OBJECTIVETo investigate protective effect of paeonol (Pae) on human umbilical vein endothelial cells (HUVECs)injured by hyperlipidemic serum and explore its possible mechanism.METHODS The injury model was induced by 20％ hyperlipidemic serum incubating HUVECs for 24 h.Pae 124,247 and 495.μmol· L-1 were given followed by administration of hyperlipidemic serum for 24 h.The morphological changes were observed under inverted microscope,cell survival rate was evaluated by MTT method,the nitric oxide (NO) content was measured by nitric acid reductase method and the endothelial nitric oxide synthase (eNOS) mRNA expression was determined by RT-PCR.RESULTSCompared with normal contorl group,most cells in model group split and exfoliated.However,the morphology was tending to normal level after intervention with Pae.Pae significantly improved cell viability(P ＜0.01).Compared with model group,the survival rate increased from (53.0 ±10.1 ) ％ to ( 68.4 ± 9.1 ) ％,( 84.5 ± 6.7 ) ％ and (98.1 ± 7.5 ) ％,respectively.The NO content and eNOS mRNA expression both increased greatly in Pae groups(P ＜ 0.01 ).Compared with model group,content of NO increased from 54 ± 4 to 79 ± 6,115 ± 5 and ( 136 ± 6) μmol · L- 1,respectively.The expression level of eNOSmRNA improved from 0.215 ± 0.060 to 0.451 ± 0.045,0.563 ± 0.013,0.704 ± 0.068,respectively.CONCLUSIONPae exerts protective effect on HUVECs injured by hyperlipidemic serum by increasing eNOS mRNA expression,which might therefore improve the content of NO.

OBJECTIVETo investigate effects of exogenous hydrogen sulfide ( H2 S) on the spatial memory disorder induced by cerebral anoxia in mice and explore related mechanism.METHODS Sodium nitrite (NaNO2) 120 mg·kg-1 was sc given to mice for4 d in model group.Sodium hydrosulfide (NaHS) 1 mg·kg-1 was ip given and NaNO2 120 mg·kg-1 simultaneously was sc given to mice for4 d in NaHS group.All drugs were given to mice immediately after Morris water maze experiment every day and escape latency.The number of crossings over the target area (NCTA) and search time in target quadrant (STTQ) were recorded.The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) level in the brain was determined with colorimetry.The morphological alterations in hippocampus slices were assessed by microscope.RESULTSOn the third and fourth days in Morris water maze experiment,compared with ( 16.1 ±9.6)s and ( 11.1 ±6.2)s in normal control group,the escape latency in model group was longer,(26.0 ±7.3)s(P＜0.05) and (23.3 ±8.7)s(P＜0.01).On the fifth day,compared with 7.2 ± 1.6 and (28 ± 8) s in normal control group NCTA and STTQ in model group were 4.1 ± 1.9and (20 ± 8 ) s ( P ＜ 0.05 ),and they were obviously less.Compared with normal control group,SOD activity and M DA content of mice in model group were reduced by 12.6％ (P ＜ 0.01 ) and increased by 43.9％ (P ＜ 0.01 ),respectively.The neuron degenerative changes including karyopyknosis,dark cytoplasm and irregular pyramidal layer were observed in model group.On the third and fourth day,compared with model group,the escape latency in NaHS group was shorter,(17.9 ±7.0)s and (15.8 ±8.5)s (P＜0.05).Compared with model group,NCTA and STTQ in NaHS group increased to 6.7 ± 2.5 and ( 30 ± 9) s ( P ＜ 0.01 ).SOD activity and MDA content in NaHS group were increased by 8.9％ ( P ＜ 0.05 ) and reduced by 29.6％ ( P ＜ 0.01 ),respectively.Neuron degeneration was significantly attenuated in NaHS group (P ＜ 0.01 ).CONCLUSIONNaHS can attenuate the spatial memory disorder induced by cerebral anoxia and the mechanism may be related to the antioxidation effect and alleviation of neuron damage of H2S.

OBJECTIVE To investigate whether the pulmanary fibrosis formed after a single PFIB exposure.METHODS A total of 70 male mice were exposed to PFIB 130 mg·m-3 for 5 min.Pulmonary edema of 10 mice was evaluated by lung indices at 24 h after PFIB exposure.Pathological changes and collagen deposition were detected by hematoxylin and eosin (HE) and Sirius red stainings in the other mice,changes in collagen content in lungs and plasma by measuring the respective hydroxyproline content at 2,4,6,8,12 and 16 weeks after PFIB exposure.RESULTS Severe pulmonary edema was observed at 24 h after PFIB exposure.At day 14 after PFIB exposure,inflammatory cell infiltration,alveolar septum thickening,interstitial and alveolar edema and protein leakage were noticed.Collagens types Ⅰ and Ⅲ on the wall of vessel and bronchi were severely damaged,but considerable amount of collagen type Ⅲ deposited on the alveolar wall.The content of hydroxyproline considerably decreased in the lungs but increased significantly in the plasma up to six weeks.Hydroxyproline in lungs and plasma began to recover at the end of 8 weeks,and then returned to normal.At 16 weeks,they recovered to normal level.At the end of 4 weeks,the lung lesions and the collagens at the wall of vessel and bronchi began to recover gradually; collagen typeⅢ at the alveolar wall was gradually absorbed,too.At 16 weeks,the lungs almost recovered to normal level.CONCLUSION At earlier phase after PFIB exposure,the excessive collagens destruction in lungs is observed,but no pulmonary fibrosis forms at the later phase.

OBJECTIVETo evaluate antithrombotic efficacy and preliminary pharmacokinetics of Bg115-2.METHODSProthrombin time (PT) and activated partial thromboplastin time (APTT) in vitro were determined using assay kits,respectively.The effect on venous thrombosis was evaluated with the model of inferior sinus venous thrombosis in mice.Bleeding reaction was measured by tail bleeding time test.Preliminary pharmacokinetic study was conducted using FXa activity assay.RESULTSBg115-2 (sc) 0.75 -3.0 mg·kg-1 prolonged PT (P＜0.05,P＜0.01) and APTT (P ＜ 0.01) dose-dependently. In the inferior sinus venous thrombosis model,Bg115-20.19 - 3.0 mg· kg-1 significantly reduced thrombus mass with ID50 of 0.19 mg· kg-1.Interestingly,Bg115-2 (ig) 1.5 -6.0 mg·kg-1 also significantly reduced venous thrombus mass (P ＜0.01 ).Bg115-2 was similar to nadroparin calcium in bleeding reaction,and ED2/ID50 reached up to 26.8.Furthermore,Bg115-2 3.0 mg· kg-1 displayed a pharmacokinetic character with a two-compartment model.t1/2,cmax and AUC were (6.18 + 1.45 ) h,(5.20 + 0.66) mg·L-1 and (43.75 +8.20)mg·L-1 ·h,respectively.CONCLUSIONBg115-2 is a potent and oral effective inhibitor of venous thrombosis in mice with slight bleeding side-effect.It has longer t1/2 and the distribution character of a twocompartment model.

Nonclinical safety evaluation plays a critical role in the process of new drug development.International Conference on Harmonization (ICH) guideline M3 (R2) provides a key direction for the nonclinical safety evaluation process.Proper strategies and toxicological studies should be considered together to move the drug candidates forward efficiently and quickly to support clinical plans and market registration.Updates on ICH guidelines,such as ICH S6 and ICH S9,have great impact on the direction of development.With the increasing cost of development and competition in the industry,elements like predictivity,animal models,and regulatory compliance are also very important in the process.Therefore,an insight into all these factors is essential to toxicologists in the safety evaluation process.The ability to use the overall knowledge will result in a quicker and better new drug development program.

OBJECTIVETo assess the DNA damage of copper 8-quinolinolate (CuQ) and to elucidate the plausible mechanisms.METHODSHepG2 cells were treated with CuQ0-4 μmol·L-1 for different time,DNA damage was measured by Comet assay.Catalase (CAT) activity,glutathione(GSH) level and thiobarbituric acid reactive substances (TBARS) were measured.NF-κB was examined using Western blotting.8-Hydroxydeoxyguanosine (8-OHdG) was measured by immunoperoxidase staining analysis.RESULTSCuQ 0.5 -4 μmol·L-1 caused significant increase of DNA migration in HepG2 cells.CuQ significantly decreased levels of GSH and activity of CAT in HepG2 cells (P ＜0.05).Moreover,CuQ significantly increased accumulation of the p65 subunit of NF-κB into nucleus,levels of lipid peroxidation product TBARS and the formation of 8-OHdG (P ＜0.05).The intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO),depletion of GSH in HepG2 cells pre-treated with BSO dramatically increased susceptibility of HepG2 cells to CuQ-induced DNA damage.CONCLUSIONCuQ exerts DNA damage by oxidative stress and increases accumulation of p65 subunit of NF-κB into nucleus in HepG2 cells.

Assessment of QTc prolongation is a critical step for small molecule drug development.ICH S7B continues to be the main frame to guide the assessment for this potential cardiovascular risk.The ICH guideline outlines a 3-step approach to QTc prolongation,including in vitro bERG inhibition,ex vivo action potential duration (APD),and in vivo animal telemetry approch.Dog,monkey,swine,rabbit,ferret,and guinea pig are the common laboratory animals used for in vivo electrophysiology studies,especially using conscious Beagle Dog. In addition to all these guideline standard studies,many newly developed approaches,such as receptor binding for hERG inhibition,ex vivo methods such as perfused rabbit heart or guinea pig heartare are useful models for this purpose.All these methods display good correlation to clinic outcomes,and are low cost and have rapid turn-around time in nature; so that,they can help rapidly and predict this potential cardiac liability,resulting into accelerating process for small molecule drug candidate development.

OBJECTIVE To investigate the blocking activities of a series of potential α1-adrenoceptor (α1-AR) antagonists (Compounds B1 -B9) on α1-AR.METHODS ① A series of potential α1-adrenoceptor (α1-AR) antagonists,indolylpiperidine derivative (IPD) and Compounds B1 -B9,with indolylpiperidine moiety and different substitutes were synthesized through the coupling of indolylpiperidine and piperazine derivatives.② Inotropic responses experiment was used to examine blocking effects of IPD and Compounds B1 - B9 in isolated rat atria by phenylephrine (PE) stimulation.③ Blocking effect of IPD and Compounds B1 - B9 on phosphorylation level of extracellular signal-regulated kinase (ERK) in PE treated HEK293 cells was tested by Western blotting.RESULTS ① Potential α1-adrenoceptor (α1-AR) antagonists with indolylpiperidine moiety and different substitutes were synthesized successfully.② PE caused a dose-dependent inotropic response which was inhibited by pre-incubation of phentolamine (Phen),a non-selective α1-AR antagonist,IPD and Compounds B1,B3,B4,B7,B8 and B9,respectively; IPD and Compounds B4 and B8 caused an obvious rightward shift of inotropic response-curve,the pA2 values for IPD and Compounds B4 and B8 were 6.72 ± 0.21,6.86 ± 0.29 and 6.67 ± 0.19,respectively.③ Phosphorylation level of ERK1/2 was inhibited by pre-incubation with Compounds B1,B2,B3,B5,B6,B7,B8 and B9 or IPD in PE treated α1A-AR stably expressed HEK293 cells; PE-stimulated phosphorylation level of ERK1/2 was inhibited by pre-incubation with Compounds B2,B4,B7 or B8 in α1B-AR stably expressed HEK293 cells.CONCLUSION Compound B4 has a selective blocking activity on α1B-AR,and Compounds B1,B3,B5,B6 and B9 or IPD have a selective blocking activity on the phosphorylation level of ERK1/2.

OBJECTIVE To validate the anticancer effect of Angong Niuhuang pill (AGNH) and pinpoint associated molecular mechanisms using human cancer cells.METHODS Human MGC-803 gastric carcinoma and human BEL-7402 hepatocarcinoma cells were incubated with AGNH 9,30,90,300 and 900 mg·L-1 for 24,48and 72 h,respectively.Cell viability was detected with 3-(4,5-dimethylthiazol-2-yl) -5-( 3-carboxymethoxyphe-nyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and colony formation assay.Apoptosis was measured with flow cytometry and Hoechst 33258/PI staining.Change in mitochondrial membrane potential (△qψ) was detected by spectrofluorophotometer.RESULTS AGNH inhibited MGC-803 cell proliferation ( for 48 h,r =0.996,P =0.002; for72 h,r=0.756,P=0.024 ) and BEL-7402 cells (for 48 h,r =0.732,P=0.030; for72 h,r=0.702,P =0.037) in a concentration-dependent manner,as showed by MTS assay.AGNH inhibited colony formation on MGC-803 cells (r =0.914,P =0.011 ) and BEL-7402 cells (r =0.871,P =0.024) in a concentration-dependent manner for 24 h.Hoechst 33258/PI staining and flow cytometry assay showed that AGNH 900 mg·L-1 for 24 h induced apoptosis of MGC-803 and BEL-7402 cells,and the apoptosis rate was 27.2％ and 19.7％,respectively.Compared with normal control group,AGNH 900 mg·L-1 for 3 min decreased the mitochondrial membrane potential of MGC-803 and BEL-7402 cells to 15.9％ and 15.0％ of control group.CONCLUSION AGNH inhibits proliferation of human cancer cells.Apoptosis and depolarization of mitochondrial transmembrane potential are probablly its mechanism.

OBJECTIVE To investigate the nuclear proteomes in mitochondrial DNA (mtDNA)-depleted A549 cells (Rho0 cells) and their parental cells (Rho+ cells),and to learn more about the nuclear responses to mitochondrial dysfunction.METHODS The nuclear proteomes of Rho and Rho + cells were characterized by two dimensional electrophoresis (2-DE) and SELDI-TOF ProteinChip technologies,the differentially expressed protein- spots were identified by MALDI-TOF mass spectrum (MS),the nucleophosmin and P53 expression were detected by Western blotting assay,and the mitochondrial memhrane potential (MMP) was measured by the laser scanning confocal microscope.RESULTS 2-DE results showed 11 protein-spots were down-regulated and 21 protein-spots were up-regulated in Rho0 cell nuclei.SELDI-TOF MS analysis with NP20 ProteinChips revealed 4 protein-peaks decreased in Rho0 cell nuclei.One down-regulated protein-spot was identified as elF-6,and 4 up-regulated proteinspots were identified as nucleophosmin,SFRS1,SFRS3 and hnRNP G,respectively.The increased expression of nucleophosmin in Rho0 cells was verified by Western blotting.Based on the clues from proteomic analysis,P53 expression in Rho0 cells was higher than in Rho + cells,and MMP was consistent in Rho + and Rho0 cells.CONCLUSION mtDNA-depletion induces nuclear proteome alteration.Rho0 cells can be used as a model to study the crosstalk between mitochondrion and nucleus.