BACKGROUND:After cerebral ischemia/reperfusion injury,as a executioner caspase, procaspase-3 and caspase-3-like activity increased significantly. We observe both the dephosphorylated and phosphorylated procaspase-3, and try to find out their variations during the processes of cerebral ischemia/reperfusion injury.OBJECTIVE:Observe the expression of procaspase-3 in hippocampus following transient forebrain ischemia.DESIGN: A randomized and controlled experiment.DEPARTMENT:Department of Biochemistry and Molecular Biology,Capital University of Medical Sciences.SETTING:Department of Hyperbaric Oxygen of Beijing Chaoyang Hospital Affiliated to Capital University of Medical Sciences.METHODS:Transient forebrain ischemia was induced by bilateral common carotid occlusion (BCCAO) for 20 minutes. Hippocampus was obtained at reperfusion time points of 6 hours, 12 hours,24 hours and 48 hours respec-tively after 20 minutes of BCCAO. Sham-operated group did not occlude bi-lateral common carotid, and hippocampus was obtained at reperfusion time point of 24 hours. Western Blotting was used to detect the level of procaspase-3.MAIN OUTCOME MEASURES:The comparison of total procaspase-3,dephosphorylated procaspase-3 and phosphorylated procaspase-3 level in hippocampus between each group.3 level: Total procaspase-3 level increased in hippocampus at reperfusion time points of 12 hours and 24 hours post-BCCAO (9 133.1 ±2 216.3,dephosphorylated procaspase-3 level increased in hippocampus at reperfusion time point of 24 hours post-BCCAO (7812.0±1625.1, 3825.8±155.6, P was not significant (P ＞ 0.05) as compared with the expression levels in sham-operated mice.CONCLUSION: Procaspase-3 is upregulated after ischemia/reperfusion.The increment of dephosphorylated form of procaspase-3 was higher than that of phosphorylated form of procaspase-3 upon cerebral ischemia/reperfusion injury, which indicates that cerebral ischemia/reperfusion injury possibly induced the dephosphorylation of procaspase-3 and promoted its transforming into activated form.

BACKGROUND: The level of blood neuron-specific enolase may help predict the severity of brain damage.OBJECTIVE: To define the optimal time window of hyperbaric oxygen(HBO) treatment for brain ischemia based on the dynamical changes in plasma neuron-specific enolase bioactivity.DESIGN: Factorial design.SETTING: Department of Biochemistry and Molecular Biology of Capital University of Medical Sciences.PARTICIPANTS: The experiment was conducted in the Laboratory of the Department of Hyperbaric Oxygen, Beijing Chaoyang Hospital Affiliated to Capital University of Medical Sciences in June 2002. Totally 54 adult female SD rats were randomized into 3 groups, namely sham operation group(n=6), ischemia-reperfusion (IR) group (n=24), and HBO group (n=24), the latter 2 groups further divided into 4 groups according to the reperfusion time of 6, 24, 48 and 96 hours, with 6 rats in each subgroup.METHODS: [1] Rat models of IR was prepared by occlusion of the 4 arteries for 20 minutes followed by reperfusion for different time. [2] The rats in the sham operation received the same operation without blocking the arteries. The rats in HBO group were subjected to HBO treatment (0.2 MPa,pure oxygen for 45 minutes), which was given after a 3-hour reperfusion inthe 6-hour subgroup and scheduled once daily at the same time point in the other 3 subgroups until blood sampling. The rats in IR group and sham operation group were kept under normal pressure without additional oxygen.MAIN OUTCOME MEASURES: Blood samples were collected at the specified time points in IR and HBO groups and at 24 hours of reperfusion in the sham operation group. Enzyme-linked immunosorbent assay (ELISA)was used to determine the activity of plasma neuron-specific enolase.RESULTS: Totally 54 rats enter result analysis after supplementary.Plasma neuron-specific enolase level was significantly lower in the sham operation group (1.97±0.09) μg/L than in 6 and 96-hour subgroups in the IR group [(2.80±0.26), (2.40±0.19) μg/L, respectively, P ＜ 0.05],and was obviously lower in 6-hour HBO subgroup than the 6-hour IR group [(2.04±0.27) μg/L, P ＜ 0.05], which was slightly increased at 24hours after HBO treatment but the difference was of no statistical significance (P ＞ 0.05).CONCLUSION: IR injury may lead to increment of plasma neuron-specific enolase level, which occurred at 6 and 96 hours respectively in IR group, possibly due to acute neuronal necrosis during brain ischemia and subsequent delayed neuronal apoptosis. HBO treatment promotes the recovery of neuron-specific enolase level, with 6 hours of reperfusion as the optimal therapeutic time window.

Objective To investigate the protective effect and mechanism of taurine on the cytotoxicity of iohexol on HK-2 cells. Methods HK-2 cells were exposed to iohexol at different dosage (25, 50, 100, 125 gI/L) for 6 h and at the dose of 100 gl/L for different time(2 h, 4 h, 6 h). Then taurine (3,12,24 mmol/L) was coincubated with iohexol (100 gI/L) for 6 h.Cell viability was assessed by CCK-8 assay. Cell apoptosis was determined by Hoechest 33342 flurescence stains,flow cytometry with Annexin V-FITC/PI double stains and caspase-3 activity by colorimetric assay. Bcl-2 and Bax expression were examined by Western blot. Intracellular ROS was detected by flow cytometry with fluorescent probe DCFH-DA. Results Iohexol decreased HK-2 cell viability and induced apoptosis in concentration-dependant and time-dependant manner (all P＜0.05). ROS was increased following iohexol (100 gI/L for 6 h) treatment (P＜0.05). Taurine increased cell viability and attenuated apoptosis in dose-dependant manner. The cell viability levels in taurine intervention (3,12,24 mmol/L) group were significantly increased compared with that in iohexol treated group respectively [(88.00±1.00)%, (91.33±0.58)%, (95.67±1.52) % vs (76.67±1.53)%, all P＜0.05]. Apoptosis rate by flow cytometry were decreased respectively [(8.84±1.75)%,(7.86±1.82)%, (6.30±1.50)% vs (11.98±0.39)%, all P＜0.05]. Caspase-3 activities were decreased respectively [(1.33±0.10), (1.27±0.06), (1.10±0.04) vs (1.42±0.13), all P＜0.05].Taurine up-regulated the expression of Bcl-2, and decreased the intracellular ROS (all P＜0.05).Conclusions Iohexol induces cell apoptosis and oxidative stress. Taurine attenuates direct cytotoxic effect induced by iohexol. The anti-oxidative stress effect and up-regulated Bcl-2 expression may partly account for the protection of taurine.

Objective To investigate the effect of erythropoietin on the proliferation,differentiation,and apoptosis of the cultured neonatal porcine islet cells in vitro.Methods Neonatal porcine islet cells were separated and pured from neonatal pigs with collagenase digestion and tissue culture,and their viability and purity were tested. The neonatal porcine islet cells were divided into a control group and an experimental group.The experimental group was treated with erythropoietin but not the control group,and the insulin secretion responsiveness induced by low and high glucose stimulation in the islet was tested after 5 days. Cells were counted and the activation of amplification was determined by MTT chromatometry. The rates of cell apoptosis were observed by ethidium bromide/acridine orange (EB/AO) of fluorescent light staining and flow cytometry,and the cell cycle was analyzed by flow cytometry. The expression of bcl-2,bax,caspase-3,glucose transporter 2 (GlUT-2),and pancreatic duodenal homeobox-1 (PDX-1) mRNA was tested by RT-PCR.Results After erythropoietin was treated in the cell culture,the neonatal porcine islet cells had normal morphology,function,and reaction of insulin secretion to the glucose stimulation. Cell count showed more cells in the experimental group than in the control group (P＜0.05). MTT chromatometry showed the optical absorbance tended to increase with time,and compared with the control group,the optical absorbance was higher in the experimental group (P＜0.05),the expression of PDX-1 mRNA was slightly up-regulated (P＜0.05). The expression of GLUT-2 mRNA had no difference in the 2 groups (P=0.34). In the experimental group,the apoptisis rate was lower than that in the control group by flow cytometry and EB/AO fluoscence staining (P＜0.01),and the expression of bcl-2 mRNA was higher. Howerer bax mRNA and caspase-3 mRNA were obviously lower than those in the control group (P＜0.01).Conclusion Erythropoietin can promote the proliferation but has no effect on the function of neonatal porcine islet cells in vitro. Erythropoietin can protect neonatal porcine islet cells from apoptosis through up-regulating bcl-2 mRNA and downreguling bax and caspase-3 mRNA.

Objective To investigate the clinical characteristics and laboratory examinations in incident dialysis effect on the prognosis of elderly patients undergoing hemodialysis.Methods Ninety-three patients aged 65 years or older initiating hemodialysis were enrolled from Hemadialysis Center of Beijing Hospital from January lst,2007 to June 30th,2016.The duration time of HD of all patients was more than three months.Patients were divided into death group and non-death group.The clinical characteristics and laboratory examinations were compared between the two groups.Cox proportional hazards regression was used for the multivariate analysis to determine independent prognosis factors.Results The average year of patients was 74.2±6.5 years old with 43 months of median time of follow-up.The first two causes of death were infection (n =25,49.0％) and cardiovascular and cerebrovascular diseases (n =16,31.4％).Cox single factor regression analysis showed that the older ages,diabetic nephropathy being the cause of end-stage renal disease (ESRD),complicating with diabetes mellitus or congestive heart failure,the higher Charlson cardiovascular diseases score,ALB being under 35 g/L were correlated with poor outcome respectively(P＜0.05).Cox multivariate regression analysis indicated that older ages (HR =1.056,P =0.021),diabetic nephropathy being the cause of ESRD (HR =2.661,P =0.001),the higher Charlson cardiovascular diseases score (HR =1.675,P =0.010),central venous catheters being vascular access(HR=1.167,P=0.048) on incident dialysis were the main risk factors for mortality in elderly patients.Conclusion The older ages,diabetic nephropathy being the cause of ESRD,the higher Charlson cardiovascular diseases score,central venous catheters being vascular access on incident dialysis are independent risk factors influencing survival of elderly patients.

Background Activation of serum complement system is involved in the pathological process of uveitis and open angle glaucoma.Pathogenesis and pathological characteristics of Posner-Schlossman syndrome (PSS) are similar to uveitis and open angle glaucoma.However,etiology of PSS remains unelucidated.The activation complement in PSS patients' serum is rarely reported.Objective The aim of this study was to investigate the activation of serum complement in PSS patients for PSS pathogenesis.Methods A prospective case-controlled study was designed.The peripheral blood simples of 79 PSS patients were collected from Shenzhen Eye Hospital during December 2013 to December 2015,and the peripheral blood simples were obtained from 83 unrelated healthy blood donors as healthy control group.Immuno-scatter turbidmetry was adopted to detect the common activated components in complement pathway in each group including complement C3 (a vital intersection molecule in the three pathways),C4 (the vital molecule both the complement classical and lectin pathways),split products C3a,soluble membrane attack complex (sC5b-9),C 1q (complement classical pathway),L-ficolin (complement lectin pathway),complement factor Bb (complement alternative pathway),IgG,IgA and IgM.The correlation between serum C3a content and sC5b-9 content in PSS group was analyzed.The serum contents of fabric binding protein 2 (FCN2) (a marker of serum classical pathway),factor Bb (a marker of complement alternative pathway),C3a (the common activation products of three complement activation pathways),and sC5b-9 were assayed by ELISA.This research protocal was approved by Shenzhen Eye Hospital and written informed consent was obtained from each PSS patient prior to any medical examination.Results Compared with normal control group,the serum levels of C3,C4,C3a,sC5b-9,C1q,FCN2,IgG,IgA and IgM were significantly higher in PSS group (Z =-4.743,-2.913,-1.985,-2.620,-2.062,-2.500,-7.010,-6.327,-3.652,all at P ＜ 0.05).The serum complement factor Bb level was 13.87 (9.24,32.00) μg/ml in PSS group,which was significantly lower than 20.51 (12.90,33.50) μg/ml in normal control group (Z =-2.515,P =0.012).Serum C3a content was positively correlated with the serum sC5b-9 content in PSS group (rs =0.832,P＜0.001).Conclusions The serum complement system is activated in PSS patients.Complement alternative pathway,classical pathway and lectin pathway might all be involved in the activative process of complement system.

The ubiquitin-proteasome system is composed by multiple enzymes which are ubiquitously expressed in mammalian cells and plays an essential role in a variety of biological processes. As key members of the protein degradation enzymatic system, the function of E3 ubiquitin ligases has been extensively investigated. BMP and TGF-? are critical molecules that regulate proliferation, differentiation and apoptosis of osteoblasts and chondrocytes through different signaling pathways. Resent findings indicate that the ubiquitin-proteasome system functions as a key regulator in bone cells and the E3 ligase mediates the proteolytic degradation of critical molecules in BMP and TGF-? signaling pathways. Recent progress on studies of HECT domain E3 ligase, Smurf, in osteoblasts and chondrocytes were summarized. The regulatory role of Smurf1 and Smurf2 in BMP and TGF-? signaling and osteoblast and chondrocyte function has been reviewed.

Objective:To analyze the association of clinical characteristics and laboratory indicators at initial maintenance hemodialysis(MHD)with long-term prognosis in advance-aged patients, and to find influencing factors for the prognosis in advance-aged MHD patients.Methods:This retrospective study was conducted at the Nephrology Department of Beijing Hospital between April 2007 and January 2018.A total of 61 patients receiving first-time hemodialysis at ≥ 80 years of age and undergone regular dialysis for 3 months or longer were enrolled.All patients were followed-up until death or the end of July 1, 2018.Patients were divided into the survivor and non-survivor groups, and differences in clinical characteristics and laboratory indicator values were compared between the two groups.Influencing factors for prognosis in advance-aged MHD patients were analyzed by using multivariate Cox regression.Results:For the 61 subjects, the median follow-up time was 25.8 months.During the follow-up, 32 patients died(52.5%). The main death causes were infectious diseases(40.6%, n=13)and cardiovascular and cerebrovascular diseases(37.5%, n=12). The 1-, 2-, 3-, 4-, and 5-year cumulative survival rates were 75.4%(46/61), 54.1%(33/61), 37.7%(23/61), 22.9%(14/61)and 16.4%(10/61), respectively.The median survival time was 25.8 months for all patients, 27.5 months for patients aged 80-84 years, and 14.9 months for patients aged 85 years and over.The non-survivor group had a higher male ratio(65.6% or 21/32 vs.37.9% or 11/29, χ2=4.678, P=0.031)and lower levels of hemoglobin(85.4±13.0 vs.95.0±17.6 g/L, t=2.867, P=0.019)and albumin(30.3±5.0 vs.34.6±4.8 g/L, t=3.039, P=0.001)than the survivor group.Kaplan-Meier curves indicated that the survival rate decreased with age, and subjects aged less than 85 years had a higher survival rate than subjects aged 85 years and older(the median survival time: 14.9 months vs.27.5 months, Log Rank P=0.006); patients who received continuous renal replacement therapy(CRRT)before dialysis had lower survival rates than patients who did not receive CRRT(the median survival time: 7.8 months vs.29.2 months, Log Rank P=0.002); patients with high serum levels of albumin(≥33 g/L)had higher survival rates than patients with low serum levels of albumin(<33 g/L)(the median survival time: 29.2 months vs.18.9 months, Log Rank P=0.003). Multivariate Cox regression analysis showed that age at initial dialysis( HR=1.136, 95% CI: 1.005-1.285, P=0.041), female( HR=0.409; 95% CI: 0.169-0.994, P=0.048), serum albumin level( HR=0.836, 95% CI: 0.772-0.906, P<0.001)and CRRT before dialysis( HR=6.161, 95% CI: 1.848-20.538, P=0.003)were independent predictors of all-cause mortality in advance-aged patients. Conclusions:Advance-aged patients undergoing hemodialysis have complicated clinical conditions and poor prognosis.Age, gender and serum albumin level at initial dialysis and CRRT before dialysis are independent predictors of prognosis in these patients.

Objective To detect the expression levels of adipose-specific phospholipase A2 (AdPLA) mRNA in orbital adipose tissue of thepatients with thyroid associated ophthalmopathy and the normals.Methods Sixteen patients with TAO of m level and stationary phase underwent orbital decompression,and 29 normals underwent ocular plastic surgery in Shenzhen Eye Hospital between August,2015 and October,2016.Orbital fat samples were collected from one eyes of these patients during surgery.The age,gender,height,weight,body mass index (BMI),exophthalmos degree,orbital fat of the patients with TAO and the normals were recorded and calculated.Using real time PCR,the AdPLA mRNA were detected from these orbital fat samples.Results There was no significant difference between the patients with TAO and the normals in age,gender,and BMI (all P ＞ 0.05).TAO group had more exophthalmos degree (20.406 ± 1.369)mm than the normals (14.207 ± 1.146) mm.TAO group had more orbital fat (32.162 ± 1.923) mL than the normals (24.279 ± 1.070) mL.The average expression level of AdPLA in patients with TAO was 0.039 42 ± 0.009 85,and 0.004 42 ± 0.001 36 in the normal.There was significant difference between two groups (P ＜ 0.05).Conclusion The patients with TAO of Ⅲ level and stationary phase have more exophthalmos degree and orbital fat than the normals.AdPLA mRNA is higher expressed in orbital adipose tissue of the patients with TAO of Ⅲ level and stationary phase than the normals.The high expression of AdPLA may reduce lipolysis in the orbital adipose tissue,lead to fat accumulation in orbits,and aggravate exophthalmos of patients with TAO.

Objective To explore the protective effect and mechanism of antioxidant N-acetyl-L-cysteine (NAC)on the cytotoxicity induced by iohexol in HK-2 cells. Methods The incubated HK-2 cells were divided into four groups:control group,iohexol group,NAC group,and NAC+iohexol group(pre-incubated with NAC and then co-incubated with iohexol).The cell viability was tested by CCK-8 assay;cell apoptosis was determined by Hoechst 33342 fluorescence staining and flow cytometry with Annexin V-FITC/PI double staining.Intracelluar ROS waft detected by flow cytometry with DCFH-DA fluorescence staining.The signaling transduction pathways were investigated by Western blotting and immunofluorescence staining. Results Iohexol decreased cell viability,and increased apoptosis in a dose-and time-dependent manner.In iohexol(100 gl/L,6 h)group,ROS was increased by 1.30-fold of control(P＜0.05).In NAC(5,10,15 mmol/L)+iohexol groups,the cell viability was increased by 104%,118%,130%respectively,and iohexol group was 63% (P＜0.05, respectively); apoptosis rate was decreased by 13.51%, 13.46%, 12.23% respectively, and iohexol group was 24.41% (P＜0.05, respectively); ROS was decreased by 1.05-fold, 0.93-fold, 0.86-fold respectively, and iohexol group was 1.3-fold (P＜0.05, respectively).Iohexol induced the increase of p53 phosphorylatian and activity, then up-regulation of Bax and down-regulation of Bcl-2 protein expression. Iohexol induced the release of cytochrome C from mitochondria to cytoplasm, all of which caused final activation of caspase-3. The expression levels of p53, Bax and caspase-3 were decreased, while Bcl-2 protein expression level was increased by NAC. Conclusions Iohexol induces the increase of apeptosis rate and ROS generation in HK-2 cells. NAC attenuates this iohexol-induced cytotoxicity by decreasing intracelluar ROS, which is mairdy through the intrinsic pathway.