Neuroprotective Effects of a Multi-Herbal Extract on Axonal and Synaptic Disruption in Vitro and Cognitive Impairment in Vivo.

Background: Alzheimer's disease (AD) is a multifactorial disorder characterized by cognitive decline. Current available therapeutics for AD have limited clinical benefit. Therefore, preventive therapies for interrupting the development of AD are critically needed. Molecules targeting multifunction to interact with various pathlogical components have been considered to improve the therapeutic efficiency of AD. In particular, herbal medicines with multiplicity of actions produce cognitive benefits on AD. Bugu-M is a multi-herbal extract composed of Ganoderma lucidum (Antler form), Nelumbo nucifera Gaertn., Ziziphus jujuba Mill., and Dimocarpus longan, with the ability of its various components to confer resilience to cognitive deficits. Objective: To evaluate the potential of Bugu-M on amyloid-ß (Aß) toxicity and its in vitro mechanisms and on in vivo cognitive function. Methods: We illustrated the effect of Bugu-M on Aß25-35-evoked toxicity as well as its possible mechanisms to diminish the pathogenesis of AD in rat cortical neurons. For cognitive function studies, 2-month-old female 3×Tg-AD mice were administered 400 mg/kg Bugu-M for 30 days. Behavioral tests were performed to assess the efficacy of Bugu-M on cognitive impairment. Results: In primary cortical neuronal cultures, Bugu-M mitigated Aß-evoked toxicity by reducing cytoskeletal aberrations and axonal disruption, restoring presynaptic and postsynaptic protein expression, suppressing mitochondrial damage and apoptotic signaling, and reserving neurogenic and neurotrophic factors. Importantly, 30-day administration of Bugu-M effectively prevented development of cognitive impairment in 3-month-old female 3×Tg-AD mice. Conclusion: Bugu-M might be beneficial in delaying the progression of AD, and thus warrants consideration for its preventive potential for AD.

Chlorella sorokiniana Extract Prevents Cisplatin-Induced Myelotoxicity In Vitro and In Vivo.

Cisplatin chemotherapy causes myelosuppression and often limits treatment duration and dose escalation in patients. Novel approaches to circumvent or lessen myelotoxicity may improve clinical outcome and quality of life in these patients. Chlorella sorokiniana (CS) is a freshwater unicellular green alga and exhibits encouraging efficacy in immunomodulation and anticancer in preclinical studies. However, the efficacy of CS on chemoprotection remains unclear. We report here, for the first time, that CS extract (CSE) could protect normal myeloid cells and PBMCs from cisplatin toxicity. Also, cisplatin-induced apoptosis in HL-60 cells was rescued through reservation of mitochondrial function, inhibition of cytochrome c release to cytosol, and suppression of caspase and PARP activation. Intriguingly, cotreatment of CSE attenuated cisplatin-evoked hypocellularity of bone marrow in mice. Furthermore, we observed the enhancement of CSF-GM activity in bone marrow and spleen in mice administered CSE and cisplatin, along with increased CD11b levels in spleen. In conclusion, we uncovered a novel mechanism of CSE on myeloprotection, whereby potentially supports the use of CSE as a chemoprotector against cisplatin-induced bone marrow toxicity. Further clinical investigation of CSE in combination with cisplatin is warranted.

Rhinacanthin C Alleviates Amyloid-ß Fibrils' Toxicity on Neurons and Attenuates Neuroinflammation Triggered by LPS, Amyloid-ß, and Interferon-γ in Glial Cells.

Neuroinflammation plays a central role in the pathophysiology of Alzheimer's disease (AD). Compounds that suppress neuroinflammation have been identified as potential therapeutic targets for AD. Rhinacanthin C (RC), a naphthoquinone ester found in Rhinacanthus nasutus Kurz (Acanthaceae), is currently proposed as an effective molecule against inflammation. However, the exact role of RC on neuroinflammation remains to be elucidated. In the present study, we investigated RC effect on modulating lipopolysaccharides (LPS), amyloid-ß peptide (Aß), or interferon-γ- (IFN-γ-) evoked pathological events in neurons and glia. Our findings demonstrated that RC prevented Aß-induced toxicity in rat hippocampal neurons and attenuated LPS-activated nitric oxide (NO) production, inducible nitric oxide synthase (iNOS) expression, and NF-κB signaling in rat glia. Likewise, RC suppressed LPS-induced neuroinflammation by reducing NO production and iNOS, IL-1ß, CCL-2, and CCL-5 mRNA levels in rat microglia. Further studies using BV-2 microglia revealed that RC inhibited LPS-, Aß-, and IFN-γ-stimulated IL-6 and TNF-α secretion. Of note, NF-κB and ERK activation was abrogated by RC in BV-2 cell response to Aß or IFN-γ. Moreover, RC protected neurons from Aß-stimulated microglial conditioned media-dependent toxicity. Collectively, these data highlight the beneficial effects of RC on neuroprotection and support the therapeutic implications of RC to neuroinflammation-mediated conditions.

4-Hydroxy-17-methylincisterol from Agaricus blazei Decreased Cytokine Production and Cell Proliferation in Human Peripheral Blood Mononuclear Cells via Inhibition of NF-AT and NF-κB Activation.

Agaricus blazei Murill is an edible and medicinal mushroom. In the previous study, we have proved that extracts of A. blazei inhibit human peripheral blood mononuclear cell (PBMC) proliferation activated with phytohemagglutinin (PHA). Currently, we purified 4-hydroxy-17-methylincisterol (4-HM; C21H33O3) from A. blazei investigated its regulatory effects on cytokine productions and cell proliferation of PBMC induced by PHA. The results indicated that 4-HM suppressed, in activated PBMC, the production and mRNA expression of interleukin-2 (IL-2), IL-4, tumor necrosis factor- α , and interferon- γ in a concentration-dependent manner. This inhibition was not related to cell viability. While 4-HM did not affect ERK phosphorylation and its downstream c-fos gene expression in PBMC induced by PHA, it decreased both NF-AT and NF- κ B activation. The upstream signaling of NF-AT and NF- κ B, intracellular calcium concentrations ([Ca(2+)]i), and protein kinase C theta (PKC θ ) activation in PHA-treated PBMC were reduced by 4-HM. The data demonstrated that the suppressant effects of 4-HM on cell proliferation in PBMC activated by PHA appeared to be mediated, at least in part, through inhibition of Ca(2+) mobilization and PKC θ activation, NF-AT and NF- κ B activation, and cytokine transcripts and productions of PBMC. We suggested that A. blazei contained a potential immunomodulator 4-HM.

3-methoxyapigenin modulates ß-catenin stability and inhibits Wnt/ß-catenin signaling in Jurkat leukemic cells.

AIMS: Aberrant activation of Wnt/ß-catenin signaling has been implicated in carcinogenesis. Identification of inhibitors of this pathway may help in cancer therapy. The purpose of this study is to investigate the inhibitory effect of 3-methoxyapigenin (3-MA) with ß-catenin/LEF reporter system. The anti-cancer mechanisms in Jurkat leukemic cells were also examined. MAIN METHODS: HEK 293-TOP/FOP reporter cells were used to determine the inhibitory effect of 3-MA on Wnt/ß-catenin pathway. We also used Jurkat-TOP reporter cells to confirm the inhibitory effect and the action mechanisms of 3-MA. Target genes and cell proliferation were analyzed by RT-PCR and (3)H-thymidine uptake assay. The effects of 3-MA on ß-catenin phosphorylation was determined by Western blotting and by in vitro kinase assays. ß-catenin translocation and its transactivation were verified by cellular fractionation and EMSA. KEY FINDINGS: 3-MA inhibited Wnt-3A-induced luciferase activity in the HEK 293-TOP/FOP reporter system. Western blotting analysis showed that phosphorylation sites in ß-catenin by glycogen synthase kinase-3ß (GSK-3ß) and casein kinase 2 (CK2) were inhibited by 3-MA in Jurkat. In parallel, in vitro kinase assays verified this effect. As a result, total ß-catenin turnover remained balanced by this dual inhibitory effect of 3-MA. Although the ß-catenin protein level remained unchanged, 3-MA did inhibit ß-catenin translocation. Finally, we found that the ß-catenin/LEF transcriptional activity, expression of c-myc and cyclin-D3, and cell proliferation were inhibited by 3-MA. SIGNIFICANCE: 3-MA modulates the turnover of ß-catenin and suppresses the Wnt/ß-catenin signaling pathway through inhibition of ß-catenin translocation. We suggested that 3-MA has potential as an anti-cancer drug.

Characterized polysaccharides from black soybean induce granulocyte colony-stimulated factor gene expression in a phosphoinositide 3-kinase-dependent manner.

Black soybean (Glycine max L. merr.) is an edible Chinese medicine for nourishment spleen. In the present study, effects of characterized polysaccharides from black soybean (PGM) on granulocyte colony-stimulated factor (G-CSF) production in human peripheral blood mononuclear cells (PBMC) were determined and their action mechanisms were examined. The results indicated that PGM concentration-dependently enhanced G-CSF production in PBMC through modulation of mRNA expression. Data from Western blotting showed that PGM significantly induced the extracellular signal-regulated protein kinase (ERK) activation in PBMC. The nuclear factor (NF)-κB activation in PBMC was increased with PGM by modulation of IκB degradation and PKC θ activation. The levels of G-CSF mRNA in PGM-treated PBMC could be reduced by ERK inhibitor U0126 and NF-κB inhibitor pyrrolidine dithiocarbamate, respectively. Furthermore, the data showed that PGM stimulated phosphoinositide 3-kinase (PI3K)-regulated Akt phosphorylation. The PI3K inhibitor, Ly294002, blocked ERK, NF-κB, and PKC θ activation and G-CSF mRNA expression in PBMC induced by PGM. Thus, we first proved that the enhancement mechanisms of PGM on G-CSF production, appeared to be mediated, at least in part, through activation of PI3K, ERK, PKC θ, and NF-κB signaling pathways in PBMC. We suggest that PGM from black soybean is a potential G-CSF stimulator.

Blockade of JAK2 activity suppressed accumulation of ß-catenin in leukemic cells.

The Wnt/ß-catenin pathway has been implicated in leukemogenesis. We found ß-catenin abnormally accumulated in both human acute T cell leukemia Jurkat cells and human erythroleukemia HEL cells. ß-Catenin can be significantly down-regulated by the Janus kinase 2 specific inhibitor AG490 in these two cells. AG490 also reduces the luciferase activity of a reporter plasmid driven by LEF/ß-catenin promoter. Similar results were observed in HEL cells infected with lentivirus containing shRNA against JAK2 gene. After treatment with 50 µM AG490 or shRNA, the mRNA expression levels of ß-catenin, APC, Axin, ß-Trcp, GSK3α, and GSK3ß were up-regulated within 12-16 h. However, only the protein levels of GSK3ß and ß-Trcp were found to have increased relative to untreated cells. Knockdown experiments revealed that the AG490-induced inhibition of ß-catenin can be attenuated by shRNA targeting ß-TrCP. Taken together; these results suggest that ß-Trcp plays a key role in the cross-talk between JAK/STAT and Wnt/ß-catenin signaling in leukemia cells.

Norcantharidin reduced cyclins and cytokines production in human peripheral blood mononuclear cells.

AIMS: To evaluate potential agents of therapeutic value in tissue inflammation, we studied norcantharidin (NCTD) and its derivatives for their effects on immune responses of human peripheral blood mononuclear cells (PBMC) in vitro. MAIN METHODS: PBMC proliferation was evaluated by tritiated thymidine uptake method. The production and gene expression of cytokines were determined with enzyme immunoassays (EIA) and reverse transcription-polymerase chain reaction (RT-PCR), respectively. KEY FINDINGS: Five derivatives from NCTD had no significant effect on cell proliferation in PBMC. NCTD inhibited PBMC proliferation induced by phytohemagglutinin (PHA) with a 50% inhibitory concentration (IC(50)) 42.1+/-2.3 microM. The inhibitory action of NCTD did not involve direct cytotoxicity. To localize the point in the PBMC proliferation where arrest occurred, a set of key regulatory events leading to the cell proliferation, including cell cycle progression, production and gene expression of interleukin-2 (IL-2), IL-4, IL-10, and interferon-gamma (IFN-gamma) and cyclins was examined. Data demonstrated NCTD arrested the cell cycle progression of activated PBMC from the G1 transition to the S phase. The cyclin D3, E, A, and B transcripts and protein production in PHA-treated PBMC was reduced by NCTD. Whereas NCTD exerted no effect on IL-4 and IFN-gamma production, it significantly alleviated the production and mRNA expression of IL-2 and IL-10 in activated PBMC. SIGNIFICANCE: The suppressant effects of NCTD on proliferation of PBMC activated by PHA therefore appear to be mediated, at least in part, through inhibition of cyclins and IL-2 production and arrest of cell cycle progression in the cells.

Cell cycle progression and cytokine gene expression of human peripheral blood mononuclear cells modulated by Agaricus blazei.

We selected Agaricus blazei Murill, used in the treatment of tissue inflammation and cancer in traditional Chinese medicine, to test its immunopharmacological activity. The effects of A. blazei extracted fractions (AB-BDM-1 to AB-BDM-10) on human peripheral blood mononuclear cell (PBMC) proliferation were determined on the basis of the uptake of tritiated thymidine. The results indicated that AB-BDM-2 fraction suppressed PBMC proliferation activated with phytohemagglutinin. The inhibitory action of AB-BDM-2 did not involve direct cytotoxicity. Cell-cycle analysis indicated that AB-BDM-2 arrested the cell-cycle progression of activated PBMCs from the G1 transition to the S phase. In an attempt to further localize the point in the cell cycle where arrest occurred, we examined a set of key regulatory events leading to the G1/S boundary, including gene expression of interleukin-2 (IL-2), interleukin-4 (IL-4), interferon-gamma (IFN-gamma), and cyclin D. AB-BDM-2 suppressed, in activated PBMCs, the production and messenger RNA (mRNA) expression of IL-2, IL-4, IFN-gamma, and cyclin D in dose-dependent fashion. AB-BDM-2 did not affect nitric oxide production or levels of inducible nitric oxide synthetase mRNA in PBMCs stimulated with PHA. The suppressant effects of AB-BDM-2 on the proliferation of PBMC activated by PHA therefore appear to be mediated, at least in part, through inhibition of early transcripts of PBMCs, especially those of important cytokines IL-2, IL-4, and IFN-gamma, cyclin D, and the arrest of cell-cycle progression in the cells. We suggest that immunomodulatory agents are contained in AB-BDM-2 separated from A. blazei.