Objective To explore protective effect of Heme oxygenase-1(HO-1) on irradiation-induced endothelial cell apoptosis.Methods Human endothelial cell line EA.hy926 were administered with or without HO-1 activator Copp and/or HO-1 inhibitor Znpp,respectively.Then,cells were treated with or without 8 Gy radiation.The HO-1 protein expression of cells were assessed with Western blotting and apoptosis of cells treated with irradiation were evaluated with flow cytometry.Moreover,cytochrome C releasing into cytosol were also determined by Western blotting. Results In PBS+R group,HO-1 protein expression of EA.hy926 was low posterior to irradiation.When cells were preconditioned with Copp and/or Znpp,then recieved with 8Gy irradiation,the HO-1 protein expression of EA.hy926 increased significantly in comparision with the PBS+R group(P

OBJECTIVETo investigate the regulatory effects of lanthanum chloride (LaCl3) on the activation of nuclear factor kappa B inhibitor (IκB) kinase beta (IKKβ) induced by tumor necrosis factor alpha (TNF-α).

METHODS(1) Hela cells were cultured routinely in vitro. One portion of cells were collected and divided into TNF-α group (cultured with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min), low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, LaCl3 group (cultured with serum-free RMPI 1640 medium containing 100 µmol/L LaCl3 for 30 min), and control group (cultured with serum-free RMPI 1640 medium for 30 min) according to the random number table. Cells in low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group were first cultured with serum-free RMPI 1640 medium containing 5, 25, 100 µmol/L LaCl3 for 4 h, and then stimulated with serum-free RMPI 1640 medium containing 20 ng/mL TNF-α for 30 min. There were 3 samples in each group. Cells were collected for detection of intracellular location of NF-κB/p65 protein by immunofluorescence staining. (2) Another portion of cells were collected and divided into TNF-α group, low-dose LaCl3 + TNF-α group, moderate-dose LaCl3 + TNF-α group, high-dose LaCl3 + TNF-α group, and control group with the same treatment as above. There were 3 samples in each group. The protein levels of NF-κB/p65 in nuclei, and the protein levels of IκBα, phosphorylated IκBα (p-IκBα) as well as IKKβ and phosphorylated IKKβ (p-IKKβ) in cytoplasm were determined by Western blotting. The binding activity between NF-κB/p65 in the nuclear and target gene was determined by NF-κB/p65 transcription factor kit (denoted as absorption value). Data were processed with analysis of variance or LSD-t test.

RESULTS(1) High expression of NF-κB/p65 was observed in cytoplasm of control group. High expression of NF-κB/p65 was observed in nuclei of TNF-α group. The expression of NF-κB/p65 in cytoplasm of LaCl3 group was lower than that of control group. In groups treated with LaCl3 and TNF-α, NF-κB/p65 expression levels in nuclei and cytoplasm were decreased along with the increase in the concentration of LaCl3, which were all lower than those in TNF-α group. (2) There was certain amount of NF-κB/p65 protein expressed in nuclei of control group. The expression of NF-κB/p65 protein in nuclei of TNF-α group was higher than that of control group. In groups treated with LaCl3 and TNF-α, the expressions of NF-κB/p65 protein in nuclei were decreased along with an increase in the concentration of LaCl3. The level of IκBα in TNF-α group was significantly decreased but that of p-IκBα increased as compared with those in control group. Along with the increase in the concentration of LaCl3, the levels of IκBα gradually increased and the levels of p-IκBα gradually decreased in groups treated with LaCl3 and TNF-α. There were no statistical differences in expression levels of IKKβ among the 5 groups. The expression of p-IKKβ could be hardly observed in control group, but it was obviously increased in TNF-α group. The expression levels of p-IKKβ in groups treated with LaCl3 and TNF-α were gradually decreased along with the increase in the concentration of LaCl3. The absorption value in TNF-α group was 0.39 ± 0.03, which was higher than that in control group (0, t = -7.23, P<0.01). The absorption values in low-dose LaCl3 +TNF-α group, moderate-dose LaCl3 + TNF-α group, and high-dose LaCl3 +TNF-α group were respectively 0.17 ± 0.03, 0.15 ± 0.03, and 0, which were obviously lower than that in TNF-α group (with t values respectively -6.54, -5.92, -7.23, P values all below 0.01).

CONCLUSIONSLaCl3 can block the activation of NF-κB signaling pathway by blocking the phosphorylation of IKKβ of Hela cells.

Culture Media ; HeLa Cells ; Humans ; I-kappa B Kinase ; metabolism ; I-kappa B Proteins ; metabolism ; Lanthanum ; pharmacology ; NF-KappaB Inhibitor alpha ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

AIMTo study the metabolites of penehyclidine hydrochloride (PH) raceme, a new anticholinerigic drug invented by the Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences.

METHODSThree healthy rat urine samples were collected within 24 h after a single i.m. dose of PH raceme and PH-d5 [(5 + 5) mg.kg-1] simultaneously. The eight metabolites of PH raceme were identified by the methods of LC-MS/MS, GC-MS, FAB-MS and the stable isotope ion cluster. Mass spectrometry was operated in the positive mode for the method of LC-MS/MS.

RESULTSM1 and M1* were identified as the oxygenated products of PH in the cyclopentyl group; M2 and M2* were as the hydroxylated products of PH in the cyclopentyl group; M3 and M3* were as the oxygented and hydroxylated products of PH at the meta-position of cyclopentyl group; M4 and M4* were identified as the dihydroxylated metabolites of PH, the hydroxylated position were at the cyclopentyl group and quiniuclidinol ring of PH. Among them, M1 and M1*, M2 and M2*, M3 and M3*, M4 and M4* were the isomers of each other.

CONCLUSIONThese characteristics can be used for future structure elucidation in studies of the metabolites of PH optical isomers. The structure data of PH metabolites provide important information for the clinical use and for developing better anticholinerigic drug.

Animals ; Cholinesterase Inhibitors ; chemistry ; metabolism ; urine ; Chromatography, High Pressure Liquid ; Male ; Molecular Structure ; Quinuclidines ; chemistry ; metabolism ; urine ; Rats ; Rats, Wistar ; Spectrometry, Mass, Electrospray Ionization ; Stereoisomerism

OBJECTIVETo observe the morphology and function changes of cochlear hair cells before and after math1 gene injection into the cochlea of deaf guinea pigs which were induced by kanamycin and furosemide. To explore the feasibility of Math1 gene for medicine-induced deafness therapy.

METHODSKanamycin (500 mg/kg) and furosemide (50 mg/kg) were given to the healthy adult guinea pigs intramuscularly and intravenously to establish the deafness model. The guinea pigs whose auditory brainstem response (ABR) threshold > 95 dB SPL were randomly divided into five groups. Blank control group (without any treatment, n = 3), operation control group (right ear scala tympani operation, n = 3), artificial perilymph group (right ear scala tympani injection artificial perilymph, n = 3), virus vector group [right ear scala tympani injection adenovirus which carrying enhanced green fluorescent protein (EGFP) gene (Ad. EGFP) , n = 4], Math1 gene therapy group [right ear scala tympani injection adenovirus which carrying Math1 and EGFP gene (Ad. Math1-EGFP), n = 6]. Each animal received ABR test before and after injection. The cochlear tissue was observed by scanning electronic microscopy.

RESULTSThe ABR thresholds of tone burst( 4, 8, 16, 20 kHz ) were not statistically significant in different groups (P > 0.05). The number of hair cells increased in some of severe deaf guinea pigs after the injection of Ad. Math1-EGFP gene. However, there was no obvious difference with morphology and numbers of cochlea hair cells in other groups.

CONCLUSIONSThe injection of Math1 gene to cochlea can regenerate or repair the hair cells of medicine-induced deaf guinea pigs, but there was no improvement on the hearing loss.

Adenoviridae ; Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Cochlea ; Deafness ; Ear, Inner ; Evoked Potentials, Auditory, Brain Stem ; Furosemide ; toxicity ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; Guinea Pigs ; Hair Cells, Auditory ; Hearing Loss ; chemically induced ; genetics ; Kanamycin ; toxicity ; Perilymph

OBJECTIVETo probe into the mechanism and methods of needle knife relaxing therapy for treatment of osteoarthritis of knee from biomechanical view.

METHODSNeedle knife relaxing therapy was given to 92 pain points around the knee joint in 14 cases of osteoarthritis of knee, and the displacement of the local pain point under the stress of 500 g (L500 g) was measured and the VAS scores were recorded before and after treatment.

RESULTSL500 g of the pain point was (4.72+/-1.03) mm before treatment and (5.39+/-1.01) mm after treatment with a very significant difference before and after treatment (P<0.01), and VAS score was (7.10+/-1.49) points before treatment and (1.49+/-1.24) points after treatment with a very significant difference before and after treatment (P<0.01), and there was a linear correlation between the changes of L500 g and VAS scores.

CONCLUSIONThere was close connection between the local pain and tension of local soft tissue in knee osteoarthritis. The needle knife relaxing therapy can relieve the neurovascular compression or traction syndrome by relaxing the local contracted, adhesive soft tissue, so as to relieve tension pain and finally recover internal force equilibrium of the knee joint.

Acupuncture Therapy ; methods ; Adult ; Aged ; Arthralgia ; therapy ; Female ; Humans ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Needles ; Osteoarthritis, Knee ; therapy ; Pain Measurement ; Pressure

Aim To investigate the role of TGF-β/Smads signaling pathway in the improvement of myo-cardial fibrosis in diabetes mellitus by curcumin. Methods A model of type 2 diabetes mellitus was in-duced by intraperitoneal injection of small dose of streptozotocin (STZ) 35 mg·kg-1with a high glucose and high fat diet, and then intervened by drinking of 300 mg·kg·d-1curcumin. The expression of myo-cardial collagen in rats was detected by Sirius red stai-ning. The expressions of Collange I and Collagen III in myocardium of rats were detected by immunofluores-cence. Cardiac fibroblasts(CFs) in neonatal rats were stimulated by different concentrations of glucose(5.5, 20,25, 30, 35, 50 mmol·L-1) for 24 h to deter-mine the optimum concentration of high glucose model, and rat CFs were stimulated for 24 h by 30 mmol·L-1 high glucose plus different concentrations of curcumin (10,25,50,100,200 μmol·L-1) to determine the optimal concentration of curcumin. The expressions of type Ⅰ and Ⅲ collagen,TGF-β1,p-Smad2,Smad2, p-Smad3,Smad3 and TβR-Ⅲin CFs were detected by Western blot. Results Compared with the control rats,the collagen deposition in the myocardium of the diabetic rats was more obvious and the expression of Collagen Ⅰ and Collagen Ⅲ significantly increased. After treatment of curcumin,the collagen deposition in the myocardium and the expression of Collagen I and CollagenⅢof diabetic rats remarkably decreased. The CFs under the condition of 30 mmol·L-1high glucose and 24 h had the highest survival rate (P <0.05);10μmol·L-1curcumin could obviously inhibit the proliferation of myocardial fibroblasts induced by high glucose (P<0.05). After induced by 30 mmol·L-1 high glucose for 24 h, the expression of Collagen Ⅰand Collagen Ⅲ, TGF-β1, p-Smad2, Smad2, p-Smad3,Smad3 and TβR-Ⅲ proteins in CFs markedly increased(P <0.05), and the expression levels of these proteins were obviously reduced when treated with 25 μmol·L-1curcumin. Conclusion Curcumin could ameliorate myocardial fibrosis in diabetic rats through TGF-β/Smads signaling pathway, exerting the protective effect on myocardium in diabetic rats.

Objective To investigate the influence of rotenone neurotoxin on dopaminergic neurons from SD rats of different months and its mechanism.Methods Forty 3-month-old,4012-month-old and 40 20-month-old SD rats were chosen in our study and equally randomized into control group and rotenone treatment group,respectively(n=20); rotenone(1.0 mg/[kg·d])was given to the rats in the rotenone treatment group for 30 consecutive d(having drug withdrawal 1 d per week)to induce rotenone neurotoxin models.Tyrosine hydroxylase immunoreactivity in the substantia nigra pars compacta(SNpc)was observed.Levels of total superoxide dismutase(SOD)and catalase(CAT)were determined by colorimetric method and protein level of caspase-3 was determined by Western blotting.Results A significant decreased number of TH-positive cells in 3-,12-and 20-month-old rats of rotenone treatment group was noted as compared with that in the control group,respectively(P＜0.05),and 20-month-old rats had the fewest number.SOD and CAT activities in 3 month-old rats of the control group enjoyed the highest level and decreased trend was noted in 20 month-old rats of the control group;as compared with those in the control group,the SOD and CAT activities in all the rats of rotenone treatment group increased significantly(P＜0.05).Western blotting revealed that the expression of cleaved caspase-3 in all the rats of rotenone treatment group obviously increased as compared with those in the control group,and 20-month-old rats showed the highest level of active caspase-3.Conclusion Rotenone can induce the decrease of TH-positive cell level following the increase of rat age; increase levels ofcaspase-3 and oxidative stress may take part in the mechanism of high sensibility of old SD rats to rotenone.

Objective To evaluate the clinical effects of multiple rewarming interventions in adult hypothermia trauma patients.Methods A systematic search of Cochrane Library,PubMed,EMBASE,Scopus,CINAHL,Chinese Biomedical Literature Database (CBM),Chinese Knowledge Infrastructure (CNKI),VIP and Wan Fang Database was carried out to identify all randomized controlled trials(RCTs) and controlled clinical trials(CCTs) that explored the effects of rewarming interventions in adult hypothermia trauma patients.The quality of the literature was evaluated using JBI 2008 RCT and quasi-experimental study evaluation criteria.Data and network plot were analyzed and drawn by ADDIS 1.16.7 software.Results Totally 6 RCTs and 1 quasi-experimental design were included,involving 10 interventions and 619 patients.There was statistically significant difference in body temperature after rewarming between the warm blankets and the forced-air blankets in all rewarming measures.The results of the top three interventions were carbon-fiber heating blanket(set to 42℃),forced-air blankets,warmed intravenous fluids plus blanket which resulted from the primary outcome indicators.The incidence of chills and cold discomfort decreased with the use of forced-air blankets and chemical heat pad as compared with traditional warm blankets,while the heart rate of the patients who used chemical heating pads and continuous heating of carbon fiber blanket were declined more than those used normal blankets.Conclusion The effects of carbon-fiber heating blanket which set to 42°C was the best method in all rewarming interventions.But this conclusion still requires randomized controlled trials with larger sample size to further verify.

OBJECTIVETo investigate the relationship of DNA methyltransferase 1 ( DNMT1 ) with hematopoietic cell phosphatase (SHP-1) gene expression and promoter 2 methylation status in cell line K562.

METHODSThe promoter sequence of SHP-1 gene promoter 2 in NCBI database was analyzed, the K562 cells were transfected with the lentiviral plasmids-the specified retroviral vector psiHIV-mU6-shDNMT1 and psiHIV-mU6-mcherryFP-control. The methylation status of SHP-1 gene promoter 2 in K562 cells was detected by methylation-specific polymerase chain reaction (MSP) and bisulfite-modified sequencing (BSP). Western blot was used to detect the protein expression level of SHP-1 and DNMT1, the SYBR Green fluorescence quantitative PCR was used to detect the expression of SHP-1 mRNA.

RESULTSIt was found that the promoter 2 of SHP-1 gene located between -577 bp to +300 bp, and 22 CpG sites contained between -353 bp-+182 bp were aberrantly hypermethylated and the SHP-1 could not be detected in K562 cells. In vitro, the detection demonstrated that the expression level of DNMT1 in K562 cells transfected with psiHIV-mU6-shDNMT1 was 0.48±0.06 significantly lower than that of psiHIV-mU6-control group (1.33±0.19)(t= 4.18, P<0.05). The expression of SHP-1 mRNA in K562 cells transfected with psiHIV-mU6-shDNMT1 was significantly higher than that in K562 cells transfected with psiHIV-mU6-shDNMT1 (14.23±3.83 vs 1.031±0.156)(P<0.01). DNMT1 silencing induced demethylation of the 22 CpG sites located in the SHP-1 promoter 2, and SHP-1 gene was re-expression in K562 cells.

CONCLUSIONThe DNMT1 in K562 cells relates with the hypermethylation and silencing of SHP-1 promoter in K562 cells.

CpG Islands ; DNA (Cytosine-5-)-Methyltransferases ; DNA Methylation ; Humans ; K562 Cells ; Promoter Regions, Genetic ; RNA, Messenger ; Real-Time Polymerase Chain Reaction