AIM: To investigate the relationship between the prevention of probucol on restenosis and vascular remodeling after percutaneous transluminal angioplasty(PTA) in rabbits. METHODS: New Zealand rabbit thoracic aorta atherosclerosis was induced by 3.5F ballon catheter injury following a 4-weeks feeding of high cholesterol diet, and PTA was performed by using 3.5F balloon catheter. Probucol(1g/d) or vitamin E (400 mg/d) was administrated one week before PTA. Two weeks after PTA, the bore and outside diameter (OD) of arteries, the area circumscribing by intimal elastic lamina (IEL), the area circumscribing by extral elastic lamina (EEL), medial area (MA), neointima area/medial area (NEA/MA) were analyzed by computerized digitizer system. Lipids of serum were measured by means of biochemical assay.RESULTS: After two weeks of PTA, the intima proliferation and lumen restenosis were observed obviously. However, with probucol treatment for 3 weeks, the restenosis of aorta was inhibited significantly by increasing bore, outside diameter, and lumen area of rabbits aortas and decreasing NEA, NEA/MA. Furthermore, probucol regulated vascular remodeling by increasing the area circumscribing by IEL [(3.50?0.20)mm 2 vs (1.59?0.23) mm 2, P

In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG2 to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG2cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transeriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG2/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG2/GFP-Daxx cells). After incubation with hydrogen peroxide (H2O2) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG2/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG2 cells and observed that the hydrogen peroxide decreased the viability of HepG2 cells in concentration-dependent pattern. The IC50 values in three groups (Normal cells, HepG2/GFP cells and HepG2/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG2/GFP-Daxx cells as compared to the other two groups. HepG2/GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG2 cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.

Aim To investigate the effects of rosmarinic acid on H2O2-induced apoptosis in rat VSMCs and its related mechanisms. Methods Flow cytometry was used to determine apoptotic rate of VSMCs. nuclear staining by acridine orange for morphologic change,MTT assay for cell viability and Western blot for expression of Bcl-2,Bax, fas and FasL. Results After being treated by H2O2(0,250,500,750 ?mol?L-1),apoptotic rate of VSMCs was 3.71%?0.56%,17.6%?6.92%,34.9%?2.55 %, and 85.6%?5.22% by FACS analysis,and VSMCs appeared nuclear condense and nuclear fragmentation after being treated by 500 ?mol?L-1 H2O2 for 24 hours,which were typical morphological changes of apoptosis. ① VSMCs treated with 500 ?mol?L-1 H2O2 for 24 hours had a significantly decrease on cell viability compared with control,and the apoptosis rate was increased to 35.7%?1.33%; Bcl-2 protein expression decreased,Bax protein expression increased, Bcl-2/Bax protein ratio decreased and Fas receptor and Fas ligand expression also increased.② Pre-incubation with rosmarinic acid(10, 20, 40 ?mol?L-1)for 30 min enhanced the cell viability, and decreased the apoptotic rate to 31.1%?1.38%,21.2%?1.18%,13.6%?0.51% in a dose-dependent manner. Moreover, Bcl-2/Bax protein ratio increased and Fas and FasL expression decreased. Conclusions ① Rosmarinic acid antagonizes H2O2-induced apoptosis of VSMCs. ② The antagonism of rosmarinic acid on apoptosis may be correlated with an increase Bcl-2/Bax protein ratio and a decrease expression of Fas and FasL protein.

Aim To study the effects of probucol on THP-1 macrophage apoptosis and CD36、Caveolin-1 expression induced by ox-LDL.Methods Apoptosis of THP-1 macrophages was determined by flow cytometry analysis.RT-PCR and immunofluorescence were used to detect CD36,Caveolin-1 mRNA level and protein expression respectively.Results Probucol had no effect on mRNA level of CD36,Caveolin-1 in THP-1 macrophages,but it attenuated Caveolin-1 protein expression.Conclusions Probucol can inhibit apoptosis induced by ox-LDL in THP-1 macrophages by down-regulating Caveolin-1 protein expression.

To produce monoclonal antibody (mAb) specifically against human thrombomodulin (hTM), an immune-tolerizing procedure was employed to generate monoclonal antibodies specific to hTM. Female BALB/c mice were first immunized with CHO cells following at 10 min, 24 h, 48 h by intraperitoneal injection of different doses of cyclophosphamide (CP) 2 times at an interval of 2 weeks, thereby tolerizing the mice to common epitopes shared between CHO and CHO-TM5 cells. Subsequently the selected mice with the lowest titer of serum polyclonal antibody by cellular enzyme-linked immunoabsorbent assay (CELISA) were immunized with CHO-TM5 cells, which have stable high level expression of hTM, to produce antibodies specific to hTM 3 times at an interval of 2 weeks. On the third day after the third immunization, mouse with the highest titer of serum polyclonal antibody was sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96 well plates for screening with CELISA. To improve probability to obtain specific mAb, CELISA was applied twice. The first CELISA was done with polyethylene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but having CHO cells monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5 +CHO- hybridoma cells. BALB/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded mAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. Detection of CELISA showed that 100 mg/kg dose of CP could tolerize the mouse to common epitopes shared between CHO and CHO-TM5 cells. And CELISA also discovered that all hybridomas positive for CHO-TM5 cells were negative for CHO cells. Five lines of positive hybridoma cells had been obtained altogether and 2F7 was selected randomly for next investigation. The Ig subclass of the mAb 2F7 was IgG1 and the titer of ascitic mAb was 1?10-6. Furthermore, the content of ascitic mAb was 19.56 g/L and chromosome numbers is 98. Flow cytometry, CELISA and Western blotting assays demonstrated that mAb 2F7 could specifically recognize hTM expressed on CHO-TM5 and human umbilical vascular endothelial cells (HUVEC). Meanwhile, the tissue specificity of mAb 2F7 was also identified by immunohistochemical ABC staining. On the other hand, Western blotting assays indicated that mAb 2F7 could recognize the antigen protein with 105 ku molecular mass under reduction condition. Moreover, the dissociation constant of mAb 2F7, 1.22? 10-9 mol/L, indicated the affinity higher than some others. The results suggest that the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms. mAb 2F7 can specifically recognize the natural hTM expressed mainly on vascular endothelial cells, which will potentially useful for investigating the functions and clinic values of hTM.

AIM: To investigate the protective effect of onychin on the endothelial cells injured by oxidative stress. METHODS: The injured model was established by endothelial cells treated with menadione. The growth inhibitory rate of endothelial cell was determined by MTT assay; NO - 2/NO - 3 concentration in the medium was determined by nitrate reductase assay; eNOS and caveolin-1 protein levels were determined by Western blot. RESULTS: Onychin significantly decreased the growth inhibitory rate of endothelial cells injured by menadione, increased NO - 2/NO - 3 concentration in the medium and eNOS activity and up-regulated caveolin-1 expression. CONCLUSION: Onychin possesses a protective effect against endothelial cell injury induced by menadione via caveolin-1/eNOS pathway.

AIM To investigate the protective action of onychin aganist the growth inhibition of endothelial cell injured by menadione and its mechanism. METHODS The injured model was established by endothelial cell treated with menadione.Protective effect of onychin aganist growth inhibition of injured endothelial cell was determined by MTT assay and cell counting method; NO concentration in the medium was determined by nitrate reductase assay; eNOS and phosph ERK1/2 protein levels were determined by Western blot. RESULT Onychin significantly decreased the growth inhibitory rate of injured endothelial cells,increased NO concentration in the medium and eNOS activity and up regulated phosph ERK1/2 expressing. CONCLUSION Onychin has protective action and against the growth inhibition of endothelial cell injured by menadione may be via NO and ERK1/2 signal pathway.

Objective To investigate the related factors on effects of uterine artery embolization (UAE) in the treatment of dysmenorrhea in patients with adenomyosis, and to construct and validate the efficacy prediction model. Methods A total of 127 cases of adenomyosis patients with symptoms of dysmenorrhea in Guangzhou No.1 People′s Hospital and Nanfang Hospital of Southern Medical University from June 1999 to December 2009 were reviewed. The evaluation standard was to improve the degree of dysmenorrhea, the related factors of efficacy were analysed. Combined with artificial neural network theory, the effect prediction model was constructed, and the effectiveness of the model was evaluated using receiver operating characteristic (ROC) curve, and the effectiveness of the cut-off point was calculated. The model was validated by 68 cases of patients with adenomyosis in the Nanfang Hospital from January 2010 to November 2014. Results (1) In 127 cases of dysmenorrhea patients, UAE treatment was effective in 98 cases, effective rate was 77.2% (98/127). (2) Age was an independent predictor of effective UAE treatment (HR=1.129, P=0.026);in the range of this study, the greater the age, the higher the UAE treatment efficiency. (3) The developing situation of ovary branches of uterine artery was an independent predictor of effective UAE treatment (HR=0.460, P=0.020), the efficiency of patients whose intraoperative bilateral uterine artery ovarian branch did not develop was 89.7%(35/39), the efficiency of patients whose unilateral uterine artery ovarian branch was developing was 84.1% (37/44) and the efficiency of patients whose bilateral uterine artery ovarian branch were developing was 59.1% (26/44). (4) Blood supply of adenomyosisis was an independent predictor of effective UAE treatment (HR=0.313, P=0.001). Type Ⅰ (bilateral predominated) patients, efficiency was 93.5%(43/46);typeⅡ(bilateral balanced) patients, efficiency was 78.0%(39/50);type Ⅲ (unilateral predominated) patients, efficiency was 51.6% (16/31). (5) UAE for the treatment of adenomyosis efficacy of artificial neural network prediction model was constructed, the model′s area under the ROC curve was 0.808, the optimal cut-off point was 0.669 13. Actual verification of the model, sensitivity was 96.5%, specificity was 81.8%, positive predictive value was 96.5%and negative predictive value was 81.8%, the total accuracy was 94.1%. Conclusions (1) Age, the developing situation of ovary branches and blood supply of adenomyosis are the independent predictors of effective UAE treatment. (2) The artificial neural network prediction model is satisfied with the accuracy and the accuracy of prediction.

Objective: To summarize the peri-operative management experience of pulmonary endarterectomy (PEA) in patients with chronic thromboembolic pulmonary hypertension (CTEPH). Methods: A total of 56 CTEPH patients received PEA in our hospital from 2015-01 to 2016-11 were retrospectively analyzed. Our study was focused on the medication in respiratory and circulatory system during ICU stay, peri-operative application of vasoactive drug and target drug to pulmonary hypertension (HP), usage of ventilators, mechanical assisted devices and other management experiences. Results: No peri-operative death occurred. There were 2/56 (3.6%) patients with lung reperfusion, 2 (3.6%) with PH crisis. Compared with pre-operation, the post-operative pulmonary artery hemodynamics parameters were improved as right heart catheter measured pulmonary artery systolic pressure (PASP) decreased from (85.05±22.40) mmHg to (36.83 ±17.21) mmHg and pulmonary vascular resistance decreased from (773.84±342.95) dyn·s·cm-5 to (293.59±214.95) dyn·s·cm-5. Post-operative oxygen saturation was maintained at (95-100) % in all patients. Echocardiography found that PASP from pre-operation (85.03±25.78) mmHg decreased to (39.44±19.24) mmHg at follow-up period, P<0.01.Conclusion: A comprehensive peri-operative management of PEA was helpful to improve pulmonary hemodynamics in CTEPH patients; meanwhile, effective prevention and treatment of severe complication could obviously reduce peri-operative mortality.

Objective To study the value of endothelin-1 (ET-1) in predicting the outcome of stable coronary artery disease (SCAD) patients.Methods A total of 3154 SCAD patients who were followed up for 24 months were divided into cardiocerebral vascular events group (n=189) and cardiocerebral vascular events-free group (n =2965).Their serum ET-1 level was measured by ELISA.The patients were further divided into ET-1 ＜0.3 pmol/L group (n=1588) and ET-1≥0.3 pmol/L group (n=1566).The value of ET-1 in predicting the end events was assessed by Cox regression analysis.The survival curve was plotted by Kaplan-Meier analysis.Results The serum ET-1 level was signify-cantly higher in cardiocerebral vascular events group than in cardiocerebral vascular events-free group (0.33 pmol/L vs 0.30 pmol/L,P=0.004).The incidence of clinical end events was significantly lower in ET-1 ≥0.3 pmol/L group than in ET-1 ＜0.3 pmol/L group (7.02％ vs 4.97％,P=0.015).Multivariable Cox regression analysis showed that ET-1 was a predictor of clinical end events (HR=1.656,95％CI:1.099-2.496,P=0.016).Kaplan-Meier analysis showed that the events-free survival rate was lower in patients with a higher serum ET-1 level than in those with a lower serum ET-1 level (P=0.016).Conclusion ET-1 is an important risk factor for the outcome of SCAD patients.Further studies are needed to confirm its long-term value in predicting the outcome of SCAD patients.