The protection mechanism of proline from D-galactosamine hepatitis involves the early activation of ROS-eliminating pathway in the liver.

The oral pre-administration of proline, one on the non-essential amino acids, has been shown to effectively protect the liver from D-galactosamine (GalN)-induced liver injury and dramatically improve the survival rate. In the previous study, we reported that protective effect of proline involves the early activation of IL-6/STAT-3 pathway, an anti-inflammatory and regenerative signaling in the liver. Reactive oxygen species (ROS) are mediator of cellular injury and play an important role in hepatic damage during GalN-induced hepatitis. The aim of this study is to investigate the effect of proline on ROS-eliminating system. The activities of major ROS-detoxifying enzymes, i.e., glutathione peroxidase (GP), glutathione reductase (GR), catalase, and the level of glutathione in the liver were determined. Catalase activity was significantly upregulated in proline group from 0 to 3 h after GalN-injection, although GP and GR were downregulated during this period, compared with control group. From 6 to 12 h, the level of reduced glutathione (GSH) was significantly higher and the ratio of GSH/oxidized glutathione (GSSG) tended to be higher in proline group. Consistently with this, at 6 h, the GR activity in the proline group was significantly higher, followed with the higher tendency of GP activity at 12 h. Catalase activity was also significantly higher at 12 h. Taken together, catalase was activated at the beginning, followed with the significant activation of glutathione redox system around 6 to 12 h in proline group. These results suggest that the elimination of ROS in the liver was accelerated in proline group compared with control group at the very early stage of GalN-induced hepatitis.

Proline protects liver from D-galactosamine hepatitis by activating the IL-6/STAT3 survival signaling pathway.

The oral administration of proline, one of the non-essential amino acids, has been shown to effectively protect the liver from D-galactosamine (GalN)-induced liver injury and to improve the survival rate. The aim of this study was to investigate the mechanism of this protective action of proline. We paid particular attention to the effect of proline on inflammatory activation, regenerative response, and the associated signal transduction in the liver. Male Fischer rats received intraperitoneal injections of GalN (1.4 g/kg) with or without the oral administration of proline (2 g/kg) 1 h before GalN treatment. Liver pathology, plasma indices of inflammation, and the level of proliferative marker in the liver were monitored. The hepatic activation of interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)-3 pathway, which is downstream of tumor necrosis factor (TNF)-α/nuclear factor-κB, was also studied. GalN induced massive inflammatory expansion in the liver, leading to a high death rate (60 %) more than 72 h after the treatment. Proline administration significantly suppressed inflammatory infiltration in the live after 48 h, which was accompanied by depletion of plasma TNF-α, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase. The mRNA expression of histone H3, a marker of proliferation, was significantly upregulated in the liver of proline-treated animals. Furthermore, IL-6/STAT-3 pathway, an anti-inflammatory and regenerative signaling pathway, was strongly activated prior to these observations, with the upregulated expression of downstream genes. These results suggest that the tissue-protective mechanism of proline involves the early activation of IL-6/STAT-3 pathway in the liver, with subsequent activation of the regenerative response and suppression of massive inflammatory activation.

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug-drug interactions via these transporters.

OBJECTIVES: We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug-drug interactions via these transporters. METHODS: Uptake studies using transporter-expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug-drug interactions. KEY FINDINGS: The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter-expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3-sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I(in,max,u) /IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug-drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. CONCLUSIONS: OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug-drug interactions appeared to be low.

Comparison of the rapidity of onset of the therapeutic effect between nateglinide and mitiglinide by PK/PD analysis in rats.

Nateglinide and mitiglinide are immediate short-acting insulinotropic agents. Both are administered preprandially to control postprandial hyperglycemia. Glinide drugs are characterized by immediate onset as well as rapid disappearance of effect as compared with sulfonylurea drugs. We examined the rapidity of onset of the therapeutic effect between nateglinide and mitiglinide by pharmacokinetic/pharmacodynamic analysis using the receptor-binding-dissociation model in rats. Nateglinide or mitiglinide was administered orally or intravenously to rats and blood samples were collected at various time-points post administration. The plasma concentrations of the unbound drug forms and the blood glucose were measured. When the simultaneous fitting of oral administration and intravenous administration was performed using the receptor-binding-dissociation model, the measured values exhibited good correspondence with the fitting curve. Moreover, the time-courses of changes of the receptor-binding rate (sulfonylurea receptor) were examined using the parameters (k (on): second-order binding association constant to the receptor, Φ: receptor-binding occupancy ratio) obtained from the analysis. The results showed that the binding rate, which is important for glinide drugs in the early phase after administration, was obviously higher for nateglinide than that for mitiglinide from 10 min after oral administration and between 0 and 30 min after intravenous administration. These results suggest a more rapid onset of the therapeutic effect of nateglinide than that of mitiglinide after the drug is distributed into the blood.

Effect of N-(p-coumaroyl)serotonin and N-feruloylserotonin, major anti-atherogenic polyphenols in safflower seed, on vasodilation, proliferation and migration of vascular smooth muscle cells.

SCOPE: The objective of this study is to investigate a vascular effect of N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS), major antioxidative indolic polyphenols in safflower seeds with anti-atherogenic properties, with emphasis on effects on vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Both CS and FS (each 10 to 100 µM) relaxed rat femoral arteries, which were pre-contracted by 10(-5) M phenylephrine or 50 mM KCl, independently of their endothelium. Both CS and FS also concentration-dependently inhibited the increase of cytosolic free Ca(2+) concentration ([Ca(2+) ](i) ) that was induced by KCl or 5-hydroxytryptamine in cultured rat VSMCs. Next, we examined the effects of CS and FS on platelet-derived growth factor (PDGF)-BB-evoked proliferation and migration of the VSMCs. Both CS and FS inhibited PDGF-BB-evoked proliferation and migration of the VSMCs in a concentration-dependent manner. They also inhibited PDGF-BB-induced phosphorylation of PDGF receptor ß and ERK1/2, and Ca(2+) release from sarcoplasmic reticulum in the VSMCs in a concentration-dependent fashion. CONCLUSION: These results indicated a possible vascular effect of CS/FS to inhibit the activation of VSMCs by blocking the increase of [Ca(2+) ](i) and/or blocking PDGF signaling. These may explain a part of anti-atherogenic mechanism that underlies their ability to improve vascular distensibility and to inhibit aortic hyperplasia.

Combined administration of (L)-cystine and (L)-theanine enhances immune functions and protects against influenza virus infection in aged mice.

Cell-mediated and humoral immune responses are attenuated with aging. Intracellular glutathione (GSH) levels also decrease with aging. Previously, we have reported that combined administration of (L)-cystine and (L)-theanine enhances antigen-specific IgG production, partly through augmentation of GSH levels and T helper 2-mediated responses in 12-week-old mice. These findings suggest that combined administration of (L)-cystine and (L)-theanine to aged mice improves immune responses via increase of GSH synthesis. Here, we examined the effects of combined administration of (L)-cystine and (L)-theanine on antigen-specific antibody production and influenza virus infection in aged mice. Combined administration of these amino acids for 14 days before primary immunization significantly enhanced the serum antigen-specific IgM and IgG levels in 24-month-old mice. Furthermore, 13-month-old mice co-treated with these amino acids orally for 10 days had significantly lower lung viral titers than controls at 6 days after influenza virus infection. In addition, this co-treatment also significantly prevented the weight loss associated with infection. Enhancement of anti-influenza-virus IgG antibodies by combined administration of (L)-cystine and (L)-theanine was seen 10 days after infection. The significantly elevated serum interleukin-10/interferon-gamma ratio and gamma-glutamylcysteine synthetase mRNA expression, which is the rate-limiting enzyme of GSH synthesis, in the spleen 3 days after infection may have contributed to the observed beneficial effects. These results suggest that combined administration of (L)-cystine and (L)-theanine enhances immune function and GSH synthesis which are compromised with advanced age, and may become a useful strategy in healthy aging.

Effects of safflower seed extract supplementation on oxidation and cardiovascular risk markers in healthy human volunteers.

We previously demonstrated that safflower seed extract (SSE) and its major antioxidant constituents, serotonin hydroxycinnamic acid amides, suppressed LDL oxidation in vitro, decreased plasma autoantibody titres to oxidized LDL and attenuated atherosclerotic lesion formation in apoE-deficient mice. In this report, we examined whether SSE, rich in serotonin derivatives, could affect markers of oxidative stress, inflammation and aortic stiffness in healthy human subjects. Twenty Japanese male volunteers were studied at baseline, after 2.1 g SSE supplementation daily (providing 290 mg serotonin derivatives/d) for 4 weeks, and after a 4-week washout period. Significant reductions in circulating oxidized LDL, autoantibody titres to malondialdehyde-modified LDL, the soluble form of vascular cell adhesion molecule-1 (sVCAM-1), and urinary 8-isoprostane were observed after a 4-week intervention. Although there were no statistically significant differences in blood pressure or brachial-ankle pulse wave velocity (baPWV), an index of arterial stiffness, baPWV was lower than baseline in eleven of twenty subjects and was accompanied by a reduction in blood pressure. Statistically significant negative correlations were observed between the extent of initial cardiovascular risk markers (autoantibody titres, 8-isoprostane, sVCAM-1 and baPWV) and the effect of intervention. This suggested that individuals with elevated oxidative stress, inflammation, and/or arterial stiffness may receive more benefit from SSE supplementation.

Enhancement of antigen-specific immunoglobulin G production in mice by co-administration of L-cystine and L-theanine.

Supplementation with both cystine and glutamic acid increases the synthesis of glutathione (GSH), which has a marked effect on immune cell function, as compared with supplementation with either amino acid alone in human macrophages in vitro. As dietary glutamic acid is metabolized during intestinal transport, oral administration of L-theanine (gamma-glutamylethylamide), which is metabolized to glutamic acid mainly in the liver, may act as a glutamic acid donor in vivo. The present study was performed to investigate the effects of oral administration of L-cystine and/or L-theanine on GSH levels and immune responses. Co-administration of L-cystine (200 mg/kg) and L-theanine (80 mg/kg) for 11 days before immunization significantly increased the levels of total GSH in the liver 6 hr after immunization as compared with the levels in control mice. To examine the effects of administration of L-cystine and/or L-theanine on the balance of T helper (Th) 1/Th2 cell responses, the serum ratios of the Th1 cytokine, interferon (IFN)-gamma, and the Th2 cytokine, interleukin IL-10, were investigated. At 24 hr after immunization, co-administration significantly increased the IL-10/IFN-gamma ratio compared with the ratios of the control and single-administration mice. Furthermore, co-administration before primary immunization significantly enhanced serum antigen-specific IgG levels. Taken together, these findings suggest that co-administration of L-cystine and L-theanine enhances antigen-specific IgG production partly through augmentation of GSH levels and Th2-mediated responses.

Serotonin derivatives, major safflower (Carthamus tinctorius L.) seed antioxidants, inhibit low-density lipoprotein (LDL) oxidation and atherosclerosis in apolipoprotein E-deficient mice.

The effects of defatted safflower seed extract and its phenolic constituents, serotonin derivatives, on atherosclerosis were studied. Ethanol-ethyl acetate extract of safflower seeds (SSE) inhibited low-density lipoprotein (LDL) oxidation induced in vitro by an azo-containing free-radical initiator V70 or copper ions. Two serotonin derivatives [N-(p-coumaroyl)serotonin, CS; N-feruloylserotonin, FS] and their glucosides were identified as the major phenolic constituents of the extract. The study with chemically synthesized materials revealed that a majority of the antioxidative activity of SSE was attributable to the aglycones of these two serotonin derivatives. Orally administered CS and FS suppressed CuSO(4)-induced plasma oxidation ex vivo. Long-term (15 week) dietary supplementation of SSE (1.0 wt %/wt) and synthetic serotonin derivatives (0.2-0.4%) significantly reduced the atherosclerotic lesion area in the aortic sinus of apolipoprotein E-deficient mice (29.2-79.7% reduction). The plasma level of both lipid peroxides and anti-oxidized LDL autoantibody titers decreased concomitantly with the reduction of lesion formation. Serotonin derivatives were detected as both intact and conjugated metabolites in the plasma of C57BL/6J mice fed on 1.0% SSE diet. These findings demonstrate that serotonin derivatives of SSE are absorbed into circulation and attenuate atherosclerotic lesion development possibly because of the inhibition of oxidized LDL formation through their strong antioxidative activity.