Objective To explore the characteristics of mycobacterium dependend antituberculous drug. Methods 137 strains of mycobacterium were tested for resistance by the absolute concentration method on Lowenstein Jensen medium. Dependent positive were the coloun that on higher concentration drug medium stronger than lower concentration than controls. Results 12(8.76%) of the 130 strains M. tuberculosis and 7 strains nontuberculous mycuberculous (NTM) were found dependent, in which strains dependend on INH, RFP and SM were 3(2.19%), 10(7.3%) and 3(2.19%) respectively, 4(2.92%) were dependend on two drugs. Conclusions Were NTM dependent, Not found all detected from multiple strains.

Objective:To investigate the effect of different ischemic preconditioning (IPC) modes on ischemia-reperfusion (IR) injury of skeletal muscle in rats.Methods:Forty male SD rats were selected to construct the model of IR injury of skeletal muscle by clamping one side of the femoral artery. In the IPC mode, the right femoral artery was clipped for 10 minutes, and reperfusion ensued for 10 minutes after the release of artery clamp. Such preconditioning procedure was repeated 3 times. The rats were divided into conventional IR group, IPC immediate group, IPC24-hour group, IPC48-hour group and sham operation group according to the random number table method, with 8 rats in each group.In conventional IR group, the right femoral artery was clipped for 3 hours and then the artery clamp was released to allow reperfusion for 3 hours. In IPC immediate group, the same treatment as conventional IR group was performed immediately after preconditioning. In IPC24-hour group, the rats received skin suturing after preconditioning and then fed in a cage for 24 hours before the same treatment as conventional IR group. In IPC48-hour group, the rats received skin suturing after preconditioning and then fed in a cage for 48 hours before the same treatment as conventional IR group. In sham operation group, the right femoral artery was bluntly separated but not clipped. At the end of reperfusion, the tibialis anterior muscle tissue, gastrocnemius muscle tissue and serum were collected. The ratio of wet weight to dry weight (W/D) of the tibialis anterior muscle tissue was calculated to evaluate tissue edema. After HE staining, histopathological changes of gastrocnemius muscle were observed and the injury severity of gastrocnemius tissue was scored. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and malondialdehyde (MDA) were determined by ELISA. Expressions of hypoxia receptor (EGLN1) and hypoxia-inducible factor-1α (HIF-1α) in the gastrocnemius tissue were detected by real-time fluorescence quantitative PCR (qRT-PCR).Results:The degree of tissue edema was reduced under different IPC modes when compared with conventional IR group (all P<0.01). The W/D in conventional IR group, IPC immediate group, IPC24-hour group and IPC48-hour group were 6.05±0.19, 5.70±0.12, 5.25±0.13 and 5.50±0.08, higher than 3.80±0.08 in sham operation group (all P<0.01). IPC24-hour group had the least degree of edema when compared with IPC immediate group and IPC48-hour group ( P<0.05 or 0.01). The muscle fibers in sham operation group were neatly arranged with clear structure, and the muscle fibers in other groups showed different degrees of injury and inflammatory infiltration. The total scores for tissue injury severity in IPC immediate group, IPC24-hour group and IPC48-hour group were (8.15±0.15)points, (6.15±0.38)points and (6.90±0.19)points, lower than (9.60±0.50)points in conventional IR group and higher than (0.16±0.16)points in sham operation group (all P<0.01). The total scores for tissue injury severity in IPC24-hour group and IPC48-hour group were not significantly different ( P>0.05), but both decreased when compared with IPC immediate group ( P<0.05 or 0.01). ELISA showed that the levels of TNF-α, IL-1β and MDA under different IPC modes were higher than those in sham operation group (all P<0.01), and the order from high to low was conventional IR group, IPC immediate group, IPC48-hour group, IPC24-hour group and sham operation group. Levels of IL-1β, TNF-α and MDA in IPC24-hour group were decreased when compared with IPC immediate group and IPC48-hour group (all P<0.05). qRT-PCR showed that the expression of EGLN1 mRNA from high to low was conventional IR group, IPC48-hour group, sham operation group, IPC immediate group and IPC24-hour group and that the expression of EGLN1 mRNA in IPC24-hour group decreased when compared with conventional IR group, IPC immediate group and IPC48-hour group (all P<0.01). The expression of HIF-1α mRNA increased under different IPC modes when compared with sham operation group (all P<0.01), and the order from high to low was IPC24-hour group, IPC immediate group, IPC48-hour group, conventional IR group and sham operation group. The expression of HIF-1α mRNA in IPC24-hour group increased when compared with conventional IR group, IPC immediate group and IPC48-hour group ( P<0.05 or 0.01), but there was no significant difference between IPC immediate group and IPC48-hour group ( P>0.05). Conclusions:Different ischemic preconditioning modes can reduce IR injury of skeletal muscle in rats by reducing tissue edema, inflammatory symptoms and oxidative stress response, among which reperfusion 24 hours after IPC has the best effect on IR injury. EGLN1 and HIF-1α may be involved in IPC to alleviate IR injury of skeletal muscle in rats.

OBJECTIVE： To establish content determination method for simultaneously determining aucubin，geniposidic acid，catechin，chlorogenic acid，asperuloside，rutin，isoquercitrin and astragalin in Eucommia ulmoides leaves. METHODS：HPLC method was adopted. The determination was performed on Agilent ZORBAX SB-C18 column with the mobile phase consisted of acetonitrile-0.1％ phosphoric acid（gradient elution） at the flow rate of 1.0 mL/min. The detection wavelength was set at 203 nm for aucubin and catechin，239 nm for geniposidic acid and asperuloside，220 nm for chlorogenic acid，354 nm for rutin and isoquercitrin，and 266 nm for astragalin. The sample size was 5 μL. RESULTS： The linear range of aucubin， geniposidic acid， catechin， chlorogenic acid， asperuloside， rutin， isoquercitrin and astragalin were 0.812-6.090 μg（r＝0.999 3），0.438-3.285 μg（r＝0.999 2），0.045-0.336 μg（r＝0.999 2），0.882-6.615 μg（r＝0.999 3），0.097-0.726 μg（r＝0.999 1），0.064-0.483 μg（r＝0.999 3），0.048-0.360 μg（r＝0.999 1） and 0.014-0.108 μg（r＝0.999 7），respectively. RSDs of precision， stability and reproducibility tests were all lower than 3.5％（n＝6）. The average recovery rates were 101.60％，103.06％，99.77％，96.93％，98.17％，96.75％，98.97％ and 99.60％，with RSDs of 1.42％，2.65％，2.78％，2.05％，2.26％，0.93％，2.79％ and 3.08％，respectively（n＝6）. The contents of aucubin， geniposide， catechin， chlorogenic acid， plantain， rutin， isoquercetin and astragaloside in 12 batches of E. ulmoides leaves from different collection time and planting varieties were 10.903-17.245， 5.578-7.892， 0.198-0.440， 13.890-19.782， 1.008-1.547， 1.102-2.396， 0.267-0.701， 0.150-0.412 mg/g， respectively. The content fluctuated greatly. The contents of aucubin， geniposide， catechin， chlorogenic acid， rutin and pinoresinol diglucoside in cortex of E. ulmoides were 0.299， 0.123， 0.580， 0.112， 0.026，1.961 mg/g， respectively. CONCLUSIONS： The method is simple， reproducible and accurate. It can be used to evaluate the quality of E. ulmoides leaves. There are obvious differences in composition and content of components in different medicinal parts （cortex， leaves） of E. ulmoides.

ObjectiveTo explore the role of saikosaponin D （SSD） targeting signal transducer and activator of transcription 3 （STAT3） in inducing apoptosis of bladder cancer cells by computer-aided drug design and experimental verification. MethodThe druggability and biotoxicity of SSD were explored by Bayesian classifier modeling. The information about SSD， the active ingredient of Bupleuri Radix， was searched against the Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform （TCMSP）. The targets of SSD were predicted by PubChem， TCMSP， a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM）， Coremine， an Encyclopedia of Traditional Chinese Medicine （ETCM）， and SwissTargetPrediction. GeneCards， Therapeutic Target Database （TTD）， and Online Mendelian Inheritance in Man （OMIM） were employed to predict the potential therapeutic targets of bladder cancer. Then， the common targets shared by SSD and bladder cancer were selected for Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analyses. Molecular docking was adopted to explore the binding affinity and structural stability of SSD with target proteins. Cytoscape 3.9.1 was used to construct the STAT3-drug regulatory network and STAT3-apoptosis regulatory network. UM-UC-3 cells were treated with 0， 5， 10， 15 μmol·L^-1 SSD for 24 h. Then， flow cytometry was used to detect the apoptosis of bladder cancer cells， and Western blot was employed to determine the protein levels of B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， Bcl-2-associated death promoter （Bad）， STAT3， and phosphorylation （p）-STAT3. ResultBayesian classifier modeling and molecular docking showed that SSD had low biotoxicity and bound well to the target protein STAT3 to form a stable protein-ligand complex. There were 282 common targets between bladder cancer and SSD， among which STAT3 was the most central target. The GO enrichment analysis showed that the potential core therapeutic targets involved 3 036 biological processes， 82 cellular components， and 171 molecular functions. The KEGG enrichment analysis showed that the potential core targets were mainly related to the C-type lectin receptor signaling pathway， Toll-like receptor signaling pathway， and cell apoptosis pathway. The STAT3-drug regulatory network and STAT3-apoptosis regulatory network showed that 29 drugs interacted with STAT3， and 27 apoptosis-related genes had a strong correlation with STAT3. Flow cytometry showed that the apoptosis rate increased with the increase in SSD concentration （P<0.05）. Western blotting results showed that SSD down-regulated the protein levels of p-STAT3 and Bcl-2 and up-regulated the protein levels of Bax and Bad in a concentration-dependent manner （P<0.05）. ConclusionSSD has good druggability and low biotoxicity. It may promote the apoptosis of bladder cancer cells by targeting STAT3.

Objective To express the monkeypox virus (MPXV) A23R protein in Escherichia coli and purify by Ni-NTA affinity column, and to prepare mouse antiserum against MPXV A23R. Methods The recombinant plasmid pET-28a-MPXV-A23R was constructed and transformed into Escherichia coli BL21 to induce the expression of A23R protein. After optimizing the conditions of expression, A23R protein was highly expressed. Recombinant A23R protein was purified by Ni-NTA affinity column and identified by Western blot analysis. The purified protein was used to immunize mice for preparing the A23R polyclonal antibody, and the antibody titer was detected by ELISA. Results The expression of A23R recombinant protein reached the peak under the induced conditions of 0.6 mmol/L isopropyl-β-D-thiogalactoside (IPTG), 37 DegreesCelsius and 20 hours. The purity of the protein was about 96.07% and was identified by Western blot analysis. The mice were immunized with recombinant protein, and the titer of antibody reached 1:102 400 at the 6th week after immunization. Conclusion MPXV A23R is expressed highly and purified with a high purity and its antiserum from mouse is obtained with a high titre.
Animals ; Mice ; Monkeypox virus ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Recombinant Proteins ; Escherichia coli/genetics*