ABSTRACT
Peroxiredoxin I (Prx I) plays a role in regulating macrophage proinflammatory cytokine production and gene expression and participates in immune regulation. However, the possible protective role of Prx I in endotoxininduced lethal shock is poorly understood. In the present study, western blot analysis, ELISA and haematoxylin and eosin staining were performed to examine the protein expression of cytoines and analyses the levels of cytokines in the serum and tissue to evaluate the tissue damage. The present study revealed that lipopolysaccharide (LPS)induced lethality in Prx I/ mice was is accelerated via the observed decreased serum IL10 levels. Results also demonstrated rapid immune cell infiltration and oxidative stress in the Prx I/mice liver after LPS injections. These phenomena increased liver apoptosis through increasing cleaved caspase3 protein expression in Prx I/ mice after LPS injections, resulting in high lethality after LPS challenges. These findings provide a new insight for understanding the function of Prx I against endotoxininduced injury.
Subject(s)
Oxidative Stress/genetics , Peroxiredoxins/genetics , Shock, Septic/genetics , Animals , Apoptosis/genetics , Caspase 3/genetics , Gene Expression Regulation/genetics , Humans , Interleukin-10/blood , Lipopolysaccharides/toxicity , Liver/drug effects , Liver/pathology , Macrophages/metabolism , Macrophages/pathology , Mice , Mice, Knockout , Shock, Septic/blood , Shock, Septic/chemically inducedABSTRACT
High concentrations of glutamate may mediate neuronal cell apoptosis by increasing intracellular reactive oxygen species (ROS) levels. Peroxiredoxin V (Prx V), a member of the Prx family, serves crucial roles in protecting cells from oxidative stress. The present study investigated the regulatory effect of Prx V on glutamateinduced effects on viability and apoptosis in HT22 cells. Western blotting was used for protein expression analysis and Annexin V/PI staining and flow cytometry for determination of apoptosis. The results demonstrated that glutamate may ROSdependently increase HT22 cell apoptosis and upregulate Prx V protein levels. Furthermore, knockdown of Prx V protein expression with a lentivirus significantly enhanced HT22 cell apoptosis mediated by glutamate, which was reversed by inhibition of ROS with NacetylLcysteine. Inhibiting the extracellular signalregulated kinase (ERK) signaling pathway with PD98059, a specific inhibitor for ERK phosphorylation, markedly decreased glutamateinduced HT22 cell apoptosis in Prx V knockdown cells, indicating the potential involvement of ERK signaling in glutamateinduced HT22 cell apoptosis. In addition, an increase in nuclear apoptosisinducing factor was observed in Prx V knockdown HT22 cells following glutamate treatment, compared with mock cells, whereas no differences in Bcell lymphoma2 and cleavedcaspase3 protein expression levels were observed between mock and Prx V knockdown cells. The results of the present study indicated that Prx V may have potential as a therapeutic molecular target for glutamateinduced neuronal cell death and provide novel insight into the role of Prx V in oxidativestress induced neuronal cell death.
Subject(s)
Apoptosis/genetics , Glutamic Acid/metabolism , Peroxiredoxins/genetics , Pyramidal Cells/metabolism , Animals , Apoptosis/drug effects , Apoptosis Inducing Factor/metabolism , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Knockout Techniques , Glutamic Acid/pharmacology , Mice , Pyramidal Cells/drug effects , Reactive Oxygen Species/metabolismABSTRACT
It has previously been reported that 16α, 17α-epoxypregnenolone-20-oxime (EPREGO) exerts an inhibitory effect on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in microglia. The present study aimed to investigate the effects of EPREGO on the lipopolysaccharide (LPS)induced inflammatory response in RAW264.7 macrophage cells, and to determine the underlying molecular mechanisms using western blot analysis, enzymelinked immunosorbent assays and fluorescenceactivated cell sorting. The present study demonstrated that LPSinduced production of NO and interleukin (IL)-6, and the protein expression levels of iNOS, were reduced by EPREGO in a dose and timedependent manner, whereas, EPREGO did not affect tumor necrosis factorα production. In addition, EPREGO suppressed LPSinduced cellular reactive oxygen species production and phagocytosis. Furthermore, EPREGO significantly inhibited the LPSinduced activation of mitogenactivated protein kinases and inhibitor of κB α degradation in LPSstimulated RAW264.7 cells, thus resulting in modulation of the production of NO and IL6. Taken together, these results suggest that EPREGO exhibits anti-inflammatory activity in macrophages, thus validating the hypothesis that EPREGO may be useful as a therapeutic agent for the treatment of macrophage-mediated inflammation.
Subject(s)
Interleukin-6/biosynthesis , Macrophages/drug effects , Macrophages/metabolism , Nitric Oxide/biosynthesis , Oximes/pharmacology , Animals , Cell Line , Cytokines/metabolism , Inflammation Mediators/metabolism , Lipopolysaccharides/immunology , MAP Kinase Signaling System/drug effects , Macrophage Activation/immunology , Macrophages/immunology , Mice , Phagocytosis/drug effects , Phagocytosis/immunology , Reactive Oxygen Species/metabolismABSTRACT
The free radical nitric oxide (NO), a main member of neuroinflammatory cytokine and a gaseous molecule produced by activated microglia, has many physiological functions, including neuroinflammation. In the present study, we evaluated the effects of serial 16-dehydropregnenolone-3-acetate derivatives on lipopolysaccharide (LPS)-induced NO production and inducible nitric oxide synthase (iNOS) expression in BV-2 microglial cells. Among the six derivatives tested, the increases in NO production and iNOS expression observed in BV-2 microglial cells after LPS stimulation were significantly inhibited by treatment with 16α, 17α-epoxypregnenolone-20-oxime. Moreover, the inhibitory effect of 16α,17α-epoxypregnenolone-20-oxime on NO production was similar to that of S-methylisothiourea sulfate (SMT), an iNOS inhibitor. Further studies showed that 16α,17α-epoxypregnenolone-20-oxime inhibited c-Jun N-terminal kinase (JNK) phosphorylation but not inhibitor kappa B (IκB)-α degradation. Our data in LPS-stimulated microglia cells suggest that 16α,17α-epoxypregnenolone-20-oxime might be a candidate therapeutic for treatment of NO induced neuroinflammation and could be a novel iNOS inhibitor.