OBJECTIVEBy using the RNAi method to inhibit Itk protein expression specificity, to observe lymphocytes proliferation and cytokines production, verify its function as a drug target.

METHODSDesigned siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA.

RESULTSItk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-gamma. They all have statistical difference (P < 0.05).

CONCLUSIONItk has an important immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines.This can supply an experimental basis to regard Itk as drug target for inflammation therapy.

Animals ; Cell Differentiation ; Cell Proliferation ; Cytokines ; genetics ; immunology ; Lymphocytes ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Protein-Tyrosine Kinases ; genetics ; immunology ; Spleen ; cytology ; immunology

OBJECTIVETo evaluate the possibility of short interfering RNA (siRNA) inhibiting Coxsackievirus B3 (CVB3) infection in vitro, and discover the mechanism initially.

METHODSWe obtained proper effective dosage of siRNA by observing cytopathic effect (CPE). Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR.

RESULTSResults showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77% and its effect can last for 48 h stably in cells. 0.6 micromol/L siRNA-3753 got a high inhibiting effect of virus and didn't show any toxicity to cells. So we consider this concentration as the experimental concentration. siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R (PKR).

CONCLUSIONThe data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.

Coxsackievirus Infections ; therapy ; virology ; Enterovirus B, Human ; genetics ; metabolism ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics

OBJECTIVETo observe the effects of jieyuwan on neuroendocrine-immune of chronic unpredictable stress rats, for identifing its mechanism of action.

METHODAnimals were randomly assigned to six groups: one non-stressed (control) group, one stressed group (CUS + vehicle) , three treatment groups (CUS + Jieyuwan 53, 106, 212 mg x kg(-1), respectively), and one group for imipramine (CUS + imipramine 10 mg x kg(-1)). We used the chronic unpredictable stress (CUS) test in rats to investigated the effects of chronic oral administration (21 days) of Jieyuwan and imipramine (ip) on the sucrose consumption, the relative weight of adrenal and thymus, the concentration of serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta measured by ELISA.

RESULTOur results showed that Sprague-Dawley rats subjected to CUS exhibited increased adrenal weight, reduction in thymus weight, less sucrose consumption, and synchronized with higher serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta concentration. Chronic treatment with Jieyuwan (53, 106, 212 mg x kg(-1), po, 21 days) could improve locomotion of stressed rat sucrose consumption, turn over the high levels of serum corticosterone, adrenocorticotropic hormone, TNF-alpha, IL-1beta concentration.

CONCLUSIONThe therapeutic actions of jieyuwan, in some degree, may be related with indirect or direct effects on HPA axis and immune system.

Adrenocorticotropic Hormone ; blood ; Animals ; Antidepressive Agents ; pharmacology ; Behavior, Animal ; drug effects ; Corticosterone ; blood ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Hypothalamo-Hypophyseal System ; drug effects ; physiopathology ; Interleukin-1beta ; blood ; Male ; Pituitary-Adrenal System ; drug effects ; physiopathology ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological ; immunology ; pathology ; physiopathology ; Thymus Gland ; pathology ; Tumor Necrosis Factor-alpha ; blood

BACKGROUNDSeveral randomized controlled trials (RCTs) have compared endoscopic and symptomatic relapses in patients with erosive gastroesophageal reflux disease (GERD). We have summarized current evidence for rabeprazole 10 or 20 mg once daily for GERD maintenance treatment over 1 or 5 years.

METHODSMEDLINE, EMBASE, and the Cochrane Central Register of Controlled Trials were searched, through August 2012, for eligible RCTs of adults with erosive GERD. The efficacies of rabeprazole 10 and 20 mg/d were compared.

RESULTSThe search identified 288 citations, and five RCTs containing 1480 patients were considered eligible. Heartburn relapse rates did not differ significantly between patients treated with rabeprazole 10 and 20 mg/d for 1 year (relative risk (RR) = 1.29; 95% confidence interval (CI): 0.97-1.72), but differed in patients treated for 5 years (RR = 1.274; 95% CI: 1.005-1.615). Endoscopic relapse rates differed significantly between rabeprazole 10 and 20 mg/d for 1 year (RR = 1.92; 95% CI: 1.21-3.06), for 5 years (RR = 1.667; 95% CI: 1.073-2.589), and in combined 1- and 5-year maintenance trials (RR = 1.785; 95% CI: 1.298-2.456).

CONCLUSIONRabeprazole 20 mg/d was superior to rabeprazole 10 mg/d in preventing endoscopic relapse of erosive GERD, but that the two dosages were equivalent in symptomatic relief over 1 year.

Dose-Response Relationship, Drug ; Gastroesophageal Reflux ; prevention & control ; Humans ; Proton Pump Inhibitors ; therapeutic use ; Rabeprazole ; therapeutic use ; Randomized Controlled Trials as Topic ; Recurrence

OBJECTIVETo elucidate the regulatory mechanism underlying proliferation and anti-apoptosis in NSCLC by overexpression of miR-21.

METHODSReal-time PCR was used to measure miR-21 abundance in non-small cell lung cancer (NSCLC) tumor samples and adjacent normal tissues, as well as NSCLC cell lines. Tumor suppressor genes as potential targets of miR-21 were predicted by sequence analysis. Luciferase assay and Western blot were used to assess the regulatory effect. The effect on A549 cell viability and apoptosis by miR-21-induced gene repression was tested by trypan-blue exclusion and flow cytometry.

RESULTSmiR-21 expression was 2.24-fold higher in the NSCLC tumor samples and 3.06-fold higher in the A549 cells than that in the adjacent normal tissues. Sequence prediction and gene expression regulation assays showed that miR-21 could reversely regulate the expression of PDCD4 (P < 0.01). Suppression of miR-21 expression is associated with an elevation of Pdcd4, resulting in a significant reduction of proliferation and the apoptosis rate (2.6%) was increased to 10.9%. Moreover, the anti-proliferation and pro-apoptotic effect by miR-21 suppression could be reversed by PDCD4 knock down.

CONCLUSIONSuppression of the tumor suppressor PDCD4 expression may be one of the important regulatory pathways of the miR-21-mediated cell proliferation and decrease of apoptosis in non-small cell lung cancer.

Aged ; Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Oligonucleotides, Antisense ; genetics ; RNA Interference ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection

To evaluate the effects of lentivirus-delivered short hairpin RNA (shRNA) on CVB3 infection in an animal model by RNA interference technique, we constructed a recombinant lentivirus expressing shRNA-3753 against the viral genome region 3753-3771, then transduced Lenti-sh3753 into mice infected with CVB3. We evaluated the antiviral ability of lenti-sh3753 by cytopathic effect (CPE), viral plaque assay and histological analysis of mice hearts. The results showed that Lenti-sh3753 exhibited a significant protective effect on cell viability and reduction of viral titers in supernatant of cell culture by specific inhibition on viral replication. Lenti-sh3753 also prolonged the mice survival and limited the viral production in mice hearts. These data proposed that Lenti-sh3753 can effectively inhibit CVB3 infection in a coxsackievirus-induced myocarditis model, suggesting its potential role in prevention and therapy of viral diseases.
Animals ; Coxsackievirus Infections ; drug therapy ; virology ; Down-Regulation ; Enterovirus B, Human ; genetics ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; drug therapy ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; RNA, Viral ; genetics ; Virus Replication

OBJECTIVETo evaluate feasibility of inhibiting coxsackievirus B3 (CVB3) infection at cellular, protein and gene levels by using small interfering RNA (siRNA).

METHODSAntiviral activities of siRNAs were evaluated by observing cytopathic effect (CPE), using plaque reduction Western blotting assays and RT-PCR.

RESULTSEight siRNAs were synthesized, among them, SiRNA-2, SiRNA-3, SiRNA-6 and SiRNA-7 which were targeted against sequences located in 2B, VP4, 2A and 3C section of CVB3 genome, were designed to have different effect of inhibiting CVB3 infection in vitro. SiRNA-2 showed the best protective effect, 95 percent inhibition of CVB3 cytopathic effect and plaque forming effect was observed at 0.0001 MOI, viral protein synthesis and replication were inhibited. SiRNA-2 showed 30 percent inhibition of virus at 0.1 MOI, 70 percent inhibition at 0.01 MOI, 88 percent inhibition at 0.001 MOI, and 99 percent inhibition at 0.0001 MOI 48 hours after CVB3 infection.

CONCLUSIONSiRNA could effectively inhibit CVB3 infection in vitro, siRNA-2, which is targeted against sequence in 2B section of CVB3 genome, seemed to be the best one among those synthesized in this study.

Coxsackievirus Infections ; therapy ; virology ; Cytopathogenic Effect, Viral ; drug effects ; Enterovirus ; genetics ; physiology ; HeLa Cells ; Humans ; RNA Interference ; RNA, Small Interfering ; genetics ; therapeutic use ; Virus Replication ; drug effects

OBJECTIVETo investigate the effects of Herbal Compound 861 (Cpd 861) on collagen synthesis and degradation in rat mesangial cells exposed to high glucose.

METHODSThe third to fifth passage of rat mesangial cells were exposed to high glucose and Cpd 861 at a concentration of 0.25-4.00 g/L for 24, 48 and 72 h, respectively. Benazepril (10(-7)-10(-3) mmol/L) was selected as positive control. The methyl thiazolyl tetrazolium colorimetric assay was used to evaluate the effect of Cpd 861 on cell proliferation. After incubation with Cpd 861 at a concentration of 2.00 g/L for 48 h, the protein secretions of collagen type IV, matrix metallopeptidase 9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), transforming growth factor beta 1 (TGF-β1), and hepatocyte growth factor (HGF) were detected by enzyme-linked immunosorbent assay method. And rat mesangial cells were harvested to determine MMP-9, TIMP-1, TGF-β1 and HGF mRNA expression by reverse transcription polymerase chain reaction.

RESULTSCpd 861 inhibited cell proliferation induced by high glucose in a dose- and time-dependent manner. Compared with high glucose, collagen type IV production was decreased significantly by Cpd 861 (P<0.01). Cpd 861 increased the protein secretions and mRNA expressions of MMP-9 and HGF, whereas the protein secretions and mRNA expressions of TIMP-1 and TGF-β1 were reduced markedly (P<0.05). The ratio of MMP-9 to TIMP-1 was enhanced by Cpd 861 significantly. There was no significant difference in all above-mentioned effects between Cpd 861 (2.00 g/L) and benazepril (10(-5) mmol/L).

CONCLUSIONThe anti-glomerulosclerosis mechanisms of Cpd 861 were partly attributed to its effects of inhibiting mesangial cell proliferation, decreasing collagen synthesis and enhancing collagen degradation.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type IV ; biosynthesis ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Glucose ; toxicity ; Hepatocyte Growth Factor ; secretion ; Matrix Metalloproteinase 9 ; metabolism ; Mesangial Cells ; cytology ; drug effects ; enzymology ; metabolism ; Polymerase Chain Reaction ; Proteolysis ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Transforming Growth Factor beta1 ; secretion

To study the inhibitory effect and function characteristics of small interfering RNA (siRNA) on cosxackievirus B3(CVB3) infection by RNA interference technique, siRNA-2B against the viral 2B region was synthesized and transfected into HeLa cell, which was then infected with CVB3. The efficiency of siRNA transfection was examined by FCM, the cell toxicity of siRNA-2B by MTT, and the antiviral ability of siRNA-2B by cytopathic effect (CPE), plaque reduction assay and RT-PCR. The results showed that siRNA-2B could be transfected efficiently into HeLa cell and lasted at least 48h. High concentration of siRNA-2B didn't show any sign of toxicity to cells. siRNA-2B exhibited a significant protective effect on cell viability by specific inhibition of viral replication. It showed a close relationship between the concentrations of siRNA-2B and the antiviral effects. siRNA-2B led to dramatical reduction of viral titers in supernatant of cell culture and weakened the reinfection ability of the virus. These data proposed that siRNA-2B, targeting 2B protein, can effectively inhibit CVB3 infection in HeLa cell and exhibits its transfection efficiency, viral inhibition specificity and adose-dependant manner, suggesting its potential role in prevention and treatement of CVB3 infection.
Enterovirus ; genetics ; growth & development ; Green Fluorescent Proteins ; genetics ; metabolism ; HeLa Cells ; Humans ; Microscopy, Fluorescence ; Plasmids ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Viral Nonstructural Proteins ; genetics ; Virus Replication ; genetics ; physiology

OBJECTIVETo study the risk factors of bronchial asthma of Li nationality in Hainan.

METHODSA total of 13 050 subjects of Li nationality were selected by random unequal ratio stratified cluster sampling method from southern, central and western part of Hainan and investigated with Hainan Epidemiological Asthma Survey Questionnaire of Li Nationality. There were 441 cases of bronchial asthma, and 1296 cases of control that were sampled by random number table method. The logistic regression method was used to analyze risk factors.

RESULTSThe asthma prevalence of Li nationality in Hainan was 3.38%(441/13 050). The main risk factors of asthma were family asthma (OR = 4.323, 95%CI = 3.259 - 5.735), hypersensitiveness (OR = 7.775, 95%CI = 5.686 - 10.632), smoking (OR = 1.494, 95%CI = 1.174 - 1.902), cooking fuels and living environment. Cold air change (OR = 1.604, 95%CI = 1.286 - 2.001) and respirable dust or irritant gas (OR = 2.123, 95%CI = 1.702 - 2.648) were the important incentives.

CONCLUSIONThe main risk factors of asthma among Li nationality were family asthma, hypersensitiveness, smoking, cooking fuels by means of fuel oil, hay or wood, living environment by means of couch grass room and human-livestock mix live, cold air change, respirable dust or irritant gas.

Adult ; Asian Continental Ancestry Group ; Asthma ; epidemiology ; Child ; China ; epidemiology ; Ethnic Groups ; Female ; Humans ; Male ; Prevalence ; Risk Factors