Nobuyuki Fukuishi

Magnolol and honokiol prevent learning and memory impairment and cholinergic deficit in SAMP8 mice

The therapeutic use of neurotrophic factors to treat neurodegenerative disorders, including Alzheimers disease, is considered feasible. Magnolol and honokiol, constituents of the Magnolia plant, are small organic compounds with neurotrophic activity. We investigated whether magnolol and honokiol can prevent age-related learning and memory impairment and cholinergic deficits in senescence-accelerated mice (SAM). Magnolol (1, 10 mg/kg) or honokiol (0.1, 1 mg/kg) were orally administered to SAMP8 mice once a day for 14 days in 2-month-old mice. Learning and memory performance were evaluated by passive avoidance tests and location and object novelty recognition tests. SAMP8 mice showed significant impairment of learning and memory at 4 and 6 months of age. This age-related learning and memory impairment was prevented by pretreatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Cholinergic neuron densities in the medial septum and vertical limb of the diagonal band of the forebrain were evaluated by an immunohistochemical analysis of choline acetyltransferase (ChAT). SAMP8 mice showed a significant cholinergic deficit at 6 months of age. These age-related cholinergic deficits were prevented by treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg). Moreover, SAMP8 mice showed decreased activity of Akt, a member of the prosurvival pathway, in the forebrain at 2 months of age. A 14-day treatment with either magnolol (10 mg/kg) or honokiol (1 mg/kg) enhanced phosphorylation of Akt in the forebrain at 2 months of age. These results suggest that magnolol and honokiol prevent age-related learning and memory impairment by preserving cholinergic neurons in the forebrain. These compounds may have potential therapeutic applications to various neurodegenerative disorders.

Bacterial components regulate the expression of Toll-like receptor 4 on human mast cells.

Abstract.Objectives and design:The aim of this study was to investigate whether the exposure of mast cells (MCs) to bacterial components affects the expression of Toll-like receptor (TLR) 4, and to elucidate the behavior of MCs during the early response to infection.Materials:Two human MC lines, HMC-1 and LAD2, were employed. Messenger RNA expression was observed by RT and real-time PCR. TLR4 expression was determined by Western blotting. TNF-α secretion was analyzed with ELISA. The degranulation ratio was measured with betahexosaminidase assay.Results:Although bacterial components increased TLR4 mRNA, only lipopolysaccharide (LPS) augmented the TLR4 protein expression. LAD2 pre-treated with LPS for 8 h resulted in 2-fold increased TNF-α secretion on LPS restimulation.Conclusion:These results suggest that the exposure of MCs to LPS may reinforce the innate immune system due to up-regulation of MC TLR4, followed by increased TNF-α release.

Does β-Hexosaminidase Function Only as a Degranulation Indicator in Mast Cells? The Primary Role of β-Hexosaminidase in Mast Cell Granules

β-Hexosaminidase, which is generally present in the lysosome, is essential for glycoprotein metabolism in the maintenance of cell homeostasis. In mast cells (MCs), large amounts of β-hexosaminidase are present in the granules as opposed to the lysosome, and the biological role of MC β-hexosaminidase has yet to be fully elucidated. Therefore, we investigated the biological role of β-hexosaminidase in MC granules. Bone marrow-derived MCs from C57BL/6 (BL/6-BMMC) or β-hexosaminidase gene–deficient (hexb−/−-BMMC) mice were transplanted into MC-deficient (WBB6F1/J-KitW/KitW-v [W/Wv]) mice to generate MC-reconstituted models. In asthma model experiments, no differences were observed in the symptoms of BL/6, W/Wv, BL/6-BMMC–reconstituted W/Wv, or hexb−/−-BMMC–reconstituted W/Wv mice. In Staphylococcus epidermidis experimental infection model experiments, the severity of symptoms and frequency of death were markedly higher in W/Wv and hexb−/−-BMMC–reconstituted W/Wv mice than in BL/6 and BL/6-BMMC–reconstituted W/Wv mice. The growth of S. epidermidis in an in vitro study was clearly inhibited by addition of BL/6-BMMC lysate, but not by addition of hexb−/−-BMMC lysate. Moreover, suppression of bacterial proliferation was completely recovered when bacteria were incubated with hexb−/−-BMMC lysate plus β-hexosaminidase. Transmission electron microscopy indicated that the cell wall of S. epidermidis was heavily degraded following coincubation of bacteria with BL/6-BMMC lysate, but not following coincubation with hexb−/−-BMMC lysate. These findings strongly suggest that MC granule β-hexosaminidase is crucial for defense against bacterial invasion, but is not involved in the allergic response. Our results also suggest that the bactericidal mechanism of β-hexosaminidase involves degradation of bacterial cell wall peptidoglycan.

Carbon Tetrachloride-Induced Nephrotoxicity in Mice Is Prevented by Pretreatment with Zinc Sulfate

Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. In addition, many studies showed that CCl4 can induce kidney damage. In the current study, we evaluated the protective effect of zinc (Zn) against CCl4-induced nephrotoxicity. We hypothesized that this protective effect would result from the ability of Zn to serve as an inducer of metallothionein (MT), a known endogenous scavenger of free radicals. We administered Zn (as ZnSO4) 50 mg/kg subcutaneously once daily for 3 successive days prior to a single intraperitoneal administration of CCl4 4 g/kg in male ddY mice. Our results showed that Zn pretreatment significantly decreased creatinine and blood urea nitrogen levels and reduced renal histopathological damage at 6 h post-CCl4 injection, observations consistent with enhanced antioxidative activity in the kidney. Moreover, kidney MT levels in the Zn+CCl4-treated group decreased by greater than 70% compared with levels in the Zn-alone group, implying that MT was consumed by CCl4-induced radicals. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute nephrotoxicity, presumably by induction of MT, which in turn scavenges radicals induced by CCl4 exposure.

Chronotoxicity of bromobenzene-induced hepatic injury in mice

The aim of the present study is to investigate whether or not bromobenzene (BB) toxicity varies with circadian periodicity. Seven-week-old male ICR mice were injected with 900 mg/kg (5.73 mmol/kg) BB intraperitoneally at 4 different time points of a day (zeitgeber time [ZT]: ZT0, ZT6, ZT12, and ZT18). Mortality was then monitored for 7 days after injection. Interestingly, mice were sensitive to BB acute toxicity at ZT6 while tolerant at ZT18. Moreover, in mice that were given a non-lethal dose of BB (540 mg (3.44 mmol)/kg), levels of alanine aminotransferase and aspartate aminotransferase, used as markers of hepatic injury, markedly increased in response to injection at ZT6, but did not increase significantly in response to injection at ZT18. In contrast, the markers of renal injury (creatinine and blood urea nitrogen), showed no significant difference in response to the two injection times. To further investigate this extreme circadian variation, we examined hepatic and renal lipid peroxidation levels, and conducted histopathological studies. Similar to our observation with alanine aminotransferase and creatinine, hepatic lipid peroxidation and histopathological changes were more pronounced than renal changes, and showed circadian variation. Our present investigation demonstrated that BB-induced mortality had clear circadian variation, and suggested that hepatic injury was one of the important factors for determination of this variation.

Roles of Superoxide Dismutase in Rat Mast Cell Granules

Background: It has been suggested that in the granules of rat mast cells there is some kind of superoxide dismutase (SOD), but details of this SOD in mast cells remain unclear. In the present study, we studied the mode of existence of SOD in mast cells and its releasing mechanism from the granules. In addition, we discussed the physiological role of SOD in allergic events. Methods: Purified rat mast cells were disrupted with a sonic disrupter and granules (sample I) were separated from supernatant (sample II) by centrifugation. The granules were treated with 1 mM Ca2+, and the supernatant (sample III) was separated from the pellet (sample IV). Sample III was applied to a heparin column and the eluate was used as sample V. SOD activity was measured in these samples. Results: SOD existed in mast cell granules as a heparin-binding and an inactive form. However, when granules were released and exposed to high Ca2+ concentration, SOD was discharged from heparin and shifted to the active form. The expression of macrophage inflammatory protein-1 alpha mRNA was enhanced when hydrogen peroxide (H2O2) or sample III with the xanthine-xanthine oxidase system were added to the culture media. Conclusions: These findings suggest that in stimulated rat mast cells, the released SOD may transform the generated superoxide anion into H2O2, and the generated H2O2 may enhance the expression of chemokine mRNA in the mast cells.

Calcium-deficient diet attenuates carbon tetrachloride-induced hepatotoxicity in mice through suppression of lipid peroxidation and inflammatory response

The aim of this study is to investigate whether a Ca-deficient diet has an attenuating effect on carbon tetrachloride (CCl4)-induced hepatotoxicity. Four-week-old male ddY mice were fed a Ca-deficient diet for 4 weeks as a part of the experimental protocol. While hypocalcemia was observed, there was no significant change in body weight. The CCl4-exposed hypocalcemic mice exhibited a significant decrease in alanine aminotransferase and aspartate aminotransferase activities at both 6 h and 24 h even though markers of renal function remained unchanged. Moreover, lipid peroxidation was impaired and total antioxidant power was partially recovered in the liver. Studies conducted in parallel with the biochemical analysis revealed that hepatic histopathological damage was attenuated 24 h post CCl4 injection in hypocalcemic mice fed the Ca-deficient diet. Finally, this diet impaired CCl4-induced inflammatory responses. Although upregulation of Ca concentration is a known indicator of terminal progression to cell death in the liver, these results suggest that Ca is also involved in other phases of CCl4-induced hepatotoxicity, via regulation of oxidative stress and inflammatory responses.

Generation of Mast Cells from Mouse Fetus: Analysis of Differentiation and Functionality, and Transcriptome Profiling Using Next Generation Sequencer

While gene knockout technology can reveal the roles of proteins in cellular functions, including in mast cells, fetal death due to gene manipulation frequently interrupts experimental analysis. We generated mast cells from mouse fetal liver (FLMC), and compared the fundamental functions of FLMC with those of bone marrow-derived mouse mast cells (BMMC). Under electron microscopy, numerous small and electron-dense granules were observed in FLMC. In FLMC, the expression levels of a subunit of the FcεRI receptor and degranulation by IgE cross-linking were comparable with BMMC. By flow cytometry we observed surface expression of c-Kit prior to that of FcεRI on FLMC, although on BMMC the expression of c-Kit came after FcεRI. The surface expression levels of Sca-1 and c-Kit, a marker of putative mast cell precursors, were slightly different between bone marrow cells and fetal liver cells, suggesting that differentiation stage or cell type are not necessarily equivalent between both lineages. Moreover, this indicates that phenotypically similar mast cells may not have undergone an identical process of differentiation. By comprehensive analysis using the next generation sequencer, the same frequency of gene expression was observed for 98.6% of all transcripts in both cell types. These results indicate that FLMC could represent a new and useful tool for exploring mast cell differentiation, and may help to elucidate the roles of individual proteins in the function of mast cells where gene manipulation can induce embryonic lethality in the mid to late stages of pregnancy.

Antiallergic effect of ardisiaquinone A, a potent 5-lipoxygenase inhibitor

The antiallergic effects of ardisiaquinone A, a potent 5-lipoxygenase inhibitor, were examined. Pretreatment with ardisiaquinone A (0.1-10 microM) significantly inhibited compound 48/80-induced production of cysteinyl-leukotrienes (cys-LTs; LTC4, LTD4 and LTE4) in rat peritoneal mast cells, but not histamine release. The IC50 value was 5.56 microM. Pre-administration with ardisiaquinone A (0.1-1 mg/kg, s.c.) dose-dependently inhibited rat homologous passive cutaneous anaphylaxis (PCA) and the maximal inhibitory ratio was 22.3 +/- 3.9% at the dose of 1 mg/kg. Ardisiaquinone A (1-5 mg/kg, s.c.) dose-dependently prevented the allergen-induced increase of tracheal pressure in ovalbumin-sensitized guinea pigs, especially during the late phase. In conclusion, the findings of this study show that 5-lipoxygenase inhibitor ardisiaquinone A partially attenuates the allergen-induced increases of vascular permeability and tracheal pressure via the inhibition of cys-LTs production in mast cells.

CD72 negatively regulates mouse mast cell functions and down-regulates the expression of KIT and FcεRIα

CD72 is a transmembrane protein belonging to the C-type lectin family that is expressed by various hematopoietic cells. When bound to its natural ligand, CD100 (semaphorin 4D), CD72 inhibits the KIT-mediated responses of human mast cells, but not IgE/FcεRI-mediated mast cell degranulation. We extended these findings to examine the role of CD72 in mouse mast cells. CD72 expression was detected in mouse bone marrow-derived mast cells (mBMMCs). As for human mast cells, an agonistic antibody against CD72 (K10.6) suppressed the KIT-mediated cell growth of, IL-6 production by and chemotaxis of mBMMCs. However, in contrast to human mast cells, the IgE-triggered degranulation of mBMMCs was suppressed by K10.6. K10.6 did not affect the phosphorylation of SHP-1 in mBMMCs, although SHP-1 mediated the inhibitory effects of CD72 in human mast cells. Administration of K10.6 induced phosphorylation of the ubiquitin ligase Cbl-b and decreased the expression of KIT and FcεRIα on the surface of murine mast cells. We also observed expression of CD72 in a mouse neoplastic cell line, P815, harboring gain-of-function mutations in KIT genes. In addition, we found that K10.6 activated Cbl-b, down-regulated KIT expression and suppressed the mutated KIT-driven growth of these cells. Thus, the mechanism by which CD72 mediates inhibitory effects in mast cells is species-dependent.