OBJECTIVE:To establish a RP-HPLC method for the determination of nitrochlodipine in rabbit plasma.METH-ODS:Chromatographic conditions and extractive conditions were optimized with orthogonal design,internal standard:felodipine,solid phase extract column:C18,column size:NOVA-PackC18(4.6mm?250mm),detection wavelength:237nm.RESULTS: The optimal chromatographic conditions were:mobile phase acetonitrile-water(55∶45),flow rate 1.0ml/min,column temperature 45℃.The extraction conditions were A1 B1 C3.The calibration curve was linear within the range of 6.25～400?g/L(r=0.9 996) and the minimum limit of detection was 2.5?g/L.The average RSD of within-day ranged from 3.81% to 5.58% and that of day-to-day from 4.60% to 8.76%.The average recovery ranged from 100.7% to 101.8%.CONCLUS-ION:The method is simple,rapid,highly accurate and precise.It can be used for the quantitative determination of nitrochlodipine in plasma.

Objective To estimate the biological effects of leptin on human HaCaT keratinocytes and explore their molecular mechanisms.Methods Cell counting kit-8 (CCK-8) was used to evaluate the proliferation of cultured HaCaT cells treated with different concentrations of leptin for 24 and 48 hours.Some HaCaT cells were classified into four groups to remain untreated,be treated with leptin (100 μg/L) and piceatannol (a specific inhibitor of STAT3 phosphorylation) alone or in combination for 24 hours,respectively,followed by the evaluation of cell proliferation using CCK-8 kit.Flow cytometry was performed to assess cell cycle of HaCaT cells treated with leptin of 100 μg/L,Western blot to determine the phosphorylation level of Erk1/2 and STAT3 in HaCaT cells treated with leptin of 100 μg/L for different durations.Statistical analysis was done by Student's t-test for unpaired data using GraphPad Prism 5 software.Results The proliferation of HaCaT cells was accelerated to different degrees after treatment with leptin of 50 and 100 μg/L for 24 and 48 hours,and the accelerating effect was in a dose-dependent manner within 24 hours (r =0.9989,P ＜ 0.05).Piceatannol apparently inhibited the promotive effect of leptin on the proliferation of HaCaT cells.There was an obvious elevation in the percentage of cells at S phase ((57.70 ± 5.88)％ vs.(42.50 ± 7.55)％,P ＞ 0.05),but a significant decrease in that at G0/G1 phase ((39.70 ± 1.57)％ vs.(45.20 ± 1.44)％,P ＜ 0.05),with a significant increase in proliferation index (0.603 ±0.0157 vs.0.564 ± 0.0144,P ＜ 0.05) in HaCaT cells treated with leptin of 100 μg/L for 24 hours compared with the untreated controls.Western blot showed that leptin of 100 μg/L markedly enhanced the phosphorylation level of STAT3 in HaCaT cells.Conclusion Leptin may upregulate the proliferation of HaCaT cells through activation of STAT3 pathway.

Purpose:To study the activity of toremifene and its synergistic effect with anti-tumor drugs on human lung adenocarcinoma cell line A549,which might be expected to provide a new mode of therapy for the clinical management of lung cancer.Methods:The cytotoxic effects of these agents on human lung cancer cell line A549 were measured by a tet- razolium-based volorimetric assay (MTT assay).Results:Toremifene can inhibit the growth of A549 cell directly.The A value of the low dose toremifene combined with anti-tumor drugs were lower than that of anti-tumor drugs alone.Toremifene combined with VCR,ADM,DDP and VP-16 showed better effects.Conclusions:Toremifene combined with chemotherapy drugs shows significant synergistic anti-tumor effect on A549 cell.This might provide experimental evidence for lung cancer therapy.

Objective: To study the expression of L selectin and its possible role in pulmonary injury in rat models undergoing cardiopulmonary bypass (CPB) and to investigate the protecive effect of fucoidin during the procedure. Methods: Models of CPB and pulmonary perfusion in rats were used to investigate the expression of L selectin pre and post operation. Its expression in antagonist group was also studied. Other indexes, including the concentration of SOD, MDA and MPO in rat lung tissues as well as PaO 2/FiO 2 and histological changes, were studied to illustrate the possible mechanism of L selectin in lung reperfusion injury. Results: The expression of L selectin increased after creating CPB but decreased after pulmonary infusion with fucoidin during the procedure, and lung injury relieved as well. Conclusion: L selectin might lead to lung injury during CPB, blocking its expression may relieve lung injury and enhance the recovery of lung function.

OBJECTIVE:To detect the contents of chlorogenic acid and rutoside in Saussurea involucrate,and to optimize the decoction and extraction technology of S. involucrate from different producing areas. METHODS:The contents of chlorogenic acid and rutoside in 10 batches of S. involucrate from different producing areas were determined by HPLC. L9(34)orthogonal experiment was used to optimize the water amount,decoction times and decoction time using comprehensive score of extraction transport rate of chlorogenic acid and rutoside as index. The verification test was also conducted. RESULTS:The contents of chlorogenic acid and rutoside in 10 batches of S. involucrate were 0.380%-0.546% and 0.334%-0.617%;the optimal decoction technology was as follows as the amout of crude material of S. involucrate 100 g,soaking for 20 min,decocting for 3 times,12,10 and 10 fold of water,decocting 45,30 and 30 min,respectively. The extraction transfer rates of chlorogenic acid and rutoside were 96.2%(RSD=2.66%,n=3)and 89.3%(RSD=3.31%,n=3)in verification test. CONCLUSIONS:For S. involucrate from different producing areas,the contents of effective components are different;optimized decoction and extraction technology is stable and feasible.

ObjectiveTo investigate the effects of postconditioning with electric vagal stimulation on myocardial ischemia-reperfusion (I/R) injury in rats.MethodsSixty male SD rats weighing 250-350 g were randomly divided into 3 groups (n ＝ 20 each):group sham operation (group S); group myocardial I/R (group I/R) and group electric vagal stimulation postconditioning (group POES).Myocardial I/R was induced by occlusion of left anterior descending branch of coronary artery for 30 min followed by 120 min reperfusion in groups I/R and POES.In group POES right cervical vagus nerve trunk was stimulated for 30 min with continuous electric rectangular pulses (2 ms,10 Hz) starting from 15 min of myocardial ischemia.The voltage of the pulses was adjusted to decrease HR by 10％ of the baseline HR before stimulation.MAP,HR and RPP (MAP× HR) were recorded before (baseline) and at 1 and 10 min of ischemia and 30,60 and 120 min of reperfusion.Arterial blood samples were collected from 10 rats in each group at 120 min of reperfusion for determination of serum concentrations of cTnI,CK-MB,TNF-a,high mobility group box 1 protein (HMGB1),ICAM-1,IL-1,IL-6 and IL-10 (by ELISA).The animals were then sacrificed and myocardial infarct size was measured by Evans blue and TTC staining.Another 10 rats were sacrificed at 120 min of reperfusion for determination of myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1,IL-6 and IL-10 (by ELISA).ResultsI/R induced myocardial infarct,significantly increased serum concentrations of cTnI,CK-MB,TNF-α,HMGB1,ICAM-1,IL-1 and IL-6 and significantly increased myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1,IL-6 and IL-10 in both ischemic and non-ischemic regions in group I/R as compared with group S.Electric vagal stimulation significantly decreased myocardial infarct size and serum concentrations of cTnI,CK-MB,TNF-α,HMGB1,1CAM-1,IL-1 and IL-6 in group POES compared with group I/R.Myocardial contents of TNF-α,HMGB1,ICAM-1,IL-1 and IL-6 were significantly decreased while myocardial IL-10 content was increased in both ischemic and non-isehemic regions in groups POES compared with group I/R.ConclusionPostconditioning with electric vagal stimulation can attenuate myocardial I/R injury by inhibiting inflammatory response in rats.

Objective To explore the regulation mechanism of X box binding protein 1 (XBP1)signal transduction pathway for TNF-α and effective approach in ischemia/reperfusion (I/R) injury of liver transplantation for short hairpin RNA (shRNA) interference used to gene therapy in liver graft.Methods Male Sprague-Dawley rats were divided into three groups: the cold ischemia transfection group (CIT), the in vivo transfection group (IVT) and the control group. Experiments of orthotopic liver transplantation were performed by two cuff method. The rats in CIT were perfused with XBP1-shRNA plasmid (pSIXBP1) during cold ischemia phase, those in IVT received the equivalent volume (2 ml) of pSIIRAK 4 after portal vein inoculation, and those in the control group were not subjected to any treatment. Rats were killed at 60 or 180 min after restoring reperfusion of hepatic portal vein.Histopathological damage degree of graft liver was observed by light microscope. The expression levels of XBP1 gene and protein were detected by RT-PCR and Western blotting. The activities of NF-κB and the serum TNF-α level were detected by ELISA. Results All the indexes including the degree of histopathological damage, the expression levels of XBP1 mRNA and protein and the TNF-α level were significantly decreased in CIT as compared with IVT and control group (P＜0. 05). However,there was no significant difference in NF-κB activity among the three groups (P＞0. 05). Conclusion The role of XBP1 pathway in TNF-α gene regulation and that of NF-κB pathway in rat liver I/R injury are two relatively independent aspects, and the depression of XBP1 expression with XBP1 shRNA through portal vein perfusion during cold ischemia phase could effectively alleviate graft hepatic I/R

Objective To investigate the effect of As2 O3 in combination with cinobufacini on proliferation and apoptosis of the K562 cells and provide theoretical basis for clinical application.Methods Cell proliferation was assayed by analyzing the growth and viability of the cells.Apoptosis was assayed by performing cell morphology,Annexin-V/PI staining,DNA-PI staining,and DNA gel electrophoresis.Results After exposure to As2O3 and cinobufacini,the growth of K562 cells was inhibited and the viability of K562 cells was decreased. After treated with 1.0μmol/L As2O3,0.125μg/ml cinobufacini,0.25μg/ml cinobufacini,1.0μmol/L As2O3 + 0.125 μg/ml cinobufacini,1.0μmol/L As2O3 + 0.25μg/ml cinobufacini for 24 and 48 hours,the proliferation inhibition rate were(24 ± 1.3)%,(21 ± 1.5)%,(38 ± 3.1)%,(57 ±2.7)%,(66 ±3.3)% and(49 ±2.9)%,(48 ±2.7)%,(61 ±2.1)%,(77 ±3.8)%,(82 ±4.2)%,the apoptosis rate of K562 cells were(4.8 ± 0.5)%,(5.6 ± 0.7)%,(9.8 ± 0.6)%,(11.9 ± 1.2)%,(15.2±1.5)% and(11.0 ±0.9)%,(12.9 ±1.1)%,(18.4 ±1.5)%,(21.0 ±2.0)%,(28.0 ±1.9)%.The percentage of apoptotic cells was a time- and dose-dependent manner.Typical DNA ladder was shown by DNA gel electrophoresis.Conclusions As2O3 combined with cinobufacini inhibited the proliferation of K562 cells and induced apoptosis of the K562 cells,the combination of the two drugs had better effect.

An electrochemical sensor has been developed for the selective determination of chlortetracycline ( CTC) using the molecularly imprinted technique. A molecular imprinted polymer ( MIP) on the surface of a glassy carbon electrode ( GCE ) was prepared by electropolymerization of o-aminophenol ( OAP ) in the presence of CTC in the sodium perchlorate ( NaClO4 ) solution using cyclic voltammetry ( CV ) . The electrochemical performance of the sensor was studied by using differential pulse voltammetry ( DPV ) . A linear relationship between the peak current difference and the CTC concentration was found in the range of 2. 0×10-8-6. 1×10-7 mol/L with the detection limit of 1. 5×10-8 mol/L (3σ). After regeneration by washing with the mixture of methanol and sulfuric acid, the sensor showed excellent reproducibility and good stability. The MIP electrode exhibited almost no response to chloramphenicol and penicillin, and very weak responses to tetracycline and oxytetracycline, proving a good selectivity. Recoveries of standard addition measured in the actual samples of milk and chicken meat were between 86 . 4% -96 . 9%. Compared with the reported methods, this sensor showed a low detection limit, simple operation without derivatization, rapid response and low cost.

AIM:To investigate the roles of feeder cells ln stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet.METHODS:Using contact feeder culture,separated feeder culture,compound feeder culture and culture without feeder cells by Air-lifting method in Transwell chamber culture system,tissue engineered corneal epithelium was reconstructed.Corneal sheets were stained with hematoxylin and eosin(HE)for histological observation.The expression of p63 and keratin 19(K19)and involucrin(IVL)was investigated by immunocytochemistry analysis.RESULTS:Stratification was limited to three to four layers in the contact feeder group,whereas separate feeder sheets were slightly more stratified.The compound feeder group produced a stratified epithelium with five to seven layers of cells.The group without 3T3 feeder cells formed only two to three layers of cells.Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer,as well as differentiation marker involucrin in all layers.CONCLUSION:The remarkable stratification as well as the Iimbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.