Objective To discuss the role of p38MAPK signal pathway in the process of TWEAK inducing rheumatoid arthritis(RA) fibroblast-like synoviocyte(FLS) to synthesize MMP-9 and look for a new target for RA treatment. Methods RA FLS were primarily cultured and stimulated with TWEAK. Western blot was used to detect the expression of p-p38MAPK and p65 in RA FLS. FLS were pretreated by SB203580 or not. ELISA was used to detect the concentration of MMP-9 in cell-cultured fluid. The mRNA expression of MMP-9 was measured by RT-PCR. Results TWEAK( 100 ng/ml) can make p38MAPK phosphorylated and increased the expression of p65 protein in the cell nucleus. SB203580 can partially inhibit the expression of MMP-9 and MMP-9 mRNA produced by RA FLS which is induced by TWEAK. Conclusion TWEAK induced RA FLS to synthesize MMP-9, in that process, the p38MAPK signal trausduction pathway was in active state, and induced the expression of NF-κB.

OBJECTIVE:To provide reference for clinical rational use of anti-epileptic drugs. METHODS:In this retrospective review,serum concentrations of anti-epileptic drugs in a total of 499 patients who were treated with anti-epileptic drugs (such as sodium phenytoin,phenobarbital,carbamazepine and valproate sodium) in our hospital during 2007 were analyzed statistically. RESULTS:Among the patients receiving one kind of anti-epileptic drugs,206 cases (61.49%) were within normal range in blood concentration versus only 45 cases (44.12%) for patients receiving combined drugs. In addition,the above four drugs (sodium phenytoin,phenobarbital,carbamazepine and valproate sodium) were detected in 59.68% of the patients who took Chinese medicines,and among them,3.23% were within normal range in blood concentration. CONCLUSION:Monitoring on serum concentrations of anti-epileptic drugs is conducive to a better control of therapeutic concentration. It is advisable to refrain from using anti-epileptic drugs in combination but to adopt individualized administration. In addition,great importance should be attached to whether there are chemo-synthetic drug components contained in Chinese medicine.

Objective To investigate the effects of Annexin-A1 ( Anxa1 ) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control ( siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9 ±2.1)%, (23.1±3.6)%and (42.4±1.7)%vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)%and control group (4.3±0.2)%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01).Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways.

Humans ; Medicine, Chinese Traditional ; methods

AIM: To investigate a new method for identification of traditional Chinese medicine injection by FTIR. METHODS: For the steadiness of spectra, the factors of effecting spectrum's information quality were all investigated scientifically over the experiment procedures and instrumental setting, such as the preparation of samples, resolution ratio, scanning times, repeating scanning times, etc. The traditional Chinese medicine injections were used as the analytical samples such as Radix Isatidis, Rhizoma Chuanxiong, Flos Carthami, Radix Astragali and Herba Houttuyniae. RESULTS: Although all these original spectrums were similar at a certain degree, the FTIR combined with computer aided analysis, such as the cluster analysis and derivative spectrometry comparability calculation could be used to identify these injections. CONCLUSION: The method of identification by FTIR is non destructive testing, cheap, clean, fast, simple and convenient. The result indicates this method is suitable for establishing identification database of traditional Chinese medicine injections.

Objective To study whether p38 mitogen-activated protein kinases (p38MAPK) signal pathway were activated in the process of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) which then induces rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) to synthesize matrix metalloproteinase-1 (MMP-1) and look for the relative mechanisms of how TWEAK was involved in the destruction of articular bones and cartilage. Methods RA FLS were primarily cultured and stimulated with TWEAK. FLS were pretreated with SB203580 or not. ELISA was used to detect the concentration of MMP-1 in cell-cultured supernatant. Western blotting was used to detect the expression of p-p38MAPK and P65 in RA FLS. Results TWEAK (100 ng/ml) could induce RA FIS to synthesize MMP-1. SB203580 could partially inhibite the expression of MMP-1 producted by RA FLS which was induced by TWEAK. TWEAK could make p38MAPK phosphorylated and increase the expression of P65 protein in the cell nucleus. Conclusion TWEAK induces RA FLS to synthesize MMP-1. In this process, p38MAPK signal transduction pathway is activated and then induce the expression of NF-κB.

AIM:To evaluate the availability and our experience of intraoperative image-guidance in endoscopic nasocular operation.METHODS:Seven cases of endoscopic nasal surgery with intraoperative image-guidance were retropectively reviewed,including 3 cases of optic nerve injury;3 cases of foreign object of optic behind the eyeball;1 case of retrobulbar tumor(angeioma).RESULTS:The preoperative preparatory time would take 15 minutes,including coordination,head holder localization,conventional instrument registration.In our cases,the localization accuracy between 3-D image landmarks of navigation system and actual anatomical landmarks was less than 1.3mm.The optic nerve and other anatomical points could be orientated accurately in intraoperative procedures.No complication occurred.CONCLUSION:Nasal endoscope combined with imageguidance systems provides accurate anatomical localization of anterior skull base with enlarged operation field.It is possible for surgeons to observe important anatomical structures during endoscopic surgery.It could increase the effectiveness and decrease surgical complications,especially in complicated cases.

Background Diabetic retinopathy (DR) is one of the most important microvascular complications of diabetes,which has become one of the leading causes of blindness.Neovascularization is the main pathological manifestations of DR,but its mechanism is unknown.There is a clear need to investigate its pathogenesis which can offer potential therapeutic targets.Objective The aim of this study was to investigate the expression and distribution of visfatin and vascular endothelial growth factor (VEGF) in diabetic model rats.Methods This study was approved by Animal Ethic Committee of Inner Mongolia Medical University.Sixty SPF 8-week-old male SpragueDawley rats were randomized into the diabetic group and control group.The rats were housed under a condition that alternated between 12 hours of light and darkness,with free access to rat food and water.Diabetes was induced by intraperitoneal injection of 60 mg/kg (0.60 ml/100 g) of streptozotocin (STZ) and control rats received equivalent volume of buffer.The models were regarded as successful when blood glucose was ≥ 16.7 mmol/L.Rats were sacrificed 12 weeks after the injection of STZ and retinal specimens were prepared to detect the expression of visfatin and VEGF.Total retinal protein was isolated from the retinas of experimental and control eyes,and the expression of visfatin and VEGF was assessed by Western blot.Frozen cross sections of retinas of 5 μm thickness were used to perform double immunofluorescence staining with anti-visfatin and anti-VEGF antibodies.Results Mean body weight of the diabetic rats was (189.02±11.34) g and that of the control rats was (489.57 ± 14.48) g at 12 weeks post-injection,showing a significant difference between them (t =5.236,P =0.003).Mean blood glucose level was (29.25±3.86) mmol/L in the diabetic group and (5.32±1.01) mmol/L in the control group,demonstrating a significant difference (t =11.778,P =0.000).Double immunofluorescence staining showed reduced expression of visfatin and VEGF in the retinal nerve fibrous layer and glial cells in the control rats.A stronger staining for visfatin and VEGF was found in the various layers of the retina in the diabetic rats,with an expression level of visfatin (A value) of 346.26±41.23,which was considerably higher than that of the control group (102.07±65.01) (t =8.291,P =0.000) in 12 weeks after injection.Furthermore,the expression of VEGF in the retina was elevated in the diabetic group compared with the control group (A value) (415.88±92.15 vs.113.06±32.06) (t=10.067,P=0.000).Conclusions Visfatin might contribute to the pathologic progression of diabetic retinal,neovascularization and it might play a synergistic role with VEGF in the pathophysiology of DR.

Background Corneal neovascularization (CNV) is one of the causes of corneal blindness.Studies showed that ADP-ribosylation factor (ARF) can regulate the growth of tumor cells,and inhibiting ARF will decrease angiogenesis.However,whether ARF antagonist plays an action on CNV is unclear.Objective The aim of this study was to explore the effect and mechanism of ARF inhibitor on alkali-burn induced CNV.Methods Sixty clean male BABL/c mice aged 7-8 weeks were divided into PBS group and ARF antagonist group according to randomized number table.CNV models were induced by NaOH burn method in all the mice.ARF at the concentration of 0.5 g/L(0.5 ml) was intraperitoneally injected 3 times per week for 1 week followed the induction of CNV in the ARF antagonist group,and 0.5 ml PBS was used in the PBS group.CNV was examined 2,4,7,14 days after injection by the slit lamp microscope and the CNV related area in the cornea was calculated.Betore modeling(0 day) and 4,7,14 days after modeling,real-time PCR and Western blot were used to analyze the expressions of ARF mRNA and protein in the corneas.Forteen days after modeling,the expression of the CD31 in the CNV was detected using immnofluorescence of corneal whole mount;the expression of vascular endothelial growth factor(VEGF) in the cornea was assayed by Western blot.Cellular wound scratch test was employed to evaluate the effects of ARF antagonist on proliferation and migration of human retinal vascular endothelial cells (RECs).All animal experiments were done in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and Guideline for the Care and Use of Laboratory Animals on the the Soochow University Animal Care Committee.Results ARF mRNA and protein were expressed in the mice corneas in both the PBS group and the ARF antagonist group at various time points.The expression of ARF mRNA in mice corneas was enhanced with the lapse of the time (Ftime =65.17,P =0.00),but no significant difference was found among the groups (Fsroup =1.98,P=0.18).There was also significant difference in the expression of ARF protein in mice corneas at different time points in the ARF antagonist group (F =10.77,P =0.00).The related CNV area was 0.45±0.05 in the ARF antagonist group,and that in the PBS group was 0.72±0.11,with significant difference between them (t =-3.87,P ＜ 0.05).The green fluorescence area of C D31 expression in the cornea was smaller in the ARF antagonist group than that of the PBS group.Expression level of VEGF in the ARF antagonist group was 1.20±0.21,and that in the PBS group was 2.47±0.33,showing a significant difference (t =-5.62,P ＜ 0.05).As the increase of ARF antagonist concentration,the inhibiting rate of cell proliferation was reinforced among 10,100 and 1 000 μg/L ARF antagonist groups (F=8.47,P =0.02).Twenty-four hours after scratch test,the migrating distance of human RECs was (5.46±1.32) μm and (5.04±1.68) μm in the 100 μg/L and 1 000 μg/L ARF antagonist groups,respectively,which were shorter than (8.49± 1.18) μm of the PBS group (t=-2.94,-2.91,both at P＜0.05).Conclusions ARF inhibitor can reduce CNV by down-regulating the expression of VEGF in alkaline burn cornea and inhibiting the proliferation and migration of vascular endothelial cells.

Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization.Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent.Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro.Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling.Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D.Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31.The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry.Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells.The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee.Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas.The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage.The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm,in contrast,the expression of CD31 was absent in corneal stromal cells.Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1 ,CCR2,CCR3 and CCR4 mRNA and the weakest expression levels in CCR9,CXCR4 and CXCR5 mRNA,while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity.Conclusions Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors,which facilitate the study of their biological properties.