all routes of administration of either curcumin or TC equally inhibited this response (). The EC50
value for IL-1β inhibition in vivo was 1.722 and 1.286 µM for curcumin and TC, respectively ().
LPS can induce brain lipid peroxidation, as measured by oxidation of arachidonic acid to F2 isoprostanes, which are elevated in AD and correlate with synaptic loss and (Montine et al., 2004
) and AD models (Frautschy et al., 2001
). Therefore, we investigated the impact of curcumin or TC on induction of F2 isoprostanes. Results showed that LPS increased F2 isoprostanes. Although acute oral delivery of curcumin or TC failed to produce brain levels sufficient to modify isoprostanes, i.p. delivery slightly reduced, and i.m. delivery greatly reduced LPS-induced F2 isoprostanes (). The EC50
value for F2 isoprostane inhibition in vivo was 1.067 and 0.501 µM for curcumin and TC, respectively ().
Superoxide reaction with nitric oxide, resulting in increased peroxynitrite, typically occurs in neuroinflammatory diseases such as AD (Castegna et al., 2003
). An indirect index of peroxynitrite can be estimated by measuring anti-nitrotyrosine (NT)-reactive proteins, formed by peroxynitrite-mediated nitration of protein tyrosine residues. Results demonstrated that LPS induced prominent 75- and 90-kDa NT-reactive bands, but gavage with either compound was not effective in modifying NT (), similar to the failure of gavage to suppress F2 isoprostane induction. In contrast, curcumin was effective in suppressing LPS-induced protein oxidation, as measured by carbonyls, regardless of route of administration (), unlike TC that was only effective in reducing carbonyl induction if injected by i.m. ().
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 (more ...)
Because it has been reported that fasting may enhance absorption or use of curcumin (Chan et al., 1998
), and because chronic treatment is likely necessary for therapeutic intervention of diseases, we evaluated the impact of food ad libitum on curcumin and TC plasma and brain levels. We increased the gavage dose to 480 µg, which is closer to the daily dietary consumption of curcumin used to reduce AD pathogenesis in the Tg2576 mouse (Lim et al., 2001
). Compared with fasted mice, mice previously fed ad libitum showed a large reduction in absorption of both curcumin and TC (by gavage) as measured by their contents in the brain (). TC was not detectable in brain if given with food, and curcumin was detectable, but reduced. The diminished plasma levels associated with food intake correlated with impaired (curcumin) or abolished (TC) efficacy in inhibition of LPS-induced brain iNOS ().
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, (more ...)
Because curcumin was not detectable with acute gavage dosing with food (nonfasted), we then investigated whether it could be detected after chronic administration in chow. We chose doses previously shown to reduce oxidative damage, neuroinflammation and plaque pathology in the APPsw
Tg2576 mouse (Lim et al., 2001
). shows that in contrast to acute gavage, chronic oral dosing in diet of curcumin or TC resulted in detectable plasma levels of 0.095 and 0.73 µM, respectively. Like acute dosing, chronic dosing resulted in plasma levels of TC that were severalfold higher than those of curcumin.
We then evaluated the relative efficacies of curcumin- and TC-fed mice on plaque pathology in the Tg2576 APPsw
mouse during plaque deposition (12–16 months of age). Immunostaining for anti-Aβ revealed that, compared with control-fed mice, curcumin-fed mice showed a marked reduction in plaques (), similar to that described previously (Lim et al., 2001
). But this was not observed with TC-fed mice (). Quantitation of plaque size () and plaque burden () confirmed that curcumin, but not TC, reduced Aβ pathology. We then analyzed the dissected cortex for Aβ levels, and we showed that curcumin-fed, but not TC-fed animals had reduced Aβ in the insoluble fraction (). The only significant impact of TC on Aβ variables was on soluble Aβ, which it reduced more than curcumin ().
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 (more ...)
GFAP was analyzed immunohistochemically to evaluate the impact of curcumin and TC on the elevated gliosis in this transgenic model of AD (). Quantitation of GFAP confirmed the known induction of GFAP in this transgenic model. Both curcumin and TC significantly attenuated the gliosis associated with the transgene (). IL-1β is an index of neuroinflammation, thought to contribute to AD pathogenesis, and it was elevated in this model. Like gliosis, IL-1β was effectively reduced by both curcumin and TC compared with Tg+ mice on the control diet (). However, although carbonyls known to be induced by this transgene (Lim et al., 2001
) were reduced by curcumin as described previously, TC showed only a nonsignificant trend for reducing carbonyls (). Given that the stress-activated pJNK has been reported to be elevated in cortical homogenates of this model (Puig et al., 2004
) and JNK is a putative target of curcumin, we also examined the relative impacts of curcumin and TC on pJNK. Western analysis showed two bands at 46 and 56 kDa, which when quantified demonstrated that both curcumin and TC effectively reduced pJNK, with TC being particularly potent (). Because of data suggesting that pJNK may influence Aβ, we evaluated the association by a regression analysis, and we showed that the degree of TBS soluble Aβ reduction was proportional to that of pJNK reduction ().
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, (more ...)
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 (more ...)
In vitro data confirmed that both TC and curcumin were potent suppressors of iNOS in primary neurons () and in BV2 microglia (data not shown). Both protected from oligomeric Aβ toxicity in a neuroblastoma model as measured by LDH (). In contrast, using a model of intraneuronal APP C99 fragment accumulation (M-65 cells, provided by L. W. Jin; APP mutation lacking leader sequence), only TC protected from intraneuronal Aβ toxicity, possibly indicating differing neuroprotective mechanisms of TC and curcumin (). We then investigated the impact of TC and curcumin on Aβ oligomer formation (). Aggregating Aβ at low concentrations was robustly attenuated by curcumin, but not by TC (), similar to previous data (Yang et al., 2005
). Although TC had no impact on aggregation of Aβ at the lower concentration, at the higher Aβ concentration, TC seemed to selectively reduce aggregates of the intermediate molecular mass oligomers (30–200 kDa; ) and also the level of preaggregated oligomers (11 µM) in a cell-free dot blot assay using oligomer-specific A11 (). Unlike curcumin, TC seemed to increase (rather than reduce) very high aggregates associated with the stack (). When curcumin (but not TC) was added to preaggregated oligomers, intense monomeric bands occurred, suggesting deaggregation (data not shown).
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 (more ...)