Effect of herbal medicine Juzentaihoto on hepatic and intestinal heat shock gene expression requires intestinal microflora in mouse.

AIM: To evaluate the role of intestinal microflora in the effects of multi-herbal medicine on gene expression in the gut and liver. METHODS: The multi-herbal medicine Juzentaihoto (JTX) was administered to five germ-free mice and regular mice for 2 wk. Among the results of the comprehensive gene chip analysis of the intestine and liver, we featured heat shock proteins (HSPs) 70 and 105 because their gene expression changed only in the presence of microflora. Real-time RT-PCR was performed to confirm the expression levels of these HSP genes. To determine whether JTX acts directly on the HSP genes, sodium arsenite (SA) was used to induce the heat shock proteins directly. To examine the change of the intestinal microflora with administration of JTX, the terminal restriction fragment polymorphism (T-RFLP) method was used. To identify the changed bacteria, DNA sequencing was performed. RESULTS: Heat shock protein gene expression, documented by gene chip and real-time RT-PCR, changed with the administration of JTX in the regular mice but not in the germ-free mice. JTX did not suppress the direct induction of the HSPs by SA. T-RFLP suggested that JTX decreased unculturable bacteria and increased Lactobacillus johnsoni. These data suggested that JTX changed the intestinal microflora which, in turn, changed HSP gene expression. CONCLUSION: Intestinal microflora affects multi-herbal product JTX on the gene expression in the gut and liver.

Toki-to protects dopaminergic neurons in the substantia nigra from neurotoxicity of MPTP in mice.

Parkinson's disease (PD) is a neurodegenerative disease of the brain characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN). No clinically proven drugs that may halt or retard the progression of PD have been reported. This study examined the anti-PD effect of a traditional Japanese/Chinese herbal remedy Toki-to (TKT) using mice treated with a neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydroxypyridine (MPTP). TKT showed improvement of MPTP-induced PD-like symptoms (bradykinesia) in a behavioral test (pole test). Histological studies of SNs from these mice demonstrated that TKT had a protective effect on dopaminergic neurons against MPTP neurotoxicity. Real-time RT-PCR analyses of mRNA from SNs demonstrated that expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) genes were decreased by MPTP treatment and that these decreases were reversed by TKT administration prior to MPTP treatment. DNA microarray analyses indicated that TKT per se suppressed gene expression of serum- and glucocorticoid regulated kinase (SGK) that is believed to be a molecule that drives the pathogenesis of PD. Hence, it is suggested that TKT may inhibit the activation of SGK at the transcriptional level and thusmay participate in halting the progression of MPTP-induced neurotoxicity.

A Kampo formula Juzen-taiho-to induces expression of metallothioneins in mice.

Juzen-taiho-to (JTX), one of the commonly prescribed traditional Japanese herbal medicines (Kampo), is indicated for adjunctive treatment of cancers and autoimmune diseases. To understand the mechanisms underlying the clinical effects of JTX, the effects of orally administered JTX on the expression of metallothioneins (MTs) were examined in the liver, spleen, small and large intestines of mice. In addition, the expression of MTs in specific pathogen free (SPF) mice was examined to understand the participation of intestinal bacteria in the expression of MTs. JTX enhanced expression of MT-I and -II significantly in the liver of SPF mice. Induction of MT-II expression was observed also in the small intestine. Intestinal bacteria appeared to have no effect on MTs expression. Neither expression of MT-III nor its induction was observed in any tissue. These findings strongly suggest that MTs should mediate at least some effects of JTX in mice.

Eugenol and its structural analogs inhibit monoamine oxidase A and exhibit antidepressant-like activity.

Eugenol (1) is an active principle of Rhizoma acori graminei, a medicinal herb used in Asia for the treatment of symptoms reminiscent of Alzheimer's disease (AD). It has been shown to protect neuronal cells from the cytotoxic effect of amyloid beta peptides (Abetas) in cell cultures and exhibit antidepressant-like activity in mice. Results from this study show that eugenol inhibits monoamine oxidase A (MAOA) preferentially with a K(i)=26 microM. It also inhibits MAOB but at much higher concentrations (K(i)=211 microM). In both cases, inhibition is competitive with respect to the monoamine substrate. Survey of compounds structurally related to eugenol has identified a few that inhibit MAOs more potently. Structure activity relationship reveals structural features important for MAOA and MAOB inhibition. Molecular docking experiments were performed to help explain the SAR outcomes. Four of these compounds, two (1, 24) inhibiting MAOA selectively and the other two (19, 21) inhibiting neither MAOA nor MAOB, were tested for antidepressant-like activity using the forced swim test in mice. Results suggest a potential link between the antidepressant activity of eugenol and its MAOA inhibitory activity.

Eugenol exhibits antidepressant-like activity in mice and induces expression of metallothionein-III in the hippocampus.

Here we show that eugenol has an antidepressant-like activity comparable to that of imipramine using a forced swim test and a tail suspension test in mice. Furthermore, we show that both eugenol and imipramine induce brain-derived neurotrophic factor (BDNF) in the hippocampus with and without induction of metallothionein-III (MT-III), respectively. It may be possible that MT-III expression is involved in the exhibition of antidepressant-like activity of eugenol, not of imipramine.

Effect of Choto-san, a Kampo medicine, on impairment of passive avoidance performance in senescence accelerated mouse (SAM).

Effect of Choto-san (TJ-47), a Kampo medicine, on impairment of learning performance was evaluated by means of a step-through passive avoidance task in SAMP8 mice, a senescence-prone substrain. Tokishakuyaku-san (TJ-23), another Kampo medicine, was also employed for comparison. SAMP8 mice at the age of 10-12 months showed a poorer passive avoidance response than SAMR1 mice, a senescence-resistant substrain, in the memory-retention test, but not in the memory-acquisition test. Improved response in the memory-retention test was observed in SAMP8 mice treated with TJ-47, and almost equal degree of improvement was also observed after treatment with TJ-23. These results suggest that a long-term administration of Choto-san could improve to some extent the impairment of memory caused by aging.

Expression of metallothionein-III induced by hypoxia attenuates hypoxia-induced cell death in vitro.

Metallothioneins (MTs) are metal-binding proteins that are expressed in many tissues including brain. MTs protect cells and organs against metal toxicity and oxidants. Among MTs, a brain-predominant subtype MT-III has prominent neuroprotective activity against various types of damage. Here we show that the expression of MT-III is induced in cultured normal human astrocytes by hypoxia, and that overexpressed MT-III protects human embryonic kidney cells from hypoxia, suggesting that MT-III can protect the brain from hypoxic damage.

Rhizoma acori graminei and its active principles protect PC-12 cells from the toxic effect of amyloid-beta peptide.

The effects of water extracts of six medicinal herbs (Radix polygalae tenuifoliae, Radix salviae miltiorrhizae, Rhizoma acori graminei, Rhizoma pinelliae ternatae, Tuber curcumae and Scletrotium poriae cocos) on the cytotoxic action of Abeta(1-40) were tested with PC-12 cells. Only the extract of R. acori graminei (RAG) significantly decreased Abeta(1-40)-induced cell death. Further, eugenol and beta-asarone were isolated and identified as the major active principles. Both purified eugenol and beta-asarone protected PC-12 cells from the toxic effect of Abeta(1-40). Eugenol was active between 1 and 100 microM, and 10 microM eugenol gave approximately a 50% response. beta-Asarone was less potent and exhibited little, if any, activity at this concentration. Both eugenol and beta-asarone inhibited Ca(2+) intake by PC-12 cells: beta-asarone mainly inhibited basal Ca(2+) intake, whereas eugenol inhibited Abeta-induced Ca(2+) intake preferentially. These results suggest that eugenol may act by blocking Abeta-induced-Ca(2+) intake and provide a strong case for further pursuit of the therapeutic and prophylactic potentials of RAG and its active principles for the management of Alzheimer's disease.

Anti-amyloid beta activity of metallothionein-III is different from its neuronal growth inhibitory activity: structure-activity studies.

Recent studies have shown that metallothionein-III (MT-III), but not MT-I or -II, antagonizes both the neurotrophic and neurotoxic effects of amyloid beta peptides (Abetas). Further, its anti-Abeta-toxicity effect was attributed to the fact that it inhibits the formation of fibrillar Abeta. MT-III alone also affects neuron survival in culture-promoting at low but inhibiting at high concentrations. To characterize these biological activities of MT-III in relation to its neuronal growth inhibitory activity discovered by Uchida et al. [Neuron 7 (1991) 337-347], we here studied effects of the P7S/P9A double mutant, and the N- and C-terminal domains of MT-III on primary cultures of rat embryonic cortical neurons in the presence and absence of Abeta. Results show that (i). only the wild-type MT-III inhibited the formation of SDS-resistant Abeta aggregates and protected cortical neurons from the toxic effect of Abeta, and (ii). both the wild type and the N-terminal domain of MT-III promote neuron survival at low concentrations but inhibited it at high concentrations. On the basis of these findings, we conclude that the anti-Abeta activity of MT-III is different from its neuronal growth inhibitory activity and suggest that the increased trophic activity of AD brain extracts could be attributed to its low MT-III content.

S-nitrosothiols react preferentially with zinc thiolate clusters of metallothionein III through transnitrosation.

Metallothionein (MT) is a two-domain protein with zinc thiolate clusters that bind and release zinc depending on the redox states of the sulfur ligands. Since S-nitrosylation of cysteine is considered a prototypic cellular redox signaling mechanism, we here investigate the reactions of S-nitrosothiols with different isoforms of MT. MT-III is significantly more reactive than MT-I/II toward S-nitrosothiols, whereas the reactivity of all three isoforms toward reactive oxygen species is comparable. A cellular system, in which all three MTs are similarly effective in protecting rat embryonic cortical neurons in primary culture against hydrogen peroxide but where MT-III has a much more pronounced effect of protecting against S-nitrosothiols, confirms this finding. MT-III is the only isoform with consensus acid-base sequence motifs for S-nitrosylation in both domains. Studies with synthetic and zinc-reconstituted domain peptides demonstrate that S-nitrosothiols indeed release zinc from both the alpha- and the beta-domain of MT-III. S-Nitrosylation occurs via transnitrosation, a mechanism that differs fundamentally from that of previous studies of reactions of MT with NO*. Our data demonstrate that zinc thiolate bonds are targets of S-nitrosothiol signaling and further indicate that MT-III is biologically specific in converting NO signals to zinc signals. This could bear importantly on the physiological action of MT-III, whose biological activity as a neuronal growth inhibitory factor is unique, and for brain diseases that have been related to oxidative or nitrosative stress.