Synaptic vesicle protein 2A (SV2A) involvement has been reported in the animal models of epilepsy and in human intractable epilepsy. The difference between pharmacosensitive epilepsy and pharmacoresistant epilepsy remains poorly understood. The present study aimed to observe the hippocampus SV2A protein expression in amygdale-kindling pharmacoresistant epileptic rats. The pharmacosensitive epileptic rats served as control. Amygdaloid-kindling model of epilepsy was established in 100 healthy adult male Sprague-Dawley rats. The kindled rat model of epilepsy was used to select pharmacoresistance by testing their seizure response to phenytoin and phenobarbital. The selected pharmacoresistant rats were assigned to a pharmacoresistant epileptic group (PRE group). Another 12 pharmacosensitive epileptic rats (PSE group) served as control. Immunohistochemistry, real-time PCR and Western blotting were used to determine SV2A expression in the hippocampus tissue samples from both the PRE and the PSE rats. Immunohistochemistry staining showed that SV2A was mainly accumulated in the cytoplasm of the neurons, as well as along their dendrites throughout all subfields of the hippocampus. Immunoreactive staining level of SV2A-positive cells was 0.483 ± 0.304 in the PRE group and 0.866 ± 0.090 in the PSE group (P < 0.05). Real-time PCR analysis demonstrated that 2(-ΔΔCt) value of SV2A mRNA was 0.30 ± 0.43 in the PRE group and 0.76 ± 0.18 in the PSE group (P < 0.05). Western blotting analysis obtained the similar findings (0.27 ± 0.21 versus 1.12 ± 0.21, P < 0.05). PRE rats displayed a significant decrease of SV2A in the brain. SV2A may be associated with the pathogenesis of intractable epilepsy of the amygdaloid-kindling rats.
Amygdala ; drug effects ; metabolism ; physiopathology ; Animals ; Anticonvulsants ; pharmacology ; Disease Models, Animal ; Drug Resistance ; Electric Stimulation ; Epilepsy ; drug therapy ; genetics ; metabolism ; pathology ; Gene Expression Regulation ; Hippocampus ; drug effects ; metabolism ; physiopathology ; Kindling, Neurologic ; drug effects ; genetics ; metabolism ; pathology ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Phenobarbital ; pharmacology ; Phenytoin ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Synaptic Transmission ; Synaptic Vesicles ; drug effects ; metabolism ; pathology

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.
8,11,14-Eicosatrienoic Acid ; analogs & derivatives ; metabolism ; pharmacology ; Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Apoptosis ; drug effects ; Aryl Hydrocarbon Hydroxylases ; genetics ; metabolism ; Atherosclerosis ; genetics ; metabolism ; pathology ; Catalase ; genetics ; metabolism ; Cytochrome P-450 CYP2C8 ; genetics ; metabolism ; Gene Expression Regulation ; Heme Oxygenase-1 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Models, Biological ; NADPH Oxidase 2 ; NADPH Oxidases ; genetics ; metabolism ; NF-E2-Related Factor 2 ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide Synthase Type III ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; metabolism ; Reactive Oxygen Species ; antagonists & inhibitors ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology

OBJECTIVETo observe effect of Shufeng Xuanfei Recipe (SXR) and Jiebiao Qingli Recipe (JQR) on mRNA and protein expressions of Toll-like receptor 7 (TLR7), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappaB (NF-kappaB) in mice infected with influenza virus FM1.

METHODSOne hundred and eight mice were randomly divided into nine groups, i.e., the normal control group, the model group, the Oseltamivir group (at the daily dose of 2.5 g/mL), the high dose SXR group (at the daily dose of 3.762 g/kg), the middle dose SXR group (at the daily dose of 1.881 g/kg), the low dose SXR group (at the daily dose of 0.941 g/kg), the high dose JQR group (at the daily dose of 4.368 g/kg), the middle dose JQR group (at the daily dose of 2.184 g/kg), and the low dose JQR group (at the daily dose of 1.092 g/kg), 12 in each group. All mice were mildly anesthetized by ether. Mice in the normal control group were treated by nasal drop of 0.05 mL normal saline, while mice in the rest groups were infected by nasal drop of 0.05 mL influenza virus strain FM1 (LD50). The successful modeling rate was 100%. All medication was performed by gastrogavage 2 h after infection. Distilled water was given by gastrogavage to mice in the normal control group and the model group at the daily dose of 0.2 mL, each time per day for 4 successive days. mRNA expressions of TLR7, MyD88, and NF-kappaB in the lung tissue were determined by Western blot.

RESULTSCompared with the normal control group, mRNA expressions of TLR7, MyD88, and NF-kappaB increased in the model group (P < 0.01). Compared with the model group, mRNA and protein expressions of TLR7, MyD88, and NF-kappaB decreased in the Oseltamivir group, the high, middle, and low dose SXR groups (P < 0.05, P < 0.01); mRNA and protein expressions of TLR7 and NF-kappaB decreased in the high and middle dose JQR groups (P < 0.05, P < 0.01); mRNA expressions of MyD88 decreased in the high and middle dose JQR groups (P < 0.05); protein expressions of MyD88 decreased in the middle dose JQR group (P < 0.05); protein expressions of TLR7 and NF-kappaB decreased in the low dose JQR group (P < 0.05). Compared with the Oseltamivir group, protein expressions of MyD88 decreased in the low dose SXR group (P < 0.05); protein expressions of NF-kappaB decreased in the middle and low dose SXR groups (P < 0.01); mRNA and protein expressions of TLR7 (P < 0.05, P < 0.01), and protein expressions of MyD88 (P < 0.01) decreased in the high, middle, and low dose JQR groups; mRNA and protein expressions of NF-kappaB decreased in the low dose JQR group (P < 0.05, P < 0.01).

CONCLUSIONSEach dose SXR and middle dose JQR could down-regulating the activity of NF-kappaB through adjusting MyD88 dependent TLR signal pathway, thus fighting against influenza virus. SXR was more effective than JQR.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Lung ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Mice ; Mice, Inbred ICR ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Orthomyxoviridae ; Orthomyxoviridae Infections ; drug therapy ; metabolism ; Pneumonia, Viral ; drug therapy ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; drug effects ; Toll-Like Receptor 7 ; genetics ; metabolism

OBJECTIVEChanges of the internal and external cellular environments can induce calcium homeostasis disorder and unfolded protein aggregation in the endoplasmic reticulum (ER). This ER function disorder is called endoplasmic reticulum stress (ERS). Severe long-term ERS can trigger the ER apoptosis signaling pathway, resulting in cell apoptosis and organism injury. Recent researches revealed that ERS-induced cell death was involved in the neurocyte retrogradation in the progress of neuron degenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease and so on. Therefore, the protection effect of the traditional Chinese drug-Tiantai No. 1 (1) on the ERS injury of AD was investigated at the molecular gene level in this study with a view to explore the gene pharmacodynamic actions and mechanisms of this drug.

METHODSPrimarily cultured marrow mesenchymal stem cells (MSCs) of rats were treated by tunicamycin (TM) in order to induce ERS. RT-PCR, fluorescence immunocytochemistry and Western blot techniques were used to determine the mRNA and protein expression levels of the protective stress protein-ER molecular chaperones GRP78 and GRP94 (which would assist cells to resist cellular stress injury), and to determine the mRNA and protein expression levels of apoptosis promoting molecule Caspase-12 on the membrane of the ER, respectively.

RESULTSProtein expression levels of GRP78 and GRP94 were significantly increased in the TM-induced MSCs, and the mRNA level of Caspase-12 was also remarkably increased in the TM-induced MSCs (P<0.05). All these proved that the ERS model was successfully established by TM in MSC. Meanwhile, the mRNA and protein levels of GRP78 and GRP94 were all significantly increased compared with the model group (P<0.05 or P<0.01) after MSCs were treated with Tiantai No.1 while the mRNA and protein expression levels of Caspase-12 were significantly decreased compared with the model group (P<0.05 or P<0.01). This effect showed a dose dependent manner.

CONCLUSIONTiantai No.1 might attenuate the cell apoptosis induced by ERS injury, and thus protect the neurons against AD.

Animals ; Anti-Bacterial Agents ; antagonists & inhibitors ; pharmacology ; Cells, Cultured ; Drug Antagonism ; Drugs, Chinese Herbal ; pharmacology ; Endoplasmic Reticulum ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Heat-Shock Proteins ; genetics ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Mesenchymal Stromal Cells ; drug effects ; metabolism ; RNA ; analysis ; drug effects ; Rabbits ; Rats ; Rats, Sprague-Dawley ; Stress, Physiological ; drug effects ; genetics ; Tunicamycin ; antagonists & inhibitors ; pharmacology

OBJECTIVETo investigate the effects of the novel proteasome subunit Adrm1 knockdown by RNA interference on proliferation of colorectal cancer cells.

METHODSThe shRNA eukaryotic expression vector against Adrm1 was constructed and transfected into colon cancer RKO cells. The Adrm1-shRNA stable transfected clones were selected. Experimental cells were divided into 3 groups: the experimental group containing stable Adrm1-shRNA transfected cells, the control group containing only RKO colon cancer cells and stable empty vector transfected control group. The Adrm1 protein expression level was analyzed by Western blot. The colony-forming ability of the three groups was assessed by soft agar assay. The cell proliferation and apoptosis were analyzed by methyl thiazolyl tetrazolium (MTT) method and in situ end labeling (TUNEL) assay. Cell cycle changes were assayed by flow cytometry.

RESULTSAdrm1-shRNA effectively suppressed Adrm1 expression in the experimental group. Silencing of Adrm1 in RKO cells significantly inhibited their anchorage-independent growth, only occasional individual colonies were formed. The apoptosis rate of experimental group was (12.4 +/- 1.1)%, significantly higher than that of the stable empty vector transfected control group. The proportion of G(0)/G(1) and S/G(2) phase cells in the experimental group was (41.2 +/- 1.1)% and (58.8 +/- 1.1)%, respectively. The cells were arrested at G(1) phase. In addition, Adrm1 RNA interference combined with 5-Fu treatment significantly suppressed colorectal cancer cell growth in vitro.

CONCLUSIONSilencing of Adrm1 by RNA interference can significantly suppress proliferation of RKO cells through inducing apoptosis and arresting the cell cycle. The combined application of Adrm1 RNA interference and chemotherapy may become as a novel therapeutic strategy for Adrm1 overexpressed colorectal cancer.

Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Drug Resistance, Neoplasm ; Fluorouracil ; pharmacology ; Genetic Vectors ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Plasmids ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection

Consistent non-nucleoside reverse-transcriptase inhibitors (NNRTIs) resistant HIV-1 strains occurred due to the clinical use for more than ten years of efavirenz (EFV), nevirapine (NVP), and delavirdine (DLV). In this study, we established nine cell-based pharmacological models according to most NNRTIs-resistant clinical tested strains, Resistant mutations were introduced into vector, pNL4-3.Luc.R-E-, by overlapping PCR. Then, pseudovirions were produced by co-transfection of VSV-G plasmid and pNL4-3.Luc.R-E- -mut. All nine recombinant VSVG/HIV-mut pseudovirions (VSVG/HIV-wt, VSVG/HIV(-K103N), VSVG/HIV(-Y181C), VSVG/HIV(-L100I,K103N), VSVG/HIV(-Y188L), VSVG/HIV(-K103N,Y181C), VSVG/HIV(-K103N,P225H), VSVG/HIV(-K103N,Y188L), VSVG/HIV(-K103N,G109A) and VSVG/HIV(-K103N,V108I)) had high efficient infectivity. Furthermore, they all showed resistant characteristics to EFV and NVP with IC50 changes consisting with clinical reports, not to nucleoside reverse-transcriptase inhibitors (AZT and d4T). This series safe cell-based model, which could be carried out in BSL-2 laboratory, can be used for evaluating NNRTIs candidates.
Anti-HIV Agents ; pharmacology ; Benzoxazines ; pharmacology ; Cell Line ; Delavirdine ; pharmacology ; Drug Evaluation, Preclinical ; methods ; Drug Resistance, Viral ; Genetic Vectors ; HIV Reverse Transcriptase ; antagonists & inhibitors ; genetics ; metabolism ; HIV-1 ; drug effects ; genetics ; Humans ; Membrane Glycoproteins ; genetics ; Nevirapine ; pharmacology ; Plasmids ; genetics ; Point Mutation ; Reverse Transcriptase Inhibitors ; pharmacology ; Stavudine ; pharmacology ; Transfection ; Viral Envelope Proteins ; genetics ; Virion ; genetics ; metabolism ; Virus Replication ; Zidovudine ; pharmacology

OBJECTIVESTo investigate the regulative effect of the nonsteroidal anti-inflammatory drug NS398 on the RECK gene in the prostate carcinoma strain DU145.

METHODSDU145 was treated with various concentrations of NS398 for 48 hours. The mRNA level was measured by RT PCR technique and the expression of the RECK protein determined by Western blot.

RESULTSThe mRNA level of the RECK gene was obviously higher, while the MMP9 level markedly lower in the treated group than in the control, and so was the expression of the RECK protein.

CONCLUSIONNS398 induces the expression of the RECK gene, which might be the mechanism of its anti-tumor effect.

Animals ; Anti-Inflammatory Agents, Non-Steroidal ; pharmacology ; Blotting, Western ; Cattle ; Cell Line, Tumor ; GPI-Linked Proteins ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Male ; Membrane Glycoproteins ; biosynthesis ; genetics ; Nitrobenzenes ; pharmacology ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sulfonamides ; pharmacology

Xanthohumol (XH), the principal prenylflavonoid of the hop plant (Humulus lupulus L.), dose-dependently inhibited isobutylmethylxanthine (IBMX)-induced melanogenesis in B16 melanoma cells, with little cytotoxicity at the effective concentrations. Decreased melanin content was accompanied by reduced tyrosinase enzyme activity, protein and mRNA expression. The levels of tyrosinase-related protein 1 and 2 mRNAs were decreased by XH. XH also inhibited alpha-melanocyte stimulating hormone- or forskolin-induced increases in melanogenesis, suggesting an action on the cAMP-dependent melanogenic pathway. XH downregulated the protein and mRNA expression of microphthalmia-associated transcription factor (MITF), a master transcriptional regulator of key melanogenic enzymes. These results suggest that XH might act as a hypo-pigmenting agent through the downregulation of MITF in the cAMP-dependent melanogenic pathway.
1-Methyl-3-isobutylxanthine/pharmacology ; Animals ; Cell Line ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Drug Antagonism ; Forskolin/pharmacology ; *Humulus ; Intramolecular Oxidoreductases/antagonists & inhibitors/biosynthesis ; Melanins/antagonists & inhibitors/*biosynthesis ; Melanocytes/*drug effects/*metabolism ; Melanoma, Experimental ; Membrane Glycoproteins/antagonists & inhibitors/biosynthesis ; Mice ; Microphthalmia-Associated Transcription Factor/antagonists & inhibitors ; Monophenol Monooxygenase/antagonists & inhibitors/biosynthesis/genetics ; Oxidoreductases/antagonists & inhibitors/biosynthesis ; Propiophenones/*pharmacology ; Signal Transduction/drug effects ; alpha-MSH/metabolism

OBJECTIVETo investigate the expression of paired immunoglobin-like receptor B (PIR-B) on dendritic cells (DCs) and its relationship with tolerogenic DCs (T-DCs) in mouse.

METHODSDC2.4 cells, an immature dendritic cell line derived from C57BL/6 mouse, were stimulated by lipopolysaccharide (LPS) for 48 h to induce the mature dendritic cells (mDC) and cultured respectively with the recombined mouse interleukin-10 (rmIL-10) or recombined human transforming growth factor beta1 (rhTGF-beta1) to develop the tolerogenic dendritic cells (T-DC). Special small interference RNA (siRNA) molecular of PIR-B was chemically synthesized and transfected into DC2.4 cells (si-DC) by lip2000. The expression of PIR-B on DC2.4 cells was measured by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot. The allogeneic lymphocyte proliferative capacity of DCs was measured by mixed lymphocyte reaction (MLR) using 3H-thymidine incorporation test. The concentration of IFN-gamma in supernatants of MLR was analyzed by ELISA.

RESULTSSemi-quantitative RT-PCR and Western blot showed that PIR-B mRNA and protein were expressed on DC2.4 cells. RmIL-10 and rhTGF-beta1 induced the higher PIR-B mRNA and protein level on T-DCs (Relative values were 0.51 +/- 0.08 and 0.58 +/- 0.23; 0.85 +/- 0.07 and 0.87 +/- 0.14; 0.79 +/- 0.10 and 0.85 +/- 0.34, respectively) (P < 0.05). LPS down-regulated the PIR-B expression on mDC (0.35 +/- 0.10 and 0.32 +/- 0.04) (P < 0.05). The PIR-B mRNA and protein expression were inhibited by siRNA transfection (decreased by 78.9% and 74.2% respectively after 48 h interference) (P < 0.05). DC2.4 cells stimulated the proliferation of BALB/c mouse allo-genetic spleen cell. The mDC enhanced alloreactivity in MLR and the IFN-gamma secretion in supernatants. The T-DCs alleviated the allo-genetic spleen cell proliferation (P < 0.05) and IFN-gamma secretion in MLR (P < 0.05). Silence of the PIR-B expression (si-DC) also promoted of alloreactivity and enhanced the IFN-gamma secretion in MLR (P < 0.05).

CONCLUSIONHigh expression of immune inhibition receptor PIR-B is one of the general features and molecular mechanism of dendritic cells to acquire immune tolerance in mouse.

Animals ; Cell Line ; Dendritic Cells ; immunology ; metabolism ; Immune Tolerance ; Interleukin-10 ; pharmacology ; Membrane Glycoproteins ; genetics ; immunology ; metabolism ; Mice ; Mice, Inbred C57BL ; RNA, Small Interfering ; genetics ; Receptors, Immunologic ; genetics ; immunology ; metabolism ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo explore the effect of Herba Epimedii flavone (HEF) on the osteoblast metabolism in vitro.

METHODOsteoblast were obtained from new born rat calvaria by digestive enzymes. MTF, PNPP and RT-PCR were used to observe the proliferation, activity of ALP and mRNA expression of OPG and RANKL of cultured osteoblasts in vitro.

RESULTIt was found that HEF had the effect on stimulating cell proliferation, activity of ALP and the mRNA expression of OPG of cultured osteoblasts (P < 0.01, P < 0.05).

CONCLUSIONHEF can promote the proliferation, the differentiation and the expression of OPG mRNA of the osteoblasts cultured in vitro.

Alkaline Phosphatase ; metabolism ; Animals ; Animals, Newborn ; Carrier Proteins ; biosynthesis ; genetics ; Cell Proliferation ; drug effects ; Cells, Cultured ; Epimedium ; chemistry ; Flavones ; isolation & purification ; pharmacology ; Glycoproteins ; biosynthesis ; genetics ; Membrane Glycoproteins ; biosynthesis ; genetics ; Osteoblasts ; cytology ; metabolism ; Osteoprotegerin ; Plants, Medicinal ; chemistry ; RANK Ligand ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Cytoplasmic and Nuclear ; biosynthesis ; genetics ; Receptors, Tumor Necrosis Factor ; biosynthesis ; genetics