Hsin-Ying Wu

A comparative study on cannabidiol-induced apoptosis in murine thymocytes and EL-4 thymoma cells.

It has been shown that leukemia and glioma cells are sensitive to cannabidiol (CBD)-induced apoptosis, whereas primary monocytes and glia cells are relatively insensitive. In the current study, the cellular events and sensitivity to CBD-induced apoptosis between murine thymocytes and EL-4 thymoma cells were compared. Cannabidiol markedly induced apoptosis in a time- and concentration-related manner in both cells. The efficacy of CBD to induce apoptosis was comparable between the 2 types of T cells, whereas CBD induced apoptosis in thymocytes with a slightly greater potency than in EL4 cells. Time-course analyses revealed CBD-mediated apoptosis occurred earlier in EL-4 cells than that in thymocytes. An increased level of cellular reactive oxygen species (ROS) was detected in both cells with the peak response at 2 h post CBD treatment. Concordantly, CBD triggered a gradual diminishment in the cellular thiols. The presence of N-acetyl-L-cysteine (NAC), a precursor of glutathione, markedly attenuated the induction of apoptosis, and restored the diminished levels of cellular thiols. The results demonstrated that both thymocytes and EL-4 thymoma cells were susceptible to CBD-induced apoptosis and that ROS played a critical role in the apoptosis induction.

Iron oxide nanoparticles suppress the production of IL-1beta via the secretory lysosomal pathway in murine microglial cells

BackgroundSuperparamagnetic iron oxide nanoparticles (IONPs) have been used as magnetic resonance imaging contrast agents for various research and diagnostic purposes, such as the detection of neuroinflammation and blood-brain-barrier integrity. As the central resident macrophage-like cells, microglia are responsible for managing foreign agents invading the CNS. The present study investigated the direct effect of IONPs on the production of pro-inflammatory cytokines by murine microglia stimulated with lipopolysaccharide (LPS).MethodsPrimary murine microglial cells were pretreated with IONPs (1–50 μg Fe/mL) for 30 min and then stimulated with LPS (100 ng/mL) for 24 h. Confocal microscopy is used to visualize the intracellular IONP distribution and secretory lysosomes after staining with LysoTracker and Rab27a, respectively. The production of interleukin (IL)-1β and tumor necrosis factor (TNF)-α was quantified by ELISA. The activity of IL-1β converting enzyme (ICE) and TNF-α converting enzyme (TACE) was measured by fluorescent microplate assay using specific substrates. The lysosomal number, alkalinity, permeability and cathepsin B activity were determined by flow cytometry with ectodermal dysplasia-1, lysosensor and acridine orange staining, and using cathepsin B specific substrate, respectively.ResultsConfocal imaging revealed that IONPs were markedly engulfed by microglia. Exposure to IONPs attenuated the production of IL-1β, but not TNF-α. Concordantly, the activity of ICE, but not the TACE, was suppressed in IONP-treated cells. Mechanistic studies showed that IONPs accumulated in lysosomes and the number of lysosomes was increased in IONP-treated cells. In addition, exposure to IONPs increased lysosomal permeability and alkalinity, but decreased the activity of cathepsin B, a secretory lysosomal enzyme involved in the activation of ICE.ConclusionsOur results demonstrated a contrasting effect of IONPs on the production of IL-1β and TNF-α by LPS-stimulated microglia, in which the attenuation of IL-1β by IONPs was mediated by inhibiting the secretory lysosomal pathway of cytokine processing.

Methamphetamine and diazepam suppress antigen-specific cytokine expression and antibody production in ovalbumin-sensitized BALB/c mice.

Methamphetamine is a widely abused psychostimulant. Abusing methamphetamine causes various adverse effects, such as immune dysfunction. The present study investigated the effect of diazepam, a central depressant, on methamphetamine-induced immunosuppression. BALB/c mice were daily administered with diazepam and methamphetamine (5mg/kg of each), either alone or in combination, for 5 consecutive days followed by sensitization with ovalbumin (OVA). Two days later the same dosing and sensitization regimen was repeated once. The production of serum anti-OVA antibodies, and the cellularity and functional activities of splenocytes were measured 7 days post the 2nd OVA sensitization. The results demonstrated that methamphetamine and/or diazepam significantly attenuated the production of OVA-specific IgM, IgG(1) and IgG(2a). Concordantly, splenocytes of mice administered with diazepam and/or methamphetamine produced less IL-4 and IFN-gamma upon ex vivo re-stimulation with OVA, as compared to the vehicle-treated control. In contrast, the cellularity and metabolic activity of splenocytes were not altered by the drug treatment. These results indicated that the central depressant diazepam did not affect methamphetamine-mediated immunosuppression. Rather, both drugs markedly suppressed antigen-specific antibody production and T-cell reactivity.

Cannabidiol hydroxyquinone-induced apoptosis of splenocytes is mediated predominantly by thiol depletion

Cannabidiol, the major nonpsychotropic phytocannabinoid, has been recently demonstrated to induce apoptosis in primary lymphocytes via an oxidative stress-dependent mechanism. Cannabidiol can be converted by microsomal enzymes to the hydroxyquinone metabolite HU-331 that forms adducts with glutathione. The present study tested the hypothesis that HU-331 could cause apoptosis via the depletion of thiols in splenocytes. Our results showed that HU-331 treatment significantly enhanced splenocyte apoptosis in a time- and concentration-dependent manner. Concordantly, a gradual diminishment in the cellular thiols and glutathione was detected in HU-331-treated splenocytes. The apoptosis and thiol diminishment induced by HU-331 were abrogated in the presence of thiol antioxidants, including N-acetyl-(L)-cysteine and N-(2-mercaptopropionyl) glycine, whereas the non-thiol antioxidants catalase and pyruvate were ineffective. In comparison, both thiol and non-thiol antioxidants were capable of attenuating H(2)O(2)-induced thiol diminishment and reactive oxygen species generation in splenocytes. Collectively, these results suggest that HU-331 might be an active metabolite of cannabidiol potentially contributing to the induction of apoptosis in splenocytes, and that the apoptosis is primarily mediated by the loss of cellular thiols.

Polyethylene glycol-coated graphene oxide attenuates antigen-specific IgE production and enhanced antigen-induced T-cell reactivity in ovalbumin-sensitized BALB/c mice

Background Graphene oxide (GO) is a promising nanomaterial for potential application in the versatile field of biomedicine. Graphene-based nanomaterials have been reported to modulate the functionality of immune cells in culture and to induce pulmonary inflammation in mice. Evidence pertaining to the interaction between graphene-based nanomaterials and the immune system in vivo remains scarce. The present study investigated the effect of polyethylene glycol-coated GO (PEG-GO) on antigen-specific immunity in vivo. Methods BALB/c mice were intravenously administered with a single dose of PEG-GO (0.5 or 1 mg/kg) 1 hour before ovalbumin (OVA) sensitization, and antigen-specific antibody production and splenocyte reactivity were measured 7 days later. Results Exposure to PEG-GO significantly attenuated the serum level of OVA-specific immunoglobulin E. The production of interferon-γ and interleukin-4 by splenocytes restimulated with OVA in culture was enhanced by treatment with PEG-GO. In addition, PEG-GO augmented the metabolic activity of splenocytes restimulated with OVA but not with the T-cell mitogen concanavalin A. Conclusion Collectively, these results demonstrate that systemic exposure to PEG-GO modulates several aspects of antigen-specific immune responses, including the serum production of immunoglobulin E and T-cell functionality.

Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-L-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

Cannabidiol-induced apoptosis in murine microglial cells through lipid raft

Cannabidiol (CBD), the major nonpsychotropic phytocannabinoid, induces apoptosis in both immortalized and primary lymphocytes and monocytes. However, contrasting effects of CBD on the apoptosis between normal and immortalized glial cells have been reported. This study investigated the proapoptotic effect of CBD on primary microglial cells. Treatment of murine primary microglial cultures with CBD resulted in a time‐ and concentration‐dependent induction of apoptosis, as shown by increase in hypodiploid cells and DNA strand breaks, and marked activation of both caspase‐8 and −9. Mechanistic studies revealed that antioxidants, including N‐acetyl‐L‐cysteine and glutathione, the G protein‐coupled receptor 55 agonist abnormal‐CBD and specific antagonists for vanilloid, and CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl‐β‐cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD‐induced microglial apoptosis and caspase activation. Furthermore, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin‐1, all of which were attenuated by MCD. Taken together, these results suggest that CBD induces a marked proapoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin‐1.

Cannabidiol induced apoptosis in human monocytes through mitochondrial permeability transition pore-mediated ROS production

Abstract Cannabidiol (CBD) has been reported to induce apoptosis in immune cells through oxidative stress‐related mechanisms. The objective of the present study was to investigate the cellular mechanisms for CBD‐induced apoptosis and oxidative stress in human monocytes. Exposure of freshly isolated human monocytes to CBD induced apoptosis in a time‐ and concentration‐dependent manner. Time‐course analyses revealed the induction of intracellular reactive oxygen species (ROS) at 1–2 h post CBD (16 &mgr;M) exposure. By comparison, the CBD treatment rapidly elicited the depolarization of mitochondrial membrane potential (MMP) within 5 min, and the oxidation of cardiolipin, a major lipid component of the mitochondrial inner membrane, within 15 min. Moreover, CBD induced the release of cytochrome c (Cyt c) from mitochondria. Mechanistic studies revealed that CBD‐induced ROS production and apoptosis were not associated with the alteration of mitochondrial superoxide dismutase activity, the electron leakage through mitochondrial respiratory chain, and Fe2+‐ and Ca2+‐mediated mechanisms. In contrast, CBD‐induced apoptosis and MMP depolarization were markedly attenuated by the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporin A (CsA), but not the calcineurin inhibitor FK506. Furthermore, CsA prevented cardiolipin oxidation and the MPTP opening induced by CBD. The present study suggests that CBD acts on the mitochondria to elicit ROS generation and apoptosis through MPTP opening and provides critical insights into the cellular mechanisms for CBD‐induced oxidative stress in apoptotic monocytes. Graphical abstract Figure. No Caption available. HighlightsMechanisms for CBD‐induced apoptosis and oxidative stress in monocytes are studied.CBD elicited MMP depolarization and cardiolipin oxidation before ROS production.CBD induced Cyt c release from mitochondria.Cyclosporin A, but not FK506, prevented CBD‐induced apoptosis and MMP depolarization.CBD‐induced ROS generation and apoptosis is mediated by opening MPTP.

HISPOLON DIFFERENTIALLY MODULATED THE PRODUCTION OF ANTIGEN-INDUCED T CELL CYTOKINES VIA THE REGULATION OF CELLULAR GLUTATHIONE

Hispolon is an active ingredient contained in the medicinal mushroom Phellinus linteus (PL) that is used in traditional Chinese medicine for various remedies, including lymphatic diseases. Previous studies reported that hispolon exhibited anti-inflammatory activities and suppressed mitogen-induced proliferation of splenic lymphocytes. It remains unclear if hispolon influences antigen-specific immunity. The present study investigated the effects of hispolon on cytokine production by antigen-activated T cells. Ovalbumin (OVA)-primed splenocytes were exposed to hispolon, followed by restimulation with OVA. Cell viability was determined by the AlamarBlue® assay and T cell cytokine production was measured by enzyme linked immunosorbent assay (ELISA). The splenocyte viability and the production of interleukin (IL)-4 were unaffected, whereas the production of interferon (IFN)-γ was significantly suppressed by treatment with hispolon (1–5 μM) in a concentration-dependent manner. The suppressive effect of hispolon on the production of IFN-γ was attenuated in the presence of thiol antioxidants, including N-acetyl-L-cysteine (NAC) and glutathione, whereas the non-thiol antioxidant pyruvate was ineffective. Taken together, these results demonstrated that hispolon induced a differential effect on antigen-induced cytokine production by T cells, in which the T helper 1 (Th1) cytokine IFN-γ was sensitive, whereas the Th2 cytokine IL-4 was unaffected. In addition, the suppressive effect of hispolon on IFN-γ production was associated with the diminishment of intracellular glutathione.