OBJECTIVES: To investigate the role of transient receptor potential cation channel subfamily M member 2 (TRPM2) in hepatic ischemia-reperfusion injury of mouse (HIRI) and the possible mechanisms. METHODS: Sixty adult male C57BL/6 mice were randomly divided into 4 groups: a sham group (S group), a HIRI model group (M group), a TRPM2 adenovirus interference vector group (T group), and a TRPM2 adenovirus control vector group (C group) (=15 in each group). The liver tissues of mice before perfusion were obtained. The efficiency of adenovirus infection was detected by fluorescence microscopy, and the silencing efficiency of adenovirus against TRPM2 was detected by real-time PCR.The abdominal aorta blood and liver tissues were collected from mice at 2, 4 and 8 h after reperfusion. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of mice were detected. Hepatic pathological changes were examined by hematoxylin-eosin (HE) staining. The protein expression of TRPM2 and Rac family small GTPase 1 (RAC1) in liver tissues was detected by Western blotting. Changes of malondialdehyde (MDA), superoxide dismutase (SOD) and myeloperoxidase (MPO) activities in liver tissues were detected by enzyme-linked immunosorbent assay. RESULTS: A strong signal of green fluorescence was observed in the liver tissues of mice in the T and C groups compared to the S or M group. Compared with the S, M or C group, the expression of TRPM2 mRNA in liver tissue in the T group was significantly down-regulated (all <0.05). The morphology of hepatocytes was normal in the S group under light microscope.Hepatic sinus dilatation, congestion, hepatocyte degeneration, central necrosis of lobule, and massive inflammatory granulocyte infiltration were observed in the M and C group, respectively. The degree of hepatocyte damage in the T group was significantly reduced compared with that in the M and C group, respectively. Compared with the S group, the serum ALT and AST activities in the M, T and C groups were significantly increased at 2, 4 and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum ALT and AST activities in the T group were significantly lower in serum of mice at 2, 4, and 8 h after reperfusion (all <0.05). Compared with the M or C group, the serum SOD activity in the T group was significantly increased at 2, 4, and 8 h after reperfusion (all <0.05), while the serum MDA and MPO activities were significantly decreased (all <0.05). The protein expression of TRPM2 and RAC1 in liver tissues in the T group were significantly lower than those in the M and C groups at 2, 4 and 8 h after reperfusion (all <0.05). CONCLUSIONS Pretreatment with TRPM2 adenovirus interference vector can effectively silence TRPM2 gene expression in liver tissues of mice and attenuate HIRI, which may be related to inhibiting oxidative stress and reducing the expression of RAC1 protein.
Alanine Transaminase ; Animals ; Aspartate Aminotransferases ; Liver ; Male ; Mice ; Mice, Inbred C57BL ; Neuropeptides ; Rats, Sprague-Dawley ; Reperfusion Injury ; TRPM Cation Channels ; genetics ; Transient Receptor Potential Channels ; rac1 GTP-Binding Protein

OBJECTIVE: To explore the effects of taurolithocholic acid (tLCA) and chenodeoxycholic acid (CDCA) on the expression of aorexigenic neuropeptide in mouse hypothalamus GT1-7 cells. METHODS: Mouse hypothalamic GT1-7 cells were treated with culture medium containing 10% FBS (control group, =3) or with 10 nmol/L, 100 nmol/L, 1 μmol/L and 10 μmol/L tLCA (tLCA group, =3) or CDCA (CDCA group, =3) for 12, 24 or 48 h. Real-time PCR was performed to determine the expression levels of proopiomelanocortin (POMC) mRNA in the cells, and the production levels of α-melanocyte-stimulating hormone (α-MSH) were assessed using an ELISA kit. Signal transduction and activator of transcription 3 phosphorylation (p-STAT3), threonine kinase phosphorylation (p-AKT), suppressor of cytokine signaling 3 (SOCS3), G protein-coupled bile acid receptor-1 (TGR5) and farnesoid X receptor (FXR) protein were detected by Western blotting. RESULTS: Western blotting results showed that mouse hypothalamic GT1-7 cells expressed two bile acid receptors, TGR5 and FXR, whose expressions were regulated by bile acids. Real-time PCR showed that the expression of POMC mRNA was significantly increased in the cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. POMC-derived anorexigenic peptide α-MSH increased significantly in GT1-7 cells after treatment with 10 μmol/L tLCA or CDCA for 24 h. Treatment of the cells with tLCA or CDCA significantly increased the expressions of intracellular signaling proteins including p-STAT3, p-AKT and SOCS3. CONCLUSIONS Mouse hypothalamic GT1-7 cells express bile acid receptors TGR5 and FXR. Bile acids tLCA or CDCA can promote the expression of POMC mRNA and increase the production of the anorexigenic peptide α-MSH. The intracellular signaling proteins p-AKT, p-STAT3 and SOCS3 are likely involved in bile acid-induced anorexigenic peptide production.
Animals ; Cell Line ; Chenodeoxycholic Acid ; pharmacology ; Gene Expression Regulation ; drug effects ; Hypothalamus ; cytology ; Mice ; Neuropeptides ; genetics ; metabolism ; Pro-Opiomelanocortin ; genetics ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Suppressor of Cytokine Signaling 3 Protein ; metabolism ; Taurolithocholic Acid ; pharmacology ; alpha-MSH ; genetics

The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of HO in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular HO. Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of HO-dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and HO-dependent Rac1 activation.
Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Animals, Newborn ; Catalase ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Fibroblasts ; Gene Expression Regulation ; drug effects ; Heart ; drug effects ; Hydrogen Peroxide ; metabolism ; pharmacology ; Isoflavones ; pharmacology ; Mice ; Myocardium ; cytology ; enzymology ; metabolism ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Neuropeptides ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; antagonists & inhibitors ; metabolism ; Transcriptional Activation ; drug effects ; rac1 GTP-Binding Protein ; metabolism

Rac1 belongs to the family of Rho GTPases, and plays important roles in the brain function. It affects the cell migration and axon guidance via regulating the cytoskeleton and cellular morphology. However, the effect of its dynamic activation in regulating physiological function remains unclear. Recently, a photoactivatable analogue of Rac1 (PA-Rac1) has been developed, allowing the activation of Rac1 by the specific wavelength of light in living cells. Thus, we constructed recombinant adeno-associated virus (AAV) of PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A under the control of the mouse glial fibrillary acidic protein (mGFAP) promoter to manipulate Rac1 activity in astrocytes by optical stimulation. Primary culture of hippocampal astrocytes was infected with the recombinant AAV-PA-Rac1 or AAV-PA-Rac1-C450A. Real-time fluorescence imaging showed that the cell membrane of the astrocyte expressing PA-Rac1 protruded near the light spot, while the astrocyte expressing PA-Rac1-C450A did not. We injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A into dorsal hippocampus to investigate the role of the activation of Rac1 in regulating the associative learning. With optical stimulation, the PA-Rac1 group, rather than the PA-Rac1-C450A group, showed slower learning curve during the fear conditioning compared with the control group, indicating that activating astrocytic Rac1 blocks the formation of contextual memory. Our data suggest that the activation of Rac1 in dorsal hippocampal astrocyte plays an important role in the associative learning.
Animals ; Astrocytes ; physiology ; Cell Membrane ; Cell Movement ; Conditioning, Classical ; Cytoskeleton ; Dependovirus ; Fear ; Hippocampus ; physiology ; Memory ; Mice ; Mice, Inbred C57BL ; Neuropeptides ; genetics ; physiology ; Optogenetics ; rac1 GTP-Binding Protein ; genetics ; physiology

OBJECTIVETo investigate the role of Rheb (mTOR activator) in AML development by measuring Rheb expression in bone marrow of adult AML patients and in AML cell line HL-60.

METHODSReal-time PCR assay was used to measure the Rheb mRNA expression in 27 AML patients and 29 ITP patients as control. The relationship between Rheb mRNA expression and age, AML subtype, fusion gene, splenomegaly, hepatomegaly and survival of AML patients was analyzed and compared. In addition, HL-60 cell line over-expressing Rheb was established, and the HL-60 cells and HL-60 cells with overexpression of Rheb were treated with Ara-C of different concentrations, the proliferation level was detected by CCK-8 method, and the IC50 was calculated.

RESULTSThe mRNA level of Rheb in AML patients was similar to that in ITP patients (control). Interestingly, higher expression of Rheb was associated with better survival and was sensitive to Ara-C treatment. However, the expression level of Rheb was not associated with age, AML subtype, fusion gene, and hepatomegaly of patients. Lower expression level of Rheb was associated with splenomegaly. In vitro analysis of HL-60 line indicated that overexpression of Rheb could increased the cell sensitivity to Ara-C treatment (IC50=0.54 µmol/L) and caused HL-60 cell apoptosis.

CONCLUSIONThe lower Rheb expression is a poor prognostic indicator for AML patients, which is associated with AML splenomegaly, the patients and HL-60 cells with low expression of Rheb are insensitive to Ara-C treatment.

Adult ; Apoptosis ; Bone Marrow ; metabolism ; Cytarabine ; pharmacology ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; pathology ; Monomeric GTP-Binding Proteins ; genetics ; metabolism ; Neuropeptides ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Ras Homolog Enriched in Brain Protein ; Real-Time Polymerase Chain Reaction ; Spleen ; pathology

BACKGROUNDSleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented, however, no animal or mechanistic studies on these disturbances exist. Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation, and may play an important role in sleep disturbances in ALS. In this study, we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.

METHODSElectroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days, and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay.

RESULTSFor the first time in SOD1-G93A transgenic mice, we observed significantly increased wakefulness, reduced sleep time, and up-regulated orexins (prepro-orexin, orexin A and B) at both 90 and 120 days. Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time, wakefulness time, rapid eye movement [REM] sleep time, non-REM sleep time, and deep sleep time) and increase in orexins (prepro-orexin, orexin A and B).

CONCLUSIONSleep/wake disturbances occur before disease onset in this ALS mouse model. Increased orexins may promote wakefulness and result in these disturbances before and after disease onset, thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS. Further studies are required to elucidate the underlying mechanisms in the future.

Amyotrophic Lateral Sclerosis ; genetics ; metabolism ; Animals ; Female ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Mice ; Mice, Transgenic ; Neuropeptides ; genetics ; metabolism ; Orexins ; Reverse Transcriptase Polymerase Chain Reaction ; Sleep ; physiology ; Superoxide Dismutase ; genetics ; metabolism ; Superoxide Dismutase-1 ; Wakefulness ; physiology

OBJECTIVEInflammation and lung function decline are the main pathophysiological features of chronic obstructive pulmonary disease (COPD). Acupuncture can improve lung function in patients with COPD, but the underlying mechanisms are not well understood. Orexins (OXs), which are found in peripheral plasma, are neuropeptides that regulate respiration and their levels are related to COPD. Therefore, we hypothesized that acupuncture might alter OXs, reduce lung inflammation and improve lung function in COPD.

METHODSCOPD was induced in rats by exposure to cigarette smoke for 8 weeks and injecting with lipopolysaccharide twice. Electroacupuncture (EA) was performed at Feishu (BL13) and Zusanli (ST36) for 30 min/d for 2 weeks. Rat lung function and morphology were assessed after EA. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid (BALF) and orexin A and B levels in the lung tissue were detected by enzyme-linked immunosorbent assay. OX receptor mRNA levels and immunopositive cells were assessed with real-time polymerase chain reaction and immunohistochemical methods, respectively. The relationships among lung function, cell factors, and OX levels were analyzed by Pearson correlation analyses.

RESULTSCompared with the control group, lung function was significantly decreased in the rats with COPD (P<0.05). There were increases in TNF-α and IL-1β levels in BALF (P<0.05 and P<0.01, respectively), orexin A level in lung tissue (P<0.01; but not orexin B) and mRNA expressions of OX (OXR1) and OX 2 (OXR2) in lung tissue (P<0.05 and P<0.01, respectively); the integrative optical densities (IODs) of both receptors were greater in the COPD group (P<0.05). For rats with COPD subjected to EA, lung function was improved (P<0.05). There were notable decreases in TNF-α and IL-1β levels (P<0.05 and <0.01, respectively) in BALF. Orexin A, but not orexinB, levels in lung tissue also decreased (P<0.01), as did mRNA expression of OX1R and OX2R in lung tissue (P<0.05 and P<0.01, respectively). Receptor IODs were also reduced after EA treatment (P<0.05). Furthermore, orexin A levels and ratio of forced expiratory volume in 0.3 s to forced vital capacity were strongly negatively correlated (P<0.01), and orexin A was positively correlated with TNF-α and IL-1β (P<0.001 and P<0.05, respectively).

CONCLUSIONEA at Zusanli and Feishu improved lung function of rats with COPD and had an anti-inflammatory effect, which may be related to down-regulation of OXA and its receptors.

Animals ; Down-Regulation ; Electroacupuncture ; Interleukin-1beta ; analysis ; Intracellular Signaling Peptides and Proteins ; analysis ; genetics ; Lung ; physiopathology ; Male ; Neuropeptides ; analysis ; genetics ; Orexin Receptors ; analysis ; genetics ; Orexins ; Pulmonary Disease, Chronic Obstructive ; physiopathology ; therapy ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; analysis

MicroRNAs (miRNAs) are endogenously expressed small, non-coding transcripts that regulate protein expression. Substantial evidences suggest that miRNAs are enriched in central nervous system, where they are hypothesized to play pivotal roles during neural development. In the present study, we analyzed miRNAs expression in mice cerebral cortex and hippocampus at different developmental stages and found miR-29a increased dramatically at postnatal stages. In addition, we provided strong evidences that miR-29a is enriched in mature neurons both in vitro and in vivo. Further investigation demonstrated that the activation of glutamate receptors induced endogenous miR-29a level in primary neurons. Moreover, we showed that miR-29a directly regulated its target protein Doublecortin (DCX) expression, which further modulated axon branching in primary culture. Together, our results suggested that miR-29a play an important role in neuronal development of mice cerebrum.
Animals ; Axons ; metabolism ; physiology ; Hippocampus ; growth & development ; metabolism ; Mice ; MicroRNAs ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; Neurogenesis ; Neurons ; metabolism ; Neuropeptides ; genetics ; Primary Cell Culture

OBJECTIVETo detect potential mutation of Doublecortin (DCX) gene in a patient featuring X-linked subcortical laminar heterotopia (X-SCLH) and epilepsy.

METHODSMutation of the DCX gene was screened by PCR and direct sequencing. Pathogenicity of the mutation was analyzed with a PolyPhen-2 software.

RESULTSA de novo missense mutation c.971T>C (p.Phe324Ser) was discovered.

CONCLUSIONA diagnostic method for X-SCLH has been established, which may facilitate diagnosis and genetic counseling of patients featuring this disease.

Agenesis of Corpus Callosum ; diagnosis ; genetics ; Base Sequence ; Brain ; pathology ; Child ; Classical Lissencephalies and Subcortical Band Heterotopias ; diagnosis ; genetics ; Electroencephalography ; Epilepsy ; diagnosis ; genetics ; Exons ; Female ; Humans ; Magnetic Resonance Imaging ; Microtubule-Associated Proteins ; genetics ; Mutation ; Neuropeptides ; genetics

OBJECTIVETo investigate the synergistic anti-inflammatory effect of Radix Platycodon in combination with herbs for cleaning-heat and detoxification and its mechanism for Fel-targeting.

METHODSForty Wistar rats were randomly divided into five groups (8 per group): the sham-operated group, model group, Radix Platycodon group, Flos Lonicera and Fructus Forsythia (LF) group, and Radix Platycodon, Flos Lonicera and Fructus Forsythia combination (PLF) group, using a random number table. A rat chronic obstructive pulmonary disease (COPD) model was established by passive smoking and intratracheal instillation of lipopolysaccharide (LPS). The treatments started from the 15th day of passive smoking for a total duration of 14 days. At the end of the treatment, changes in the following measurements were determined: lung histopathology, inflammatory cytokines including tumor necrosis factor α (TNF-α), transforming growth factor β (TGF-β) and interleukin IL-1β (IL-1β) in bronchoalveolar lavage fluid (BALF), and mRNA expression of endogenous active substance intestinal trefoil factor 3 (TFF3) in the lung tissue.

RESULTSLight microscopy showed that compared with the sham-operated group, rats in the COPD model group had disrupted alveolar structure, collapsed local alveoli, significantly widened or even fused alveolar septa, and massive infiltration of inflammatory cells in the alveolar wall and interstitium. In addition, significant bronchial epithelium hyperplasia, partially shed epithelia, and marked inflammatory cell infiltration in the bronchial wall and its surrounding tissues were noticed. Electron microscopy showed that rats in the model group had degeneration of alveolar type II epithelial cell; reduction, breakage or even loss of cell surface microvilli; swollen mitochondria with disappearing cristae and vacuole-like structure; and, increased secondary lysosomes in alveolar macrophages. The TNF-α, TGF-β and IL-1β levels and white blood cell (WBC) count in BALF were significantly increased (P < 0.01 or P < 0.05) and TFF3 mRNA expression in the lung tissue was significantly reduced (P < 0.01). After treatment, the pathological morphology of lung injury was less severe in all three treatment groups. In addition, TGF-β and IL-1β and WBC count in BALF were decreased (P < 0.01 or P < 0.05), and TFF3 mRNA expression in the lung tissue was significantly increased in the PLF group (P < 0.01). Compared with the LF group, the IL-1β in BALF was significantly decreased P < 0.05), and TFF3 mRNA expression was significantly increased (P < 0.05) in the PLF group.

CONCLUSIONSRadix Platycodon synergizes with herbs for cleaning-heat and detoxification in reducing inflammatory injury in a rat model of COPD. The synergistic anti-inflammatory effect is reflected in the improvement in pathological changes and in the reduction of IL-1β levels in BALF. The mechanism of such synergistic action may be related to its effect on maintaining the TFF3 mRNA expression and Fel-targeting function.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Disease Models, Animal ; Drug Synergism ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; therapeutic use ; Immunohistochemistry ; Lung ; drug effects ; pathology ; Male ; Microscopy, Electron ; Neuropeptides ; genetics ; metabolism ; Phytotherapy ; methods ; Plant Preparations ; therapeutic use ; Platycodon ; Polymerase Chain Reaction ; methods ; Pulmonary Disease, Chronic Obstructive ; drug therapy ; pathology ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Sensitivity and Specificity ; Trefoil Factor-3