The interplay between food components and gut microbiota has been considered an important factor affecting the functionality of health-promoting foods. In this study, the effects of the probiotic ...Lactobacillus paracasei A221 on the functionality and bioavailability of kaempferol-3-o-sophroside (KP3S), a kaempferol-glucoside contained in kale, were investigated in vitro and in vivo. Unlike the type strain NBRC15889, the A221 strain converted standard KP3S as well as the kaempferol-glucosides in kale extract into kaempferol (KP). Using an intestinal barrier model, treatment with A221 significantly improved the effects of kale extract on the barrier integrity in vitro. KP, but not KP3S, clearly induced similar effects, suggesting that KP contributes to the functional improvement of the kale extract by A221. Pharmacokinetics analyses revealed that the co-administration of A221 and KP3S significantly enhanced the amount of deconjugated KP in murine plasma samples at 3 h post-administration. Finally, the oral administration of KP to Sod1-deficinet mice, which is a good mouse model of age-related disease, clearly ameliorated various pathologies, including skin thinning, fatty liver and anemia. These findings suggest that Lactobacillus paracasei A221 is effective for enhancing the anti-aging properties of kaempferol-glucosides by modulating their functionality and bioavailability through the direct bioconversion.
Objectives
This work aimed at evaluating the effect of fermented ginseng (FG) and fermented red ginseng (FRG) against rat liver injury caused by paracetamol (acetaminophen (APAP)).
Methods
Aspartate ...aminotransferase (AST) and alanine aminotransferase (ALT) in the serum and histopathological changes in the liver were analysed to determine the degree of liver injury. Deoxyribonucleic acid (DNA) microarray analysis was performed to compare gene expression levels altered in the rat livers. Phosphorylated Jun‐N‐terminal kinase (JNK) in human hepatocellular carcinoma (HepG2) cells were detected using western blot analysis to investigate the anti‐inflammatory activity of compound K.
Key findings
Pretreatment with FG, containing compound K at high concentration, attenuated AST as well as ALT levels in rats, while no obvious effect was observed in the group that received FRG, whose content of compound K was lower than that of FG. In addition, the results of our histopathological analysis were consistent with changes in the serum biochemical analysis. DNA microarray analysis indicated that JNK‐ and glutathione S‐transferase (GST)‐related genes were involved in the hepatotoxicity. Notably, compound K, a major ginsenoside in FG, inhibited the phosphorylation of JNK in HepG2 cells.
Conclusions
FG was shown to possess hepatoprotective activity against paracetamol (APAP)‐induced liver injury better than FRG. Compound K might play an important role for an anti‐inflammatory activity of FG by inhibiting JNK signalling in the liver.
Neurotrophins such as NGF promote neuronal survival and differentiation via the cell surface TrkA neurotrophin receptor. Compounds with neurotrophic actions that are low in molecular weight and can ...permeate the blood–brain barrier are promising therapeutic agents against neurodegenerative diseases such as Alzheimer’s disease. Carnosic acid (CA), an electrophilic compound in rosemary, activates antioxidant responsive element (ARE)-mediated transcription via activation of Nrf2. In the present study, we discovered that CA strongly promotes neurite outgrowth of PC12h cells. NGF as well as CA activated Nrf2, whereas CA and NGF-mediated neuronal differentiation was suppressed by Nrf2 knockdown. On the other hand, CA activated TrkA-downstream kinase Erk1/2 independently of Nrf2. CA-induced p62/ZIP expression in an Nrf2-dependent manner, while the CA-induced neural differentiation was suppressed by p62/ZIP knockdown. Furthermore, CA-induced ARE activation was attenuated both by p62/ZIP knockdown and a Trk signal inhibitor. These results suggest that the CA induction of p62/ZIP by Nrf2 enhances TrkA signaling which subsequently potentiates Nrf2 pathway. This is the first demonstration that activation of the Nrf2-p62/ZIP pathway by a low-molecular natural electrophilic compound plays important roles in TrkA-mediated neural differentiation and may represent the common molecular mechanism for neurotrophic activities of electrophilic compounds.
In a previous study, we found that carnosic acid (CA) protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by
S-alkylation of the critical cysteine thiol of ...the Keap1 protein by the “electrophilic”
quinone-type of CA T. Satoh, K. Kosaka, K. Itoh, A. Kobayashi, M. Yamamoto, Y. Shimojo, C. Kitajima, J. Cui, J. Kamins, S. Okamoto, T. Shirasawa, S.A. Lipton, Carnosic acid, a
catechol-type electrophilic compound, protects neurons both in vitro and in vivo through activation of the Keap1/Nrf2 pathway via
S-alkylation of targeted cysteines on Keap1. J Neurochem., in press. In the present study, we used HT22 cells, a neuronal cell line, to test CA derivatives that might be more suitable for
in vivo use, as an electrophile like CA might react with other molecules prior to reaching its intended target. CA and carnosol protected the HT22 cells against oxidative glutamate toxicity. CA activated the transcriptional antioxidant-responsive element of phase-2 genes including hemeoxygenase-1, NADPH-dependent quinone oxidoreductase, and γ-glutamyl cysteine ligase, all of which provide neurorprotection by regulating cellular redox. This finding was confirmed by the result that CA significantly increased the level of glutathione. We synthesized a series of its analogues in which CA was esterified at its catechol hydroxyl moieties to prevent the oxidation from the
catechol to
quinone form or esterified at those moieties and its carbonic acid to stop the conversion from CA to carnosol. In both cases, the conversion and oxidation cannot occur until the alkyl groups are removed by an intracellular esterase. Thus, the most potent active form as the activator of the Keap1/Nrf2 pathway, the
quinone-type CA, will be produced inside the cells. However, neither chemical modulation potentiated the neruroprotective effects, possibly because of increased lipophilicity. These results suggest that the neuroprotective effects of CA critically require both free carboxylic acid and
catechol hydroxyl moieties. Thus, the hydrophilicity of CA might be a critical feature for its neuroprotective effects.
Peroxisome proliferator activated receptor (PPAR) is a nuclear receptor that is one of the transcription factors regulating lipid and glucose metabolism. Fermented ginseng (FG) is a ginseng fermented ...by Lactobacillus paracasei A221 containing minor ginsenosides and metabolites of fermentation. DNA microarray analysis of rat liver treated with FG indicated that FG affects on lipid metabolism are mediated by PPAR-α. To identify a PPAR-α agonist in FG, PPAR-α transcription reporter assay-guided fractionation was performed. The fraction obtained from the MeOH extract of FG, which showed potent transcription activity of PPAR-α, was fractionated by silica gel column chromatography into 16 subfractions, and further separation and crystallization gave compound 1 together with four known constituents of ginseng, including 20(R)- and 20(S)-protopanaxadiol, and 20(R)- and 20(S)-ginsenoside Rh1. The structure of compound 1 was identified as 10-hydroxy-octadecanoic acid by (1)H- and (13)C-NMR spectra and by EI-MS analysis of the methyl ester of 1. Compound 1 demonstrated much higher transcription activity of PPAR-α than the other isolated compounds. In addition, compound 1 also showed 5.5-fold higher transcription activity of PPAR-γ than vehicle at the dose of 20 μg/mL. In the present study, we identified 10-hydroxy-octadecanoic acid as a dual PPAR-α/γ agonist in FG. Our study suggested that metabolites of fermentation, in addition to ginsenosides, contribute to the health benefits of FG.
Colorectal cancer cells are unique in that they escape Fas-mediated cell death in the presence of Fas ligand, and we recently reported that AMP-activated protein kinase-related kinase 5 (ARK5) ...suppresses cell death signaling mediated by cell death receptor in Akt-dependent manner. In the current study, therefore, we examined whether ARK5 is involved in the escape from Fas-mediated cell death of colorectal cancer cells. Among 10 cell lines, ARK5 mRNA expression was observed in LoVo, SW480, and SW1116 cell lines. Interestingly, SW480 and SW1116 cell lines, but not LoVo cell line, showed expressions of both Fas ligand (FasL) and Fas mRNAs. SW620 cell line also showed FasL mRNA; however, Fas and ARK5 mRNAs were not detected. Furthermore, well-coincided expression among ARK5, FasL, and Fas mRNAs was observed in tumor tissues from patients with colorectal cancer, suggesting the suppression of FasL/Fas system-induced cell death by ARK5 in colorectal cancer cell lines. Intensive cell death, which was dependent on the FasL/Fas system was encountered when ARK5 antisense RNA (ARK5/AS) was introduced into SW480 cells. FLIP was expressed in only ARK5 mRNA-expressing cell lines, and ARK5/AS induced FLIP cleavage in a caspase-6-dependent manner. Amino-acid sequence analysis of caspase-6 revealed two putative sites of phosphorylation by ARK5 at Ser80 and Ser257. Although active caspase-6 overexpression induced cell death in SW480 and DLD-1 cell lines, SW480 cells, but not DLD-1 cells, exhibited strong resistance to procaspase-6 overexpression. Moreover, mutant caspase-6, in which the Ser257 was substituted by Ala (caspase-6/SA), induced cell death and FLIP degradation, even in SW480 cells. Active ARK5 was found to phosphorylate wild-type caspase-6 in vitro, but not caspase-6/SA, and the prevented activation of caspase-6 was promoted due to its phosphorylation by active ARK5 in vitro. On the basis of the results of this study, we propose that ARK5 negatively regulates procaspase-6 by phosphorylation at Ser257, leading to resistance to the FasL/Fas system.
Because survival and growth of human hepatoma cells are maintained by nutrient, especially glucose, glucose starvation induces acute cell death. The cell death is markedly suppressed by hypoxia, and ...we have reported involvement of AMP-activated protein kinase-α (AMPK-α), Akt, and ARK5 in hypoxia-induced tolerance. In the current study we investigated the mechanism of hypoxia-induced tolerance in human hepatoma cell line HepG2. ARK5 expression was induced in HepG2 cells when they were subjected to glucose starvation, and we found that glucose starvation transiently induced Akt and AMPK-α phosphorylation and that hypoxia prolonged phosphorylation of both protein kinases. We also found that hypoxia-induced tolerance was partially abrogated by blocking the Akt/ARK5 system or by suppressing AMPK-α expression and that suppression of both completely abolished the tolerance, suggesting that AMPK-α activation signaling and the Akt/ARK5 system play independent essential roles in hypoxia-induced tolerance. By using chemical compounds that specifically inhibit kinase activity of type I-transforming growth factor-β (TGF-β) receptor, we showed an involvement of TGF-β in hypoxia-induced tolerance. TGF-β1 mRNA expression was induced by hypoxia in an hypoxia-inducible factor-1α-independent manner, and addition of recombinant TGF-β suppressed cell death during glucose starvation even under normoxic condition. AMPK-α, Akt, and ARK5 were activated by TGF-β1, and Akt and AMPK-α phosphorylation, which was prolonged by hypoxia, was suppressed by an inhibitor of type I TGF-β receptor. Based on these findings, we propose that hypoxia-induced tumor cell tolerance to glucose starvation is caused by hypoxia-induced TGF-β1 through AMPK-α activation and the Akt/ARK5 system.
Electrophilic compounds are a newly recognized class of redox-active neuroprotective compounds with electron deficient, electrophilic carbon centers that react with specific cysteine residues on ...targeted proteins via thiol (S-)alkylation. Although plants produce a variety of physiologically active electrophilic compounds, the detailed mechanism of action of these compounds remains unknown. Catechol ring-containing compounds have attracted attention because they become electrophilic quinones upon oxidation, although they are not themselves electrophilic. In this study, we focused on the neuroprotective effects of one such compound, carnosic acid (CA), found in the herb rosemary obtained from Rosmarinus officinalis. We found that CA activates the Keap1/Nrf2 transcriptional pathway by binding to specific Keap1 cysteine residues, thus protecting neurons from oxidative stress and excitotoxicity. In cerebrocortical cultures, CA-biotin accumulates in non-neuronal cells at low concentrations and in neurons at higher concentrations. We present evidence that both the neuronal and non-neuronal distribution of CA may contribute to its neuroprotective effect. Furthermore, CA translocates into the brain, increases the level of reduced glutathione in vivo, and protects the brain against middle cerebral artery ischemia/reperfusion, suggesting that CA may represent a new type of neuroprotective electrophilic compound.
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