Objective To study the protective effect and Bcl-2 expression of salvia miltiorrhiza pretreatment on retinal ischemia-reperfu-sion injury ( RIRI) . Methods One hundred and thirty two Wistar rats were randomly divided into the normal control group, the ischemia-reperfusion group and the salvia miltiorrhiza pretreatment group. The model of retinal ischemia-reperfusion injury was constructed by increas-ing the intraocular pressure. The ischemia-reperfusion and salvia miltiorrhiza pretreatment group were divided into five subgroups according to the different reperfusion time (6 h, 12 h, 24 h, 48 h and 72 h). Observe the histological changes in retina by HE staining. The SABC ( strept avidin-biotin complex) and Western-blot were used to measure changes of Bcl-2 protein levels in retinal. Results The positive ex-pression of Bcl-2 protein was weak in normal group. In the ischemia-reperfusion group and salvia miltiorrhiza pretreatment group, the expres-sion of Bcl-2 protein began to increase at 6 hours after reperfusion, reached the peak at 24 hours after reperfusion, began to decrease at 48 hours after reperfusion, and started to weaken at 72 hours after reperfusion. The variation tendency of the two groups were the same, however, the expression of Bcl-2 was significantly stronger in the salvia miltiorrhiza pretreatment group compared with ischemia-reperfusion group, and there was significant difference between the two groups (P<0. 01). Conclusion Salvia miltiorrhiza pretreatment can protect the retina by reducing retinal ganglion cells apoptosis in retinal ischemia-reperfusion injury. The mechanism may be achieved by regulating the expression of Bcl-2 protein.

Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods:The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was transfected into SP2/0 cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Western blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-? of spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results:RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P

Objective:To research on protective immunity of omph DNA vaccine against avian Pasteurella multocida in mice.Methods: The omph gene fragment amplified by PCR from avian Pasteurella multocida was cloned into pMD18-T.Subsequently it was subcloned into the eukaryotic expression vector pcDNA3.1(+),and the recombinant plasmid pOMPH was obtained.Then the recombinant plasmid was trans fected into SP2/O cells in vitro.The transcription and expression of target gene were analyzed by RT-PCR,Westem blot analysis and indirect immunofluorescence.Three groups of BALB/c mice(n=16) named pOMPH,pCDNA3.1(+) and PBS were intramuscularly vaccinated with the recombinant plasmid,control vector and PBS respectively.The serum antibodies were detected by indirect ELISA.The spleen lymphocyte proliferation (SLP) and secreted IFN-γof spleen were tested by MTT.The mice were challenged with virulent of avian Pasteurella multocida on week 2 post the third immunization,the protection rate were counted.Results: RT-PCR,Western blot analysis and indirect immunofluorescence showed that the omph gene could be,transfected into SP2/0 cells in vitro and expressed the target protein.Indirect ELISA showed that the levels of antibodies in pOMPH group were most significantly higher than in the other groups(P＜0.01).Spleen lymphocyte proliferation by MTT assay indicated that the SI value induced with avian Pasteurella multocida Omps in pOMPH group was higher than those in pCDNA3.1 (+) and PBS groups (P＜0.05).The IFN-γexperiments(Double-antibodies-sandwich-ELISA)showed that the levels of IFN-γ induced with Omps in the group of pOMPH was mostly higher than in the other control groups apperent(P＜0.01 ).The protection rate of pOMPH(70%) was better than in the other groups.Conclusion: The omph DNA vaccine against avian Pasteurella multocida had been constructed successfully.The DNA vaccine could enhance the immunity level and the protective effect of the vaccinated mice.Present study may be useful for the development of avian Pasteurella multocida vaccine.

Objective To analyze the management and outcomes of patients with ST-segment elevation myocardial infarction (STEMI) in Liaoning province.Methods The data were collected from a prospective and multicenter registry study including 8 tertiary hospitals and 12 secondary hospitals in Liaoning province.Total 1429 patients with acute STEMI admitted to hospitals from June 2009 to June 2010 were included in the study.A unified follow-up questionnaire was applied on patient discharged.Results The average age of patients was (63 ± 13)years.37.4％ of patients recognized the disease as heart disease and 39.7％ were transported by emergency ambulance with a median symptom-to-door time of 150 min.52.9％ patients underwent emergency reperfusion therapy,including fibrinolytic therapy (24.4％) and primary percutaneous coronary intervention (PCI,28.1％).The in-hospital treatment included aspirin (99.6％),clopidogrel (81.9％),statins (90.1％),low molecular weight heparin (89.5％),β-blocker (66.0％),angiotensin converting enzyme inhibitor (ACEI)/ angiotensin receptor blocker (ARB)(66.6％).The in-hospital mortality was 10.7％ ; the mortality in females was higher than that in males (18.3％ vs.7.9％,P ＜ 0.01) and the mortality in older patients (≥ 65 years) was higher than that in younger patients (＜65 years)(17.0％ vs.5.2％,P ＜0.01).The follow-up treatment included:aspirin (81.1％),clopidogrel (45.0％),statins (61.0％),β-blocker (48.3％),ACEI/ARB (42.4％).The follow-up mortality was 5.0％ after hospital discharge.Conclusions Longer pre-hospital delay is commonly seen in STEMI patients.There is still certain gap of emergency reperfusion therapy and the evidence-based medication with related clinical guidelines of STEMI management in Liaoning.

Objective:To investigate the effect of mitochondrial division of GABAergic neurons in substantia nigra pars reticulata(SNr)on motor dysfunction in mice with acute hepatic encephalopathy(AHE).Methods:AHE mice model was established by intraperitoneal injection of thioacetamide(TAA).The changes of liver lobules in AHE mice were observed by hematoxylin-eosin(HE)staining.The changes of serum aspartate aminotransferase(AST),alanine aminotransferase(ALT)and blood ammonia in AHE mice were detected by biochemical detection kit.Then,the motor function of AHE mice was observed by rod fatigue test,elevated cross maze test and open field test.Furthermore,the changes of mitochondrial area,perimeter,roundness and other morphological indicators in SNr of AHE mice were ob-served and analyzed by transmission electron microscopy.The expression of mitochondrial division and fusion related molecules in SNr of AHE mice was observed by Western Blot.Then,the expression of mitochondrial dynamic related protein 1(DRP1)in SNr of AHE mice was targeted by AAV virus.The mitochondrial membrane potential(MMP),ATP and reactive oxygen species(ROS)in SNr were detected by fluorescence enzyme marker,and the changes of motor function of mice were observed.Results:Compared with the control group,the motor function of AHE mice was signifi-cantly decreased,the mitochondrial division of SNr was significantly enhanced,and the expression of mitochondrial divi-sion related proteins was significantly increased.The MMP in SNr of AHE mice was significantly decreased,the ATP of cells was decreased,and the ROS was increased.After targeted inhibition of DRP1 expression in SNr of AHE mice,the movement was improved;further observation found that after the mitochondrial division in SNr of AHE mice was inhibi-ted,the MMP was significantly increased,the ATP of cells was increased,and the ROS was decreased,which demon-strated that the mitochondrial function was significantly improved.Conclusion:Targeted inhibition of mitochondrial di-vision of GABAergic neurons in SNr of AHE mice can improve mitochondrial morphology and function,thus alleviating their movement disorders.

OBJECTIVE：To stud y the effects of different processed products of Whitmania pigra on hemorheology and coagulation indexes in acute blood stasis model rats. METHODS ：SD rats were randomly divided into blank group ，model group ， aspirin group ，W. pigra hang-dried product low- ，medium- and high-dose groups ，W. pigra talcum powder-ironed product low- ， medium- and high-dose groups ，W. pigra wine bran-processed product low- ，medium- and high-dose groups ，with 6 rats in each group. Except for blank group ，other groups received subcutaneous injection of epinephrine hydrochloride and ice water bath for 15 d to induce acute blood stasis model. From the 8th day of modeling ，rats in aspirin group were given aspirin 0.2 g/kg intragastrically. Rats in each dose group of W. pigra processed products were given relevant medicine 0.35，1.4，3.5 g/kg intragastrically（calculated by crude drug ）. Rats in blank group and model group were given constant volume of normal saline intragastrically， once a day ， for consecutive 8 days. Hemorheology indexes as whole blood viscosity （high， medium and low shearrate ），plasma viscosity ，erythrocyte com deformation index ，erythrocyte aggregation index ，hematocrit， and blood coagulation indexes as prothrombin time （PT）， mail：wcl19960125@163.com activated partial prothrombin time （APTT），thrombin time （TT）were determined. RESU LTS：Compared with blank group ，whole blood viscosity under different shear rates ，plasma viscosity ， erythrocyte aggregation index and hematocrit of model group were increased significantly ，while erythrocyte deformation index was significantly decreased ，PT，TT and APTT were significantly shortened （P＜0.01）. Compared with model group ，whole blood viscosity under different shear rates ，plasma viscosity ，erythrocyte aggregation index and hematocrit of aspirin group and W. pigra hang-dried product ，talcum powder-ironed product ，wine bran-processed product high-dose groups were decreased significantly ， while erythrocyte deformation index were significantly increased ，and PT （only W. pigra talcum powder-ironed products high-dose group），APTT（except for W. pigra hang-dried products high-dose group ）and TT were prolonged significantly. The whole blood viscosity of W. pigra hang-dried product medium-dose group under low shear rate ，and those of W. pigra talcum powder-ironed product low-dose ，wine bran-processed product medium-dose groups under low and medium shear rates were decreased significantly. Erythrocyte deformation index of W. pigra talcum powder-ironed product medium-dose group was increased significantly ，while erythrocyte aggregation index was decreased significantly ，and PT ，TT were prolonged significantly. APTT of W. pigra hang-dried product medium-dose group was prolonged significantly. Hematocrit of W. pigra wine bran-processed product low-dose group was decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ： W. pigra hang-dried， talcum powder-ironed and wine bran-processed product can effectively improve hemorheology indexes and prolong blood coagulation time.

OBJECTIVE：To develop a method for simultaneous determination of 5 components in classical formula Huaihua san，including rutin ，naringin，neohesperidin，quercetin and pulegone. METHODS ：HPLC wavelength switching method was adopted. The determination was performed on Cosmosil C 18 column with mobile phase consisted of acetonitrile- 0.05％ phosphoric acid solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelengths were set at 257 nm for rutin ，283 nm for naringin and neohesperidin ，254 nm for quercetin ，252 nm for pulegone ，respectively. The column temperature was set at 30 ℃， and sample size was 10 μL. RESULTS：The linear range was 21.7-2 170 μg/mL for rutin，46-4 600 μg/mL for naringin，22.3- 2 230 μg/mL for neohesperidin，0.96-96 μg/mL for quercetin，2.7-270 μg/mL for pulegone（all r＞0.999），respectively. RSDs of precision，stability（24 h）and reproducibility tests were all lower than 2％（n＝6）. Average recoveries were 100.70％，99.31％， 101.10％，100.03％ and 99.63％（all RSD ＜2％，n＝9）. Among 3 batches of Huaihua san samples ，the contents of above 5 components were 20.055-22.615，25.557-27.806，11.428-13.250，0.350-0.478，2.372-4.011 mg/g，respectively. CONCLUSIONS ： Established method is simple ，accurate and reproducible ，and could be used for the simultaneous determination of 5 components in Huaihua san.

Objective : To investigate the characteristic genes of Programmed cell death protein 1 ( PD-1) immuno- therapy sensitivity in Hepatocellular carcinoma ( HCC) ． Methods : The common differential genes in GSE202069 and ERP117672 data sets were investigated by Weighted Gene Co-expression Network Analysis ( WGCNA) and Difference analysis，and the characteristic genes of PD-1 immunotherapy sensitivity were screened through Lasso regression．The expression levels of characteristic genes in HCC were predicted by GEPIA and Ualcan databases， and their expression was verified by real-time quantitative reverse transcription polymerase chain reaction ( RT- qPCR) ，Western blot (WB) and Immunohistochemistry (IHC) ．3-hydroxybutyrate dehydrogenase 1 (BDH1) over- expressed cell line was constructed，followed by cell counting kit-8 ( CCK-8) ，EdU，cell scratches and Transwell experiment to investigate the effects of BDH1 on the proliferation，migration and invasion of HCC cells. Results: Total of 118 common differentially expressed genes were identified in two datasets by WGCNA and differential anal- ysis．The characteristic genes associated with PD-1 immunotherapy sensitivity screened through Lasso regression in- cluding Flavin containing dimethylaniline monoxygenase 3 ( FMO3 ) ，Peroxisomal trans-2-enoyl-CoA reductase (PECR) ，BDH1 ，Solute carrier family 7 member 1 ( SLC7A1 ) ，Cytochrome b5 type A ( CYB5A) and Phos- phoenolpyruvate carboxykinase 1 (PCK1) ． Survival analysis showed that BDH1 was most associated with HCC ( Overall survival : P＜0. 001，Recurrence : P = 0. 007) ．GEPIA and Ualcan databases showed low expression of BDH1 in HCC tissues，while RT-qPCR , WB，and IHC further confirmed this．CCK-8，plate cloning assay，EdU staining，cell scratch，and Transwell experiments showed that compared with the Hep3B pCDH group，overexpres- sion of BDH1 resulted in a decrease in the absorbance of HCC cells (t = 4. 766，P＜0. 01) ，a decrease in the num- ber of clone formation (t = 16. 02，P＜0. 000 1) ，a decrease in the proportion of proliferating cells (t = 23. 13，P ＜0. 000 1) ，a decrease in cell migration rate (t = 25. 28，P＜0. 000 1) ，and a decrease in the number of small compartments (t = 10. 78，P = 0. 004) ． Conclusion BDH1 is a characteristic gene for observing the sensitivity of PD-1 immunotherapy in HCC patients． BDH1 could inhibit the proliferation ，migration ，and invasion ability of HCC cells in vitro.

Ultra-performance liquid chromatography-quadrupole-electrostatic field Orbitrap mass spectrometry(UHPLC-Q-Exactive Orbitrap MS/MS) was used for rapid identification of the chemical components in Kaixin San substance benchmark. The gradient elution was performed through a Waters ACQUITY~(TM) BEH C_(18) column(2.1 mm×150 mm, 1.7 μm) with water-acetonitrile as mobile phase, a column temperature of 30 ℃, a flow rate of 0.3 mL·min~(-1), and a sample size of 1 μL. The scanning was performed in the negative ion mode. The complex component groups in Kaixin San substance benchmark were quickly and accurately identified and clearly assigned based on the comparison of the retention time and MS data with those of the reference substance as well as the relative molecular weight of the same or similar components in the mass spectrum database and literature. A total of 77 compounds were identified, including 26 saponins, 13 triterpenoid acids, 20 oligosaccharide esters, 5 xanthones, and 13 other compounds. The qualitative method established in this study can systematically, accurately, and quickly identify the chemical components in Kaixin San substance benchmark, which can provide a basis for the further analysis of its active components in vivo and the establishment of its quality control system.
Benchmarking ; Chromatography, High Pressure Liquid/methods* ; Drugs, Chinese Herbal/chemistry* ; Tandem Mass Spectrometry/methods*