Background Sulforaphane (SFN) is an effective chemopreventive agent and can regulate the biological molecular mechanisms to inhibit the overgrowth of cells.Autophagy is a biological process of maintaining cellular internal environment.Understanding the affection of SFN to biological behavior of human lens epithelial cells (LECs) and the association of SFN with autophagy is helpful for the prevention and target treatment of posterior capsule opacification (PCO).Objective This study was to investigate the eradication effeccts of SFN on residual lens cell population in vitro posterior capsule opacification (PCO) model and evaluate the mechanism of SFN-induced cell death.Methods In vitro human capsular bag models were generated from fresh donor eyes by phacoemulsification and were cultured in EMEM containing 2％ fetal bovine serum (FBS).Different concentrations of SFN (0,1,10 and 100 μ mol) were added in the medium for 30 days respectively according to grouping,and the growth of LECs was observed by optical microscope and immunofluorescence technique.FHL124,a human LEC line,was cultured with EMEM containing 5％ FBS and divided into 0,1,10,30 and 100 μmol SFN groups.Lactate dehydrogenase (LDH) release rate in the medium was detected to evaluate cell damage/death.The migration of the cells on capsular bags was assessed by scratch test.The ultrastructure and number of autophagosomes were examined under the transmission electron microscope.The expression of LC3 in the cells were detected using Western blot in the presence or absence of autophagy inhibitors.Results The cell coverage rates on the capsular bags were significantly lower in the 10 and 100 μ mol/L SFN groups than those in the 0 and 1 μmol/L SFN groups,with a statistically significant difference among the groups (F =48.57,P ＜ 0.01).Immunofluorescence showed that the density of F-actin-and Vimentin-positive cells was evidently decreased in the 10 and 100 μmol/L SFN groups compared with 0 and 1 μ mol/L SFN groups.The releasing levels of LDH (absorbancy) were 0.19± 0.03,0.39±0.06,0.56±0.07,0.68±0.08 and 0.89±0.09 in the 0,1,10,30 and 100 μ mol/L SFN groups,respectively,and the releasing level of LDH was gradually increased in the 10 and 100 μ mol/L SFN groups in comparison with the 1 μmol/LSFN group (all at P＜0.01).With the increase of SFN concentration,the reduction rate of scratched area decreased with the increase of SFN concentration,and the decrease of scratch area was significantly lower than that of adjacent low mass concentration group and the differences were statistically significant (P＜0.05).The relative expressions of LC3-Ⅱ protein were 0.423±0.003,14.543±0.024,0.668±0.024 and 0.576±0.056 in the blank control group,SFN group,SFN + 3-MA group and 3-MA group,respectively,and the relative expressions of LC3-Ⅱ protein were significantly lower in the SFN+3-MA group and 3-MA group than those in the SFN group (all at P＜0.01).The number of autophagosomes was 4.07±0.32,4.13±0.34,9.21 ±0.53 and 21.02± 1.34 in the blank control group,and 1,10,100 μmol/L SFN groups,and the number of autophagosomes in the 10 and 100 μ mol/L SFN groups was significantly higher than that in the blank control group and 1 μmol/L SFN group (all at P＜0.01).Conclusions SFN mediates LECs death by promoting autophagy in ex vivo capsular bags,and SFN may be a novel agent of potential chemopreventive and target treatment for PCO.

Aim To assess the effects of N -[2-p-bromo-cinnamylamino-ethyl]-5-isoquinoline-sulfonamide (H-89), a potentially selective inhibitor of Protein Kinase A (PKA), on cardiac membrane ion channels and transporters, which will further fulfill our understanding of pharmacology of PKA inhibitors.Methods Whole-cell patch clamp was used to investigate the effects of H-89 on cardiac L-type Ca~(2+) current (I_(Ca-L)), Na~+ current (I_(Na)), inward rectifier K~+ current (I_(K1)), transient outward K~+ current (I_(to)) and Na~+-Ca~2+ exchanger current (I_(Na/Ca)) in enzymatic dissociated SD rat ventricular myocytes.Results H-89 at 1～10 μmol·L~(-1) could inhibit I_(Ca-L) , I_(Na) , and Ito in a concentration-relative manner (P <0.05). At low concentra-tion (5 μmol·L~(-1)), H-89 completely inhibited I_(K1) (P <0.05) just as the action of 0.5 mmol·L~(-1) BaCl_2.Further, H-89 at 1～10 μmol·L~(-1) had no significant effect on I_(Na/Ca) (P >0.05).Conclusion The direct or PKA-mediated indirect action maybe involved in the effects of H-89 on ion currents and transporter.

Objective To investigate the reasonable time limit for stopping the aspirin treatment in preoperative pa-tients with general surgery and the effects on platelet function in postoperative patients with recovering the therapy of aspirin. Methods A total of 121 patients undergoing elective general surgery were divided into stopping aspirin treatment 5 d group (n=59) and stopping aspirin treatment 7 d group (n=62). Fifty healthy volunteers were used as the control group. The arachi-donic acid (AA)-induced platelet aggregation test was used to detect the platelet agglutination rate in all groups. Aspirin was reused 24~48 h after surgery. The level of urinary 11-dehydro-thromboxane B2 (11-DH-TXB2) was assayed by ELISA 7 and 10 d after retreatment. Results The levels of the PAgT (5 min, 8 min and 10 min) were decreased significantly in pa-tients with stopping aspirin treatment 5 d group compared with those of patients with stopping aspirin treatment 7 d group and control group (P<0.05). There was no significant difference in the level of PAgT between patients with stopping aspirin treatment 7 d group and control group. The platelet aggregation showed two different characteristic curves after stopping aspi-rin treatment for 5 d. And the normal curve of platelet aggregation was found after stopping aspirin treatment for 7 d. The lev-els of PAgT and urinary 11-DH-TXB2 were significantly lower in patient recovered the aspirin treatment for 7 d and 10 d than that of control, and which was significantly higher in 7 d group than that of 10 d group (P>0.05). Conclusion The platelet aggregative function returned to normal level in patients with 7-d preoperative stopping aspirin. The laboratory moni-toring of aspirin therapy should be more than 7 d after postoperative reusing aspirin.

0.05).Zacopride at concentration of 1.0 ?mol/L showed the most potent activity on IK1 with approximately 30% increment both in inward current and outward current(P

Objective:To observe the effects of active immunization with a synthesized repetitive peptides in the extracellular loops of Na/Ca exchanger(NCX)?1 on cardiac structure and function in rats.Methods:A repetitive peptide of 124 HNFTAGDLGPSTIVGSAAFNMF145 was synthesized,which was in line with the extracellular loops of Na/Ca exchanger(NCX)?1.Healthly male Wistar rats of 2 month age were immunized actively with the synthesized peptide as antigen repeated for 12 weeks.The control group was given Freund's adjuvant only.Specific antibodies were detected by ELISA.The cardiac function was observed by Langendorff isolated heart-perfusing assay and the hearts were prepared for routine histological evaluation.Results:All rats immunized with the peptide developed highly positive autoimmunities,indicated by the antibody titers.After 12 weeks of peptide inoculation,the cardiac functioning indexes including LVSP-LVDP,+dp/dtmax and -dp/dtmax increased much more significantly in immunized group than in control.Histological evaluation showed that the myofilaments of the control group arranged regularly and densely with better continuity,whereas the myofilaments of the immunized group were lined with disorder.Some of those were ruptured.The interstitial lymphocyte infiltration was observed.Conclusion:The results indicate that long term immunization with the synthesized repeatitive peptide in line with the extracellular parts of Na/Ca exchanger(NCX)?1 can enhance both systolic and diastolic function of rat heart,but it can also induce injury in the heart structure.This may relate with an increase of myocardial oxygen consumption owing to a long time and continued excitement of membrane ion transporters as well as their active effect in heart contraction to a larger extent.

Aim To investigate the effects of 5-HT_4 receptor agonist and 5-HT_3 receptor antagonist 2-[1-(4-piperonyl)piperazinyl]benzothiazole on rat heart rhythm and the involved ionic mechanisms.Methods Langendorff-perfused rat hearts were subjected to 0.1～10 μmol·L~(-1) 2-[1-(4-piperonyl)-piperazinyl]benzothiazole for 15 minutes with simultaneous ECGs recording.The whole-cell patch-clamp electrophysiology was used to record effects of 2-[1-(4-piperonyl)piperazinyl]benzothiazole on inward rectifier K~+ current(I_(K1)),transient outward K~+ current(I_(to)),resting membrane potential(RMP)and action potential(AP)in enzymatic dissociated rat ventricular myocytes.Results In ex vivo Langendorff-perfused hearts,0.1～10 μmol·L~(-1) 2-[1-(4-piperonyl)piperazinyl]benzothiazole elicited singnificant rhythm disturbances.In the presence of 10 μmol·L~(-1) agent,the total of PVB were 236±37,87.5%(7/8)hearts exhibited VT,and 62.5%(5/8)hearts exhibited VF(P<0.01).At the concentration of 0.1～10 μmol·L~(-1),2-[1-(4-piperonyl)piperazinyl]benzothiazole could inhibit I_(K1)(EC50=0.74 μmol·L~(-1))and I_(to)(EC50=2.16 μmol·L~(-1)),decrease RMP and prolong action potential duration(APD)in concentration-dependent manners(n=6,P<0.01).Conclusion Inhibition of IK1,Ito and resultant prolongation of APD,depolarization of RMP might be the critical causes for induction of arrhythmias by 2-[1-(4-piperonyl)piperazinyl]benzothiazole in rat.

A cross-sectional study was conducted on 1619 youngsters aged 13-15.They were divided into two groups:one with family history of diabetes (FHD~+) and another without a family history of diabetes (FHD~-).Measurements included height,weight,waist circumference (WC) and hip.FHD~+ group had significantly higher WC measurement,waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) when compared with FHD~-group (P ＜0.01).The abdominal obesity rate defined by WC measurement in FHD+ group was higher than that in FHD~-group (P＜0.01).The rate of overweight and obesity defined by body mass index (BMI) were no significant difference between two groups.After adjusting the gender and age,logistic regression analysis showed that the odds ratios of WC alone and BMI+WC for FHD~+ were 2.029 and 1.364 (95% CI:1.211-3.400,1.043-1.784,P＜0.05) respectively.Our data suggest that teenage with a family history of type 2 diabetes has a tendency of overweight characterized by the abdominal obesity.

Objective:To construct a eukaryotic expression vector of leptin gene,and to transfect it into human placental mesenchy-mal stem cells(HPMSCs)and appraise its expression.Methods:Primers were designed and the leptin gene was obtained by RT-PCR from human adipose tissue.The aimed segments were inserted into the eukaryotic expression vector pIRES2-EGFP,plasmid pIRES2-EGFP-LEP was constructed and identified by restricted enzymatic resection,and then transfected it into HPMSCs by liposome.The ex-pression of leptin in the transfected cells was detected by RT-PCR and Western blotting,the multi-differentiation potential of the cells was indentified.Results:The length of specific fragment was 500 bp,the recombinant plasmid pIRES2-EGFP-LEP presented 5.3 kb and 500 bp bands by restriction enzyme digestion.Leptin gene was expressed in transfected HPMSCs and the transfected HPMSCs maintained multi-directional differentiation potential.Conclusion:The eukaryotic expression vector of leptin can be transfected and ex-pressed in HPMSCs.

Objective To establish a method of PKH26 labeled bone marrow-derived endothelial progenitor cells (EPCs) into rats with liver fibrosis and observe cell immigration and differentiation in the liver after transplantation.Methods Bone marrow-derived EPCs were isolated and cultured from rats with liver fibrosis,and then labeled with PKH26 in vitro.Under the scanning confocal microscopy and flow cytometry,PKH26 fluorescent labeling rate and cell survival rate were measured.EPCs of PKH26 fluorescent labeling were transplanted into rats with liver fibrosis via the tail vein,and the migration situation was observed in the liver.Endothelial cell markers CD31 and von willebrand factor (vWF) were detected by using immunofluorescence.Results The PKH26-labeled EPCs appeared red fluorescence and the labeling rate was 96.65 ％.As compared with unlabeled cells,the labeled cells grew well,and had no significant changes in the growth curve.After transplantation into the liver of rats,the PKH26 labeled cells were mainly distributed in blood vessel endothelium along fibers and hepatic sinusoids in hepatic lobule.Endothelial cell-specific antigens such as CD31 and vWF could be detected along the vascular walls.Conclusion PKH26 could be used to label and track EPCs in the liver of rats with liver fibrosis in vitro.The PKH26-labeled cells may migrate to the surrounding of the hepatic vessels and differentiate into mature endothelial cells.

BACKGROUND:Theoretically, bone marrow-derived endothelial progenitor cells (EPCs) from liver fibrosis rats could be filtered by the pathological environment in vivo. These EPCs would be more adapted to the micro-environment of liver fibrosis, and easier to differentiate into mature endothelial cells participating in the intrahepatic vascular remodeling after transplanted into the liver.OBJECTIVE:To explore the effectiveness of transplantation of bone marrow-derived EPCs from the liver fibrosis environment in liver fibrosis rats.METHODS:Twenty-eight Wistar rats were randomly divided into three groups as follows:normal group (n=8) were injected with olive oil, twice per week; model group (n=10) were infused with carbon tetrachloride at a dose of 3 mL/kg body weight (double doses for the first time), twice per week, and infused with normal saline through the tail vein at 2, 3 and 5 weeks; EPCs transplantation group (n=10) were infused with carbon tetrachloride at a dose of 3 mL/kg body weight (double doses for the first time), twice per week, and infused with EPCs suspension through the tail vein at 2, 3 and 5 weeks. Six weeks after final injection, the angiogenesis, hepatocyte proliferation and pathological changes in the liver tissues were observed. The liver function and coagulation function were tested.RESULTS AND CONCLUSION:(1) The pathological changes of the liver:in the model group, fatty degeneration and hepatocyte necrosis in the liver tissue were serious, inflammatory cells were infiltrated around the portal and central vein,the portal areas expanded, and fibrous tissues overgrew. Compared with the model group, these changes were significantly relieved in the EPCs transplantation group (P < 0.05). (2) The expressions of liver-related proteins:compared with the normal group, the levels of hyaluronic acid, laminin, type III procollagen, vascular endothelial growth factor, epidermal growth factor were significantly increased in the model group (P < 0.05). Compared with the model group, the levels of hyaluronic acid, laminin and type III procollagen were decreased significantly (P < 0.05), and the levels of vascular endothelial growth factor and epidermal growth factor were increased in the EPCs transplantation group (P < 0.05). (3) Liver function and coagulation function:compared with the normal group, the liver function and blood blotting function of rats were seriously damaged in the model group (P < 0.05). Compared with the model group,the liver function and coagulation function were obviously improved in the EPCs transplantation group (P < 0.05). To conclude, transplantation of bone marrow-derived EPCs from the liver fibrosis environment is effective for liver fibrosis in rats. The mechanism may be associated with the promotion of angiogenesis in the liver.