Hepatotoxicity of the antidiabetic drug metformin has been reported, but the underlying mechanisms remain unclear. We here investigated the effect of metformin in immune-mediated liver damage. While ...not hepatotoxic alone, metformin (200 mg/kg) aggravated concanavalin A (Con A, 12 mg/kg)-induced hepatitis, an experimental model of T cell-mediated liver injury, in both relatively resistant BALB/c and highly susceptible C57Bl/6 mice. Metformin + Con A-treated mice had elevated serum levels of pro-inflammatory cytokines TNF-α and IFN-γ, accompanied by a massive mononuclear cell infiltration in the liver. This was associated with the higher numbers of CD4
+
T cells producing TNF-α, IFN-γ and IL-17, CD4
+
T cells expressing chemokine receptor CXCR3 and activation marker CD27, CD4
+
CD62L
−
CCR7
−
and CD8
+
CD62L
−
CCR7
−
effector memory cells, IFN-γ producing NK cells, IL-4 and IL-17 producing NKT cells and IL-12 producing macrophages/dendritic cells. The percentage of CD4
+
CXCR3
+
Tbet
+
IL-10
+
and CD4
+
CD69
+
CD25
−
regulatory T cells was reduced. Metformin stimulated inducible nitric oxide synthase (iNOS) expression in the liver and spleen, and genetic deletion of iNOS attenuated the hepatotoxicity of metformin. Metformin increased the autophagic light chain 3 conversion and mRNA expression of important autophagy-inducing (beclin-1, Atg5 and GABARAP) and pro-apoptotic (p21, p27, Puma, Noxa, Bax, Bad, Bak1, Bim and Apaf1), but not anti-apoptotic molecules (Bcl-xL, survivin and XIAP), which correlated with the apoptotic caspase-3/PARP cleavage in the liver. The autophagy inhibitor chloroquine (20 mg/kg) prevented liver injury and apoptotic changes induced by metformin. Therefore, metformin aggravates immune-mediated hepatitis by promoting autophagy and activation of immune cells, affecting effector, as well as liver-specific regulatory T cells and iNOS expression.
To explore combined antiglioma effect of nitric oxide (NO) and hyperthermia, the rat C6 and human U251 glioma cells were exposed to NO-releasing agents sodium nitroprusside(SNP), S-nitrosoglutathione ...or PAPA-NONOate, followed by hyperthermia (1 h, 43 °C). While each treatment alone showed only moderate efficiency, a synergistic cytotoxicity of NO donors and hyperthermia was clearly demonstrated by crystal violet and MTT cytotoxicity assays. The flow cytometric analysis with the appropriate reporter fluorochromes confirmed that hyperthermia and SNP cooperated in inducing oxidative stress, mitochondrial depolarization, caspase activation and DNA fragmentation, leading to both necrotic and caspase-dependent apoptotic cell death. The acridine orange staining of intracellular acidic compartments revealed that SNP completely blocked hyperthermia-induced autophagy, while the inhibition of autophagy by 3-methyl adenine mimicked SNP-triggered oxidative stress, caspase activation and cell death in hyperthermia-exposed cells. Therefore, the synergistic cytotoxicity of SNP and hyperthermia could result from NO-mediated suppression of protective autophagic response in glioma cells.
In the present study, we compared the effects of nanocrystalline fullerene suspension (nanoC
60) on tumour cell growth in vitro and in vivo. NanoC
60 suspension was prepared by solvent exchange using ...tetrahydrofuran to dissolve C
60. In vitro, nanoC
60 caused oxidative stress, mitochondrial depolarization and caspase activation, leading to apoptotic and necrotic death in mouse B16 melanoma cells. Biodistribution studies demonstrated that intraperitoneally injected radiolabeled (
125I) nanoC
60 readily accumulated in the tumour tissue of mice subcutaneously inoculated with B16 cells. However, intraperitoneal administration of nanoC
60 over the course of two weeks starting from melanoma cell implantation not only failed to reduce, but significantly augmented tumour growth. The tumour-promoting effect of nanoC
60 was accompanied by a significant increase in splenocyte production of the immunoregulatory free radical nitric oxide (NO), as well as by a reduction in splenocyte proliferative responses to T- and B-cell mitogens ConcanavalinA and bacterial lipopolysaccharide, respectively. A negative correlation between NO production and splenocyte proliferation indicated a possible role of NO in reducing the proliferation of splenocytes from nanoC
60-injected mice. These data demonstrate that nanoC
60, in contrast to its potent anticancer activity in vitro, can potentiate tumour growth in vivo, possibly by causing NO-dependent suppression of anticancer immune response.
The present study investigated the hemolytic properties of fullerene (C(60)) nanoparticles prepared by solvent exchange using tetrahydrofuran (nC(60)THF), or by mechanochemically assisted ...complexation with macrocyclic oligosaccharide gamma-cyclodextrin (nC(60)CDX) or the copolymer ethylene vinyl acetate-ethylene vinyl versatate (nC(60)EVA-EVV). The spectrophotometrical analysis of hemoglobin release revealed that only nC(60)THF, but not nC(60)CDX or nC(60)EVA-EVV, was able to cause lysis of human erythrocytes in a dose- and time-dependent manner. Atomic force microscopy revealed that nC(60)THF-mediated hemolysis was preceded by erythrocyte shrinkage and increase in cell surface roughness. A flow cytometric analysis confirmed a decrease in erythrocyte size and demonstrated a significant increase in reactive oxygen species production in red blood cells exposed to nC(60)THF. The nC(60)THF-triggered hemolytic activity was efficiently reduced by the antioxidants N-acetylcysteine and butylated hydroxyanisole, as well as by serum albumin, the most abundant protein in human blood plasma. These data indicate that nC(60)THF can cause serum albumin-preventable hemolysis through oxidative stress-mediated damage of the erythrocyte membrane.
Purpose
The fullerene (C
60
/C
70
mixture—C
60/70
) nanocrystalline suspension prepared by solvent exchange method using tetrahydrofyran (THF/
n
C
60/70
) and polyhydroxylated C
60/70
C
60/70
(OH)
n
...were compared for their ability to modulate cytotoxicity of the proinflammatory cytokine tumor necrosis factor (TNF).
Materials and Methods
TNF-induced cytotoxicity was assessed in L929 fibrosarcoma cells by crystal violet assay. The type of cell death (apoptosis/necrosis), production of reactive oxygen species, mitochondrial depolarization and caspase activation were determined by flow cytometry using the appropriate reporter dyes.
Results
THF/
n
C
60/70
augmented, while C
60/70
(OH)
n
reduced the cytotoxicity of TNF. The numbers of cells undergoing apoptosis/necrosis, as well as of those displaying the activation of apoptosis-inducing enzymes of caspase family, were respectively increased or reduced by THF/
n
C
60/70
or C
60/70
(OH)
n
. The antioxidant
N
-acetylcysteine and mitochondrial permeability transition inhibitor cyclosporin A each partly blocked the cytotoxic action of TNF, indicating the involvement of oxidative stress and mitochondrial dysfunction in the TNF cytotoxicity. Accordingly, THF/
n
C
60/70
or C
60/70
(OH)
n
potentiated or suppressed, respectively, TNF-triggered oxidative stress and mitochondrial depolarization.
Conclusion
The ability of different fullerene preparations to modulate TNF-induced oxidative stress and subsequent cell death suggests their potential value in the TNF-based cancer therapy or prevention of TNF-dependent tissue damage.
The protective ability of novel arylpiperazine‐based dopaminergic ligands against nitric oxide (NO)‐mediated neurotoxicity is investigated. The most potent neuroprotective arylpiperazine identified ...during the study was N‐{4‐2‐(4‐phenyl‐piperazin‐1‐yl)ethyl‐phenyl}picolinamide, which protected SH‐SY5Y human neuron‐like cells from the proapoptotic effect of NO donor sodium nitroprusside (SNP) by decreasing oxidative stress, mitochondrial membrane depolarization, caspase activation and subsequent phosphatydilserine externalization/DNA fragmentation. The protective effect was associated with the inhibition of proapoptotic (JNK, ERK, AMPK) and activation of antiapoptotic (Akt) signaling pathways, in the absence of interference with intracellular NO accumulation. The neuroprotective action of arylpiperazines was shown to be independent of dopamine receptor binding, as it was not affected by the high‐affinity D1/D2 receptor blocker butaclamol. These results reported support the further study of arylpiperazines as potential neuroprotective agents.
Guarding the grey matter! Arylpiperazine dopaminergic ligands protect neurons from nitric oxide cytotoxicity. Their ability to modulate cell survival signaling pathways, stabilize mitochondrial membrane, and ultimately prevent neuronal apoptosis, makes them plausible candidates for treatment of neurodegenerative diseases.
The purpose of this study was to examine the effects of ghrelin on protein kinase B (Akt) and mitogen-activated protein kinase p42/44 (ERK1/2) activation as well as ghrelin effects on inducible ...nitric oxide (NO) synthase (iNOS; for gene
Nos2
) activity/expression in rat hearts. Male Wistar rats were treated with ghrelin (0.3 nmol/5 μl) or an equal volume of phosphate-buffered saline, injected every 24 h into the lateral cerebral ventricle for 5 days and 2 h after the last treatment the animals were sacrificed. Serum NO,
l
-arginine (
l
-Arg), and arginase activity were measured spectrophotometrically. For phosphorylation of Akt, ERK1/2, and iNOS protein expression, Western blot method was used. The expression of
Nos2
mRNA was measured by the quantitative real-time polymerase chain reaction (qRT-PCR). Treatment with ghrelin significantly increased NO production in serum by 1.4-fold compared with control. The concentration of
l
-Arg was significantly higher in ghrelin-treated rats than in control while arginase activity was significantly lower in ghrelin-treated than in control hearts. Ghrelin treatment increased phosphorylation of Akt by 1.9-fold and ERK1/2 by 1.6-fold and increased iNOS expression by 2.5-fold compared with control. In addition, ghrelin treatment increased
Nos2
gene expression by 2.2-fold as determined by qRT-PCR. These results indicate that ghrelin regulation of iNOS expression/activity is mediated via Akt/ERK1/2 signaling pathway. These results may be relevant to understanding molecular mechanisms underlying direct cardiovascular actions of ghrelin.
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