ABSTRACT
METHOD: Mice were randomly assigned to the sham, I/R, Oxy, and I/R with Oxy groups. Oxy was injected intraperitoneally 30 min before tourniquet placement. Morphological changes of the gastrocnemius muscle in these mice were assessed by hematoxylin-eosin (HE) staining and electron microscopy. Expression levels of TLR4, NF-κB, SIRT1, and PGC-1α in the skeletal muscles were detected by western blot. Blood TNF-α levels, gastrocnemius muscle contractile force, and ATP concentration were examined. RESULTS: Compared with the I/R group, Oxy pretreatment attenuated skeletal muscle damage, decreased serum TNF-α levels, and inhibited the expression levels of TLR4/NF-κB in the gastrocnemius muscle. Furthermore, Oxy treatment significantly increased serum ATP levels and the contractility of the skeletal muscles. SIRT1 and PGC-1α levels were significantly reduced in gastrocnemius muscle after I/R. Oxy pretreatment recovered these protein expression levels. CONCLUSION: Tourniquet-induced acute limb I/R results in morphological and functional impairment in skeletal muscle. Pretreatment with Oxy attenuates skeletal muscle from acute I/R injury through inhibition of TLR4/NF-κB-dependent inflammatory response and protects SIRT1/PGC-1α-dependent mitochondrial function.
Subject(s)
Muscle Contraction , Muscle, Skeletal/metabolism , Receptors, Opioid/metabolism , Reperfusion Injury/metabolism , Tourniquets , Adenosine Triphosphate/metabolism , Animals , Inflammation , Male , Mice , Mice, Inbred C57BL , Microscopy, Electron , Muscle, Skeletal/injuries , NF-kappa B p50 Subunit/metabolism , Perfusion , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha/metabolism , Sirtuin 1/metabolism , Toll-Like Receptor 4/metabolismABSTRACT
Inflammatory responses involving microglia and astrocytes contribute to the pathogenesis of neurodegenerative diseases (NDs). In addition, inflammation is tightly linked to iron metabolism dysregulation. However, it is not clear whether the brain inflammation-induced iron metabolism dysregulation contributes to the NDs pathogenesis. Herein, we demonstrate that the expression of the systemic iron regulatory hormone, hepcidin, is induced by lipopolysaccharide (LPS) through the IL-6/STAT3 pathway in the cortex and hippocampus. In this paradigm, activated glial cells are the source of IL-6, which was essential in the iron overload-activated apoptosis of neurons. Disrupting astrocyte hepcidin expression prevented the apoptosis of neurons, which were able to maintain levels of FPN1 adequate to avoid iron accumulation. Together, our data are consistent with a model whereby inflammation initiates an intercellular signaling cascade in which activated microglia, through IL-6 signaling, stimulate astrocytes to release hepcidin which, in turn, signals to neurons, via hepcidin, to prevent their iron release. Such a pathway is relevant to NDs in that it links inflammation, microglia and astrocytes to neuronal damage.
Subject(s)
Apoptosis/drug effects , Astrocytes/metabolism , Hepcidins/metabolism , Lipopolysaccharides/pharmacology , Neurons/metabolism , Animals , Astrocytes/drug effects , Astrocytes/pathology , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Encephalitis/metabolism , Encephalitis/pathology , Hippocampus/drug effects , Hippocampus/metabolism , Hippocampus/pathology , Interleukin-6/metabolism , Iron/metabolism , Male , Mice , Mice, Inbred BALB C , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Neurons/drug effects , Neurons/pathology , Rats , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effectsABSTRACT
Oxidative stress plays an important role in neuronal injuries caused by cerebral ischemia. It is well established that free iron increases significantly during ischemia and is responsible for oxidative damage in the brain. However, the mechanism of this ischemia-induced increase in iron is not completely understood. In this report, the middle cerebral artery occlusion (MCAO) rat model was performed and the mechanism of iron accumulation in cerebral ischemia-reperfusion was studied. The expression of L-ferritin was significantly increased in the cerebral cortex, hippocampus, and striatum on the ischemic side, whereas H-ferritin was reduced in the striatum and increased in the cerebral cortex and hippocampus. The expression level of the iron-export protein ferroportin1 (FPN1) significantly decreased, while the expression of transferrin receptor 1 (TfR1) was increased. In order to elucidate the mechanisms of FPN1 regulation, we studied the expression of the key regulator of FPN1, hepcidin. We observed that the hepcidin level was significantly elevated in the ischemic side of the brain. Knockdown hepcidin repressed the increasing of L-ferritin and decreasing of FPN1 invoked by ischemia-reperfusion. The results indicate that hepcidin is an important contributor to iron overload in cerebral ischemia. Furthermore, our results demonstrated that the levels of hypoxia-inducible factor-1α (HIF-1α) were significantly higher in the cerebral cortex, hippocampus and striatum on the ischemic side; therefore, the HIF-1α-mediated TfR1 expression may be another contributor to the iron overload in the ischemia-reperfusion brain.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Infarction, Middle Cerebral Artery/metabolism , Iron/metabolism , Animals , Antimicrobial Cationic Peptides/deficiency , Antimicrobial Cationic Peptides/genetics , Brain/metabolism , Cation Transport Proteins/metabolism , Ferritins/metabolism , Gene Knockdown Techniques , Hepcidins , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Infarction, Middle Cerebral Artery/complications , Infarction, Middle Cerebral Artery/genetics , Interleukin-6/genetics , Iron Overload/complications , Iron Overload/genetics , Iron Overload/metabolism , Mice , Rats , Receptors, Transferrin/metabolism , Reperfusion Injury/complications , Up-RegulationABSTRACT
This study investigates whether activation of p38 MAPK by the up-regulation of HSP 70 participates in the induction of brain ischemic tolerance by limb ischemic preconditioning (LIP). Western blot and immunohistochemical assays indicated that p38 MAPK activation occurred earlier than HSP 70 induction in the CA1 region of the hippocampus after LIP. P-p38 MAPK expression was up-regulated at 6h and reached its peak 12h after LIP, while HSP 70 expression was not significantly increased until 1 day and peaked 2 days after LIP. Neuropathological evaluation by thionin staining showed that quercetin (4 ml/kg, 50mg/kg, intraperitoneal injection), an inhibitor of HSP 70, blocked the protective effect of LIP against delayed neuronal death that is normally induced by lethal brain ischemic insult, indicating that HSP 70 participates in the induction of brain ischemic tolerance by LIP. Furthermore, SB 203580, an inhibitor of HSP 70, inhibited HSP 70 activation in the CA1 region of the hippocampus induced by LIP either with or without the presence of subsequent brain ischemic insult. Based on the above results, it can be concluded that activation of p38 MAPK participates in the brain ischemic tolerance induced by LIP at least partly by the up-regulation of HSP 70 expression.
Subject(s)
Brain Ischemia/metabolism , HSP70 Heat-Shock Proteins/biosynthesis , Hindlimb/blood supply , Ischemic Preconditioning , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Blotting, Western , Brain Ischemia/pathology , Brain Ischemia/prevention & control , CA1 Region, Hippocampal/metabolism , CA1 Region, Hippocampal/pathology , Disease Susceptibility , Enzyme Activation , HSP70 Heat-Shock Proteins/antagonists & inhibitors , Imidazoles/pharmacology , Immunohistochemistry , Male , Pyridines/pharmacology , Quercetin/pharmacology , Rats , Rats, Wistar , Up-Regulation , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitorsABSTRACT
AIM: To establish a tumor model in HLA-A2.1 transgenic mice to examine the efficacy of MAGE-3 vaccine, a cell line coexpressing HLA-A 0201/K(b) and MAGE-3 is established. METHODS: B16-HLA-MAGE-3 melanoma was obtained by means of cotransfection of HLA-A 0201/K(b) and MAGE-3 to B16 melanoma. RT-PCR, FCM analysis and Western blot were used to detect the mRNA or protein of HLA-A 0201/K(b) or MAGE-3 expression in B16-HLA-MAGE-3. The ability of MAGE-3 antigen to be processed and presented in the B16-HLA-MAGE-3 cell line were observed by CTL activity detection and tumor challenge test. RESULTS: Transcription and protein expression of HLA-A 0201/H-2k(b) and MAGE-3 were demonstrated in B16-HLA-MAGE-3 cells. CTL activity of splenocytes in immunized mice against B16-HLA-MAGE-3 was detected and the growth of B16-HLA-MAGE-3 in immunized mice was also inhibited. CONCLUSION: MAGE-3 antigen is able to be processed and presented efficiently by B16-HLA-MAGE-3 melanoma cells and this cell can be employed to test HLA-A2 restricted epitope immunogenicity in the A2-transgenic mice.
Subject(s)
Antigens, Neoplasm/immunology , Cancer Vaccines/immunology , HLA Antigens/immunology , Melanoma, Experimental/immunology , Neoplasm Proteins/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/metabolism , Blotting, Western , Cancer Vaccines/genetics , Cancer Vaccines/metabolism , Cell Line , HLA Antigens/genetics , HLA Antigens/metabolism , Melanoma, Experimental/metabolism , Mice , Mice, Transgenic , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM: To prepare rabbit anti-rat P450scc antibody, and to detect the expression of rP450scc in rat brain tissue and nerve cells. METHODS: An eight-branched polypeptide, rP450scc-16 was synthesized using solid phase synthesis (Fmoc) method. A Newzealand rabbit was immunized with rP450scc-16 and the serum was separated from the whole blood 5 days after the last immunization. The titer and specificity of the antiserum were evaluated, and the expression of rP450scc in normal rat brain and primary rat astrocytes was detected by using ELISA, Western blot and immunocytochemcal staining. RESULTS: The titer of the antiserum was 1:6,400. Western blot analysis showed that the rP450scc protein in rat brain, testis and adrenal gland homogenate was recognized by the antiserum as a single band at M(r) being 50,000, which indicated a high specificity of the antiserum. Hypothalamus, cerebral cortex, and hippocampus of rat brain and the cytoplasm of cultured rat astrocytes were positively stained by the antiserum. CONCLUSION: A highly specific anti-rP450scc antibody was prepared. The antibody can be used to study the expression and distribution of rP450scc in rat brain.
Subject(s)
Antibodies/analysis , Antibodies/immunology , Cholesterol Side-Chain Cleavage Enzyme/immunology , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/metabolism , Antibody Specificity , Astrocytes/metabolism , Brain/cytology , Brain/metabolism , Gene Expression Regulation , Immunohistochemistry , Male , Molecular Sequence Data , Peptides/analysis , Peptides/chemical synthesis , Peptides/chemistry , Rabbits , Rats , Rats, Sprague-DawleyABSTRACT
AIM: To investigate the effects of morphine dependence and withdrawal on the concentrations of neurosteroids in rat brain. METHODS: A method of simultaneous quantification of neurosteroids by gas chromatography-mass spectrometry (GC-MS) had been established. RESULTS: The chronic morphine administration (ip) resulted in a marked decrease in the brain concentrations of pregnenolone (PREG), progesterone (PROG), and pregenenolone sulfate (PREGS) in rats killed 6 h after the last treatment. In contrast, there were no significant effects of morphine dependence on the brain concentrations of allopregnanolone (AP), dihydroepiandrosterone (DHEA), and dihydroepiandrosterone sulfate (DHEAS). Naloxone-induced withdrawal produced a significant increase in the concentrations of PREG, PROG, AP, DHEA, PREGS, and DHEAS as compared with the control group. CONCLUSION: Morphine dependence and withdrawal affected the concentrations of neurosteroids in rat brain, which suggests that endogenous neurosteroids in brain might be related to the development of morphine dependence and withdrawal.
