Objective To investigate the mechanisms by which nuclear factor-κB (NF-κB)translocates to nucleus of human glioblastoma U-87MG cells. Methods The levels of NF-κB translocating to nucleus in U-87MG after activation or inhibition ofphospholipase C-γ1 (PLCγ1) were first determined by electrophoretic mobility shift assay (EMSA), and then the levels of NF-κBtranslocating to nucleus in U-87MG after activation or inhibition of protein kinase-α (PKCα) were determined by EMSA. Results The level of NF- κB translocating to nucleus of U-87MG peaked 60min after EGF stimulation, which could be reversed by the pretreatment of PLCγ1 specific inhibitor U-73122. Moreover, PKC specific agonist phorbol myristate acetate (PMA) stimulation for 60 min promoted the nuclear translocation of NF-κB to the peak, which could be also effectively reversed by the pre-treatment of PKCα specific antagonist Ro 31-8220. Conclusions Epidermal growth factor can mediate the nuclear translocation of NF-κB in U-87MG through the signal transduction of PLCγ1-PKCα,thereby regulating the transcription of related invasion and metastasis genes.

OBJECTIVETo study the effects of pentobarbital sodium in generation and modulation of rhythmical respiration in neonatal rats.

METHODSThe effects of pentobarbital sodium were examined on hypoglossal nerve (XII) rootlets and inspiratory neurons in the medullary preparations including the medial region of the nucleus retrofacialis, pre-Bötzinger complex and the dorsal respiratory group of neonatal rats aged 0-3 days. The electrical activity of XII nerve rootlets and inspiratory neurons were recorded. Different doses of pentobarbital sodium (20, 40, 60, 80 micromol/L) were added into modified Krebs solution to observe changes in the discharge activity of XII nerve and inspiratory neurons. Bicuculline was used to further investigate the mechanisms that pentobarbital sodium suppresses respiration.

RESULTSThe discharge activity inhibition of XII nerve was increased as pentobarbital sodium doses increased from 20 to 60 micromol/L, but no significant difference was observed between the doses of 60 and 80 micromol/L. Bicuculline can partly restore the rhythmical respiration discharge activity.

CONCLUSIONPentobarbital sodium can suppress respiration partly via GABAA receptors.

Adjuvants, Anesthesia ; pharmacology ; Animals ; Animals, Newborn ; Dose-Response Relationship, Drug ; Medulla Oblongata ; cytology ; drug effects ; physiology ; Neurons ; drug effects ; physiology ; Pentobarbital ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, GABA-A ; physiology ; Respiration ; drug effects ; Respiratory Center ; drug effects ; physiology

Objective To investigate the adverse effect of different doses of high power microwave(HPM) irradiation on oxidative stress in the brain of Wistar rats in order to contribute to establishing an animal model to evaluate protective agents which will be used for protection against microwave radiation.Methods Eighty male Wistar rats were randomly divided into 16 groups according to factor analysis.The average power density was 0,10,30 and 100 mW/cm2 and the sampling time was 6 h,1,3 and 7 d .The duration of exposure was 6 minutes for each radiation group.After exposure, the rats were sacrificed at each sampling time.Colorimetric method was used to measure the content of malondialdehyde(MDA) and protein carbonyl, the activity of GSH-px, SOD and CAT.Results The content of MDA and protein carbonyl of each radiation group was increased with the radiation dose, but decreased with the sampling time prolonged.The activity of superoxide dismutast(SOD),glutathion peroxidase(GSH-px) and catalase(CAT) in each radiation group was decreased with the radiation dose increased, and with the sampling time prolonged, but increased later.Conclusion Microwave radiation can cause oxidative stress in rats brain, as shown by the oxidative damage of lipid and protein and the decrease in the activity of antioxidant enzymes.Besides, the effect also depends on the radiation dose and sampling time.

OBJECTIVETo explore the role of protein kinase D3 (PKD3) in the regulation of matrix metalloproteinases 7 (MMP-7) expression in prostate cancer cells.

METHODSPC-3 cells were either stimulated with 100 nmol/L PMA to activate PKD3 kinase activity, or transiently transfected with PKD3 siRNA, and the relative expression level of MMP-7 mRNA were analyzed by real-time PCR using 2(-delta delta Ct) method. MMP-7 mRNA levels were also analyzed and quantified in HEK293 cells with over-expression of wild-type PKD3, PKD3 knockdown (using PKD3 siRNA), or over-expression of wild-type PKD3 followed by PKD3 knockdown.

RESULTSMMP-7 mRNA expression in PC3 cells was significantly decreased after PMA-induced PKD3 kinase activation. In contrast, PKD3 knockdown by siRNA transfection markedly increased MMP-7 mRNA level (P<0.01). MMP-7 mRNA level in HEK293 cells was significantly decreased by PKD3 over-expression, whereas obviously increased by PKD3 knockdown. Down-regulation of MMP-7 mRNA level in HEK293 induced by PKD3 over-expression was rescued by PKD3 knockdown.

CONCLUSIONPKD3 may contribute to the malignant progression of prostate cancer cells through negative regulation of MMP-7 expression.

Cell Line, Tumor ; Down-Regulation ; Gene Knockdown Techniques ; Humans ; Male ; Matrix Metalloproteinase 7 ; metabolism ; Prostatic Neoplasms ; enzymology ; metabolism ; Protein Kinase C ; metabolism ; Signal Transduction

Objective:To investigate the clinical efficacy of oblique lateral interbody fusion (OLIF) combined with lateral plate (LP) fixation and posterior lumbar interbody fusion (PLIF) combined with pedicle screw fixation in the treatment of adjacent segment disease (ASDis).Methods:Data of 21 ASDis patients treated with OLIF-LP from August 2016 to October 2019 were selected, including 9 males and 12 females; age was 59.3±7.0 years (range, 46-71 years). Target segments: L 2, 3 1 cases (4.8%), L 3, 4 16 cases (76.2%), L 4, 5 4 cases (19.1%). Twenty-one ASDis patients matched with age, sex and surgical segment and treated with PLIF were selected as the control group. The operation time, intraoperative bleeding, postoperative hospital stay, visual analogua scale (VAS), Oswestry disability index (ODI), disc height (DH), intervertebral foramen height (IFH) and lumbar lordosis (LL) were compared between the groups were tested by t-test. VAS score, ODI, DH, IFH and LL were compared within the group by ANOVA, and Bonferroni's test was used for pairwise comparison. Results:All of 42 patients were followed up for 23.7 ±7.4 months (range, 12-36 months). The operation time (97.6 ± 18.0 min) and interpretative bleeding (38.5±62.7 ml) in OLIF-LP group were significantly lower than those in PLIF group (operation time 154.6±42.4 min) and interpretative bleeding (288.6±55.3 ml). There were significant differences between two groups ( t=5.66, P<0.001; t=8.23, P<0.001); the postoperative hospital stay 4.4±1.3 d in OLIF-LP group was longer than that in PLIF group 5.1±1.2 d, but there was no significant difference ( t=1.93, P=0.061); VAS score in OLIF-LP group at 1 month and 3 months after operation (1.6 ± 0.9 points, 1.4 ± 0.8 points), and the ODI index (29.4%±4.7%) after one month operation was improved better than that of PLIF group ( t=2.48, P=0.017; t=2.35, P=0.024; t=2.28, P=0.029), but there was no significant difference between the 12 months after operation of two groups ( t=0.99, P=0.329; t=0.86, P=0.395). The immediately after operation, 3 months after operation and 12 months after operation of DH, IFH and LL in the two groups were significantly improved compared with those before operation ( P<0.05). The immediately after operation, 3 months after operation and 12 months after operation of DH and IFH in the OLIF-LP group were better than those in the PLIF group ( P<0.05), while LL had no significant difference ( P>0.05). There were 2 cases (9.52%) in each group with cage sinking, but no clinical symptoms occurred. In the OLIF-LP group, there was no injury of blood vessels, nerves or abdominal organs during operation, and 2 patients had transient lower limb pain after operation; In the PLIF group, 2 cases (9.52%) of dural rupture were repaired during operation, and no cerebrospinal fluid leakage occurred after operation; Postoperative lower limb pain was aggravated in 3 cases, and improved after dehydration, anti-inflammatory and analgesic treatment; 2 cases of incision exudation healed after symptomatic treatment. Conclusion:OLIF combined with LP fixation has the same clinical effect as PLIF in the treatment of lumbar ASDis, but OLIF combined with LP fixation has more advantages in surgical trauma, postoperative recovery and related complications.

OBJECTIVETo To establish a method of phosphoprotein affinity profiling for identifying phosphoproteomic differences between Thp-1 cells with or without lipopolysaccharide (LPS) stimulation, aiming to screen potential regulators involved in LPS pathway.

METHODSThp-1 cells were stimulated with 100 ng/ml PMA for 48 h to induce differentiation into mature macrophages, which, after culture for another 48 h in the absence of PMA, were either treated with 100 ng/ml LPS for 30 min or left untreated. After desalting procedure with ultrafiltration, the phosphoproteins enriched by phosphoprotein metal affinity column (PMAC) of both groups were run on 2-D electrophoresis to find the spots with different phosphorylation status. Finally, some of these spots were identified by mass spectrometry (MS) and subsequent bioinformatic analysis.

RESULTSCompared to untreated Thp-1 cells, LPS stimulated Thp-1 cells showed 29 spots with reproducible alterations on the 2-D map, including 8 representing up-regulated spots, 7 new spots, 10 down-regulated spots, and 4 absent spots. The newly emerged and absent protein spots were subjected to MS analysis, and 4 of them were identified to be involved in various cellular processes such as proteolysis, signal transduction and protein folding. Among these, phosphorylation of proteasome C2 subunit was dramatically up-regulated in LPS-stimulated cells, as was consistent with previous reports; the phosphorylation of Z-DNA-binding protein 1 has not been reported so far and needs further confirmation.

CONCLUSIONPhosphoprotein affinity profiling is an attractive method for screening novel regulators involved in LPS signaling pathways and can be widely used in systemic study of signal transduction.

Cell Line ; Electrophoresis, Gel, Two-Dimensional ; Humans ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; metabolism ; Mass Spectrometry ; Phosphoproteins ; analysis ; metabolism ; Phosphorylation ; Proteomics ; methods ; Signal Transduction

The aim of the present study is to investigate the differences in cardiac function, and the expression and activity of calcium regulatory proteins between rabbit systolic heart failure (SHF) and diastolic heart failure (DHF) models. New Zealand white rabbits were randomly divided into three groups: sham operation (SO) group, DHF group (receiving abdominal aortic constriction) and SHF group (receiving aortic valve destruction and abdominal aortic constriction). The cardiac function was detected by echocardiographic and hemodynamic assays. The mRNA expression levels of sarcoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a) and phospholamban (PLB) were evaluated by RT-PCR. The protein expression levels of SERCA2a, PLB, phosphoserine 16-PLB (pSer-16-PLB) and protein kinase A (PKA) were evaluated by Western blot, and the phosphorylation status of PLB was determined by the ratio of pSer-16-PLB protein level to that of PLB. The activity of SERCA2a was measured through inorganic phosphate. The activity of PKA was measured by gamma-(32)P ATP-binding assays. Compared with SO group, there were significantly increased ventricular wall thickness, raised left ventricular end diastolic pressure (LVEDP), reduced diastolic function in DHF group (P<0.05 or P<0.01), and significantly increased ventricular cavity size and LVEDP, reduced systolic function in SHF group (P<0.05 or P<0.01). The expression levels of SERCA2a in DHF and SHF groups were lower than that in SO group (P<0.05), while the expression and activity of PKA in DHF and SHF groups were higher than that in SO group (P<0.05 or P<0.01), and there was no significant difference between DHF and SHF groups. The expression levels of PLB and pSer-16-PLB as well as the phosphorylation status of PLB and activity of SERCA2a in SHF group were lower than those in DHF and SO groups respectively. Posing a contrast, the phosphorylation status of PLB and activity of SERCA2a in DHF group were higher than that in SO group (P<0.05). These results indicate that the SHF and DHF models were successfully established, and there are some differences in the expression and activity of calcium regulatory proteins between two models.
Animals ; Calcium-Binding Proteins ; metabolism ; Disease Models, Animal ; Heart Failure, Diastolic ; metabolism ; Heart Failure, Systolic ; metabolism ; Rabbits ; Sarcoplasmic Reticulum ; metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; metabolism

OBJECTIVETo evaluate the efficacy and optimal re-implantation time of two-stage revision for management of periprosthetic infection following hip arthroplasty.

METHODSWe retrospectively analyzed the clinical data of 15 patients (15 hip joints) undergoing two-stage ipsilateral total hip arthroplasty (THA) revision from January, 2006 to January, 2010. In the first stage, after surgical debridement and thorough removal of all the implants, a self-made Vancomycin-loaded cement spacer was implanted. The second stage operation was performed 3-6 months later for debridement and removal of the antibiotic-loaded spacer, followed by re-implantation of Vancomycin-loaded bone cement prosthesis in 9 cases and cementless prosthesis in 6 cases. The patients were followed up for 9-46 months (mean 25 months) after the operation.

RESULTSNo reinfection or prosthesis loosening/displacement was found in these cases after the operation. The Harris score increased from 40.3 before the operation to 54.0 after the first-stage operation, and to 88.2 at the last follow-up.

CONCLUSIONTwo-stage revision is effective for treatment of periprosthetic infection following hip arthroplasty, and 3-6 months can be the optimal interval between the two the first-stage and second-stage operation for re-implantation.

Adult ; Aged ; Arthroplasty, Replacement, Hip ; methods ; Female ; Hip Prosthesis ; Humans ; Male ; Middle Aged ; Prosthesis-Related Infections ; surgery ; Reoperation ; Retrospective Studies ; Treatment Outcome

Objective: To determine protein binding characteri stic and signal transmission pathway of melatonin（Mel） receptor(MR) in human e mbryonic peripheral organ tissues. Methods: MR was measured by radio ligand-binding assay and the effect of GTPγS on melatonin specific bindi ng was studied. Results: Mel specific binding sites were det ermined in 16 kinds of human embryonic tissue and this binding could be inhibit ed by GTPγS, supporting the theory that MR is coupled to inhibitory G-proteins system. Conclusion: MR is measured in human embryo tissue, the se results provide experimental data for elucidating the mechanism of the effect of Mel.