Effect of Fufang Huangbai Fluid Paint on Virulence and Biofilm of Methicillin-resistant Staphylococcus aureus / 中国实验方剂学杂志

ObjectiveTo study the virulence and biofilm inhibition effect of Fufang Huangbai Fluid Paint （FFHBFP） on methicillin-resistant Staphylococcus aureus （MRSA）， and to explore the antibacterial effect of FFHBFP on MRSA， which provides a theoretical basis and reference for clinical medication. MethodFirstly， the microdilution method and time–growth curve were used to determine the minimum inhibitory concentration （MIC） of FFHBFP and vancomycin （VAN） against MRSA and the effect on bacterial growth. The effects of FFHBFP and VAN on the inhibition of MRSA virulence factor lipase and restoration of hydrogen peroxide （H2O2） sensitivity were detected under sub-minimum inhibitory concentration （sub-MIC）. The inhibitory effect of FFHBFP and VAN on MRSA biofilm formation and maturation was detected by the microplate method. The morphological changes of mature biofilms before and after administration were observed under a scanning electron microscope （SEM）. Real-time polymerase chain reaction （Real-time PCR） was utilized to detect the effect of 50.600 g·L-1 concentration of FFHBFP on the expression of MRSA virulence gene crtM and biofilm-forming genes fnbA and icaA. Finally， molecular docking technology was used to predict the mechanism of potential antibacterial active ingredients of FFHBFP in inhibiting the virulence and biofilm of MRSA. ResultThe MIC of VAN was 2 mg·L-1， and VAN below 1 mg·L-1 exerted no effect on MRSA growth. The MIC of FFHBFP was not determined， while the 101.200-202.400 g·L-1 original solution inhibited MRSA growth. Compared with the blank group and the VAN group， sub-MIC （25.300-50.600 g·L-1 original solution） inhibited lipase and recovered MRSA sensitivity to H2O2 （P<0.01）. The results of the microplate method showed that FFHBFP （25.300-202.400 g·L-1 original solution） inhibited biofilm formation and maturation （P<0.05， P<0.01）. The SEM exhibited that FFHBFP made the structure of biofilm loose and the size of the bacteria varied. FFHBFP at 50.600 g·L-1 concentration can inhibit the expression of related virulence genes and biofilm-forming genes （P<0.05， P<0.01）， and molecular docking results also showed that the main antibacterial active ingredients in FFHBFP have good binding ability to the target. ConclusionFFHBFP that cannot directly kill MRSA exerts clinical efficacy by impairing virulence expression， biofilm formation， and other pathogenic properties.

Analysis of Inhibitory Mechanism of Reyanning Mixture Combined with Linezolid Against MRSA and Its Biofilm Based on Metabonomics / 中国实验方剂学杂志

Objective:To investigate the inhibitory effects and mechanism of Reyanning mixture （RYN） combined with linezolid （LNZ） against methicillin-resistant <italic>Staphylococcus aureus</italic> （MRSA） and its biofilm. Method:The minimum inhibitory concentrations （MICs） of RYN and LNZ against MRSA were determined by microdilution assay. The microplate method was used to detect the changes in viable count before and after MRSA administration at four time points （0， 6， 12， 24 h） in the process of biofilm growth. The morphological changes of MRSA after 24 h were observed by scanning electron microscope. Metabonomic technique was applied to analyze the changes in terminal metabolites of endogenous small molecules from MRSA treated by the two drugs at four time points. Result:The MICs of RYN and LNZ were 1/2 of the stock solution concentration and 4 mg·L^-1， respectively. The inhibitory effect of LNZ （2 mg·L^-1） against viable bacteria at 0 h was better than that of 1/16 RYN. At 6， 12， 24 h， 1/16 RYN was superior to LNZ in inhibiting MRSA. The inhibitory effects of RYN combined with LNZ were better than those of RYN or LNZ alone at the four time points. RYN combined with LNZ caused more severe damages to the morphological structure of MRSA biofilm at 24 h than RYN or LNZ alone. Cyclic adenosine monophosphate （cAMP）， adenosine diphosphate （ADP）-<italic>D</italic>-ribose and 2-methylbutanoyl-coenzyme A （2M-CoA）， as the metabolites related to biofilm formation， were immune to LNZ， but 2M-CoA and ADP-<italic>D</italic>-ribose were influenced by RYN at 12 h and 24 h. The combined use of RYN and LNZ interfered with the three metabolites at 24 h. <italic>L</italic>-tryptophan， phenylpyruvic acid， cytidine and sebacic acid were the pharmacometabolic markers of LNZ， and the related biological pathways were phenylalanine， tyrosine and tryptophan biosynthesis and phenylalanine metabolism. Four metabolites such as<italic> L</italic>-histidine， uric acid， and <italic>L</italic>-lysine were the pharmacometabolic markers of RYN， with phenylalanine metabolism and aminoacyl-transfer ribonucleic acid （tRNA） biosynthesis confirmed as the related biological pathways. Nine metabolites such as <italic>L</italic>-tryptophan，<italic> L</italic>-lysine， and sphingosine-1-phosphate were responsible for the efficacy of RYN combined with LNZ. The related biological pathways involved aminoacyl-tRNA biosynthesis， phenylalanine， tyrosine and tryptophan biosynthesis， novobiocin biosynthesis， and tyrosine metabolism. Conclusion:RYN combined with LNZ better exerts the inhibitory effects against MRSA at each time point of its biofilm formation， which is attributed to cAMP metabolism. The synergistic effect resulted from aminoacyl-tRNA biosynthesis and phenylalanine， tyrosine and tryptophan biosynthesis. RYN combined with LNZ can serve as a potentially effective solution to MRSA infection.

Study on Sub-minimal Inhibitory Concentration of Tanreqing Injection in Inhibiting Hemolytic Activity of Staphylococcus aureus α-hemolysin / 中国实验方剂学杂志

Objective:To study whether Tanreqing injection （TRQ） can alleviate the body injury in the process of infection by inhibiting the production and release of <italic>α</italic>-hemolysin of <italic>Staphylococcus aureus</italic> under sub-minimal inhibitory concentration， and to provide experimental basis for better guidance of clinical medication. Method:The effects of TRQ on the minimum inhibitory concentration （MIC） and bacterial growth of <italic>S.aureus</italic> were determined firstly by microplate method and time-growth curve. The different sub-minimal inhibitory concentrations of TRQ were co-cultured with bacteria or bacterial supernatants， and then co-incubated with defibrillated rabbit blood to detect the inhibitory and neutralizing effects of TRQ on <italic>S.aureus</italic> <italic>α</italic>-hemolysin. Cell counting kit-8 （CCK-8） cell viability assay was used to detect the protective effect of TRQ on <italic>S. aureus</italic>-mediated damage to human alveolar epithelial cells （A549）. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to detect the effect of sub-minimal inhibitory concentration of TRQ on the mRNA expression of <italic>S.aureus</italic> <italic>α</italic>-hemolysin regulatory genes hla and agrA. Result:The MIC of TRQ to <italic>S.aureus </italic>was 1/8 of the stock solution， and the sub-minimal inhibitory concentration （1/64MIC-1/16MIC） TRQ used in this study did not affect the growth of bacteria. 1/64MIC-1/16 MIC TRQ had the effect of inhibiting and neutralizing the hemolytic activity of <italic>α</italic>-hemolysin， with a protective effect on <italic>S.aureus</italic> supernatant-mediated A549 cell damage， and its inhibitory effect on <italic>α</italic>-hemolysin was closely related to the inhibition of hla and agrA mRNA expression. Conclusion:The sub-minimal inhibitory concentration TRQ can inhibit and neutralize the hemolytic activity of <italic>α</italic>-hemolysin of <italic>S.aureus</italic>， with a protective effect on A549 cell damage mediated by <italic>S.aureus</italic> infection， and its mechanism of inhibiting <italic>α</italic>-hemolysin is closely related to the interference with agr regulatory system.

Effect of Tanreqing Injection and Antibiotics on Multi-drug Resistant Pseudomonas aeruginosa Efflux Pump / 中国实验方剂学杂志

Objective:To study the interaction between Tanreqing injection and commonly used antibiotics against multi-drug resistant Pseudomonas aeruginosa (MDR-PA) and the effect on bacterial efflux pump. Method:Antibiotic susceptibility test was performed with bacteria. Paper diffusion method (Kirby-Bauer, KB) combined with efflux pump inhibitor (50 mg·L-1) was used to measure the diameter of the inhibition zone, and real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect gene expression of efflux pump Positive efflux pump strain. KB method was used to observe the changes of Tanreqing (final concentration 3 g·L-1) and antibiotics on the diameter of the zone of inhibition. Strains were co-cultured with Tanreqing and antibiotic sub-inhibitory concentrations for Real-time PCR detection. KB method was used to observe the effect of Tanreqing on the diameter of bacteriostatic ring after the continuous use of efflux pump-positive bacteria. Result:Two MDR-PA efflux pump-positive strains were identified and screened. Tanreqing has synergistic antibacterial effect with aloxicillin, aztreonam, meropenem, ceftazidime, cefoperazone, and Shupushen. In inhibiting the expression levels of bacterial efflux pump genes, the four drugs were compared by the effect: cefoperazone>Tanreqing>ceftazidime>Shupushen. After Tanreqing continued to act on efflux pump-positive strains, it could have a better effect in combination with ceftazidime, cefoperazone, and Shupushen. Conclusion:Tanreqing, ceftazidime, cefoperazone, and Shupushen can reduce the drug resistance of bacteria by down-regulating the expressions of bacterial efflux pump genes, and reducing the clinical dose of antibiotics, and thus play a bacteriostatic effect.

Establishment of a New Evaluation Method for Identification of Drug Resistance of Staphylococcus aureus Based on Rapid Identification System of Microorganisms / 中国实验方剂学杂志

Objective::To explore the feasibility of the rapid identification system(MALDI-Biotyper System) of microorganisms for rapid identification of Pseudomonas aeruginosa and clinical isolation of Staphylococcus aureus. Method::Identification quality control and clinical isolation were conducted for drug resistance of S. aureus by microbial rapid identification system and broth dilution method. The scores of microbial rapid identification system were compared with the MIC value of broth dilution method. The drug resistance of P. aeruginosa was simultaneously identified to determine the accuracy and applicability of the rapid identification system of microorganisms. Result::The scores of the microbial rapid identification system showed that the score of sensitive quality control strain S. aureus was higher than 2.000, and the that of resistant strain of methicillin-resistant S. aureus(methicillin-resistant S. aureus, MRSA)was between 1.700 and 2.000.The score of clinically isolated S. aureus was between 1.700 and 2.000, which suggested the drug resistance and was consistent with the MIC value of the broth dilution method. At the same time, the systemic identification value of the P. aeruginosa, which is independent of the quality control sensitive strain, was greater than 2.000, showing sensitivity and it was a sensitive strain itself, which was consistent with the results. Conclusion::The microbial rapid identification system scoring method can be used for the rapid identification of the drug resistance of S. aureus and P. aeruginosa.

Effect of Tanreqing Injection Combined with Imipenem-cilastatin in Inhibiting Extensively-drug Resistant Pseudomonas aeruginosa / 中国实验方剂学杂志

Objective:To observe the effect of combination of Tanreqing injection(Tanreqing) and imipenem-cilastatin on extensively-drug resistant Pseudomonas aeruginosa (XDPA), and study the mechanism of the combination. Method:The minimum inhibitory concentrations (MICs) of Tanreqing and imipenem-cilastatin against planktonic XDPA strain isolated in clinic were determined by the broth microdilution method. The checkerboard method was used to evaluate the combination effect. The bacterial metabolic activity in mature biofilm was studied by microtiter-plate test. The destructive effect of combination drugs on dynamic biofilm was observed by using BioFlux system, and viable cells were examined by confocal laser scanning microscope (CLSM) after treatment. The scanning electron microscopy (SEM) was used for observing Pseudomonas aeruginosa and length measurement. Result:The MIC values of imipenem-cilastatin and Tanreqing were 512 mg·L-1 and more than 16 500 mg·L-1. The checkerboard analysis showed that Tanreqing could enhance the sensitivity of imipenem-cilastatin, while the combination drugs synergistically inhibited the growth of bacteria. Compared with the control group or the imipenem-cilastatin individual group, the combined drugs significantly reduced the amount of living bacteria in the biofilm (PPPConclusion:Tanreqing and imipenem-cilastatin synergistically inhibit the bacterial growth in planktonic and biofilm states, and destruct biofilms.

Role of the Th17/Treg functional imbalance on the development of atherosclerosis in apo E knockout mice / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the role of the helper T cells (Th) 17/Treg cell imbalance on the development of atherogenesis in apo E knockout mice.</p><p><b>METHODS</b>Apo E(-/-) mice were examined at age of 6, 12, 24 and 48 weeks (n = 10 each). Age matched C57/B6 mice served as controls. The number of Th17, Treg and dendritic cell (DC) was detected by flow cytometry. The levels of interleukin(IL)-6, IL-17A and transforming growth factor(TGF)-β1 were detected by ELISA. The suppression ability of Treg was evaluated by mixed lymphocyte reaction.</p><p><b>RESULTS</b>With increasing ages, the frequencies of Th17 and Treg in CD4(+) T cells were increased (Th17 ratio from 1.00% to 3.14%; Treg ratio from 8.08% to 27.80%) and the level of IL-17A was up-regulated [from (87 ± 15) pg/ml to (191 ± 26) pg/ml], but the rate of Th17/Treg cell and the level of TGF-β1 remained stable during atherogenesis in apo E knockout mice. Furthermore, the phenotype of splenic DC was matured and the blood level of IL-6 was up-regulated [from (43 ± 5) pg/ml to (104 ± 11) pg/ml] with aging in apo E(-/-) mice. Addition of IL-6 to T cells reversed the ability of Treg to suppress the proliferation of effective T cells.</p><p><b>CONCLUSION</b>DC overactivation, subsequent increased secretion of IL-6, inhibition of Treg cell function and the Th17/Treg cell imbalance play key roles on the atherogenesis in apo E(-/-) mice.</p>

Identification of major impurity in gemcitabine hydrochloride for injection by HPLC-QTOF / 中国药学杂志

OBJECTIVE: To elucidate the structure of the major impurity in gemcitabine hydrochloride for injection. METHODS: HPLC-QTOF-MS was adopted to analyze the structure of this impurity based on the fragmentation behavior of gemcitabine and this impurity. The separation was performed on a ZORBAX Eclipse XDB-C18(4.6 mm×250 mm, 5 μm) column. A mixture of 10% methanol and 90% 10 mmol·L-1 ammonium acetate(pH 5.7) was used as mobile phase. Flow rate was 1.0 mL·min-1. MS was performed under positive mode with 4.0 kV capillary voltage, 65 V skimmer, 310 kPa spray pressure, 11 L·min-1 drying gas (nitrogen) flow rate, 350°C drying gas temperature and 150 V fragmentor. CONCLUSION: This study offers scientific data for studying the origin of the impurity of gemcitabine hydrochloride and improving its quality.

Effects of different pacing algorithms on cumulative ventricular pacing proportion in patients with pacemakers / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It is well known that increased cumulative ventricular pacing proportion (CumVP%) is one of the most important causes for adverse cardiovascular events. Therefore, how to reduce CumVP% has been a treatment issue in recent years. This study aimed to investigate the effects of different pacing algorithms on CumVP% in patients with pacemakers.</p><p><b>METHODS</b>Pacemakers with three pacing algorithms, i.e., conventional dual chamber rate adaptive pacing (DDDR), search atrioventricular conduction plus (SAV+) and managed ventricular pacing (MVP), were implanted in 42 patients including 41 with bradycardia arrhythmias and one with ventricular tachycardia. Pacemakers were programmed to work in conventional DDDR, SAV+ and MVP during the follow-up periods of the first, the second and the third month. In each pacing algorithm, the time percentages of four pacing and sense status including atrial sense-ventricular sense (AS-VS), atrial sense-ventricular pacing (AS-VP), atrial pacing-ventricular sense (AP-VS) and atrial pacing-ventricular pacing (AP-VP) were calculated. Cumulative ventricular pacing proportions were compared in the three pacing algorithms in the first, the second and the third month postoperatively.</p><p><b>RESULTS</b>In the DDDR algorithm AS-VS, AS-VP, AP-VS and AP-VP were 2.4%, 52.3%, 2.5% and 42.8% respectively, while in SAV+ they were 19.3%, 34.9%, 33.9% and 12.0%, in MVP they were 38.9%, 13.2%, 41.6% and 6.4%. In the above the DDDR, SAV+ and MVP algorithms, cumulative ventricular pacing proportions were 95.1%, 46.9% and 19.6%, respectively (P < 0.05) and the percentages of CumVP% < 40% in patients were 0, 23.8% and 95.2.0% (P < 0.05).</p><p><b>CONCLUSIONS</b>Compared with the conventional DDDR algorithm, both SAV+ and MVP significantly reduced the CumVP%, especially the MVP algorithm. Patients may benefit from MVP algorithm due to reduced CumVP%.</p>

Determination of ginsenosides Re, Rb1 in Panax quinquefolius by micellar electrokinetic chromatography / 中国中药杂志

<p><b>OBJECTIVE</b>A Micellar electrokinetic chromatography (MEKC) technique for the determination of ginsenosides Re, Rb1 in Panax quinquefolius was developed and validated.</p><p><b>METHOD</b>The MEKC was performed in a mixed buffer solution containing 20 mmol x L(-1) boric acid, 20 mmol x L(-1) sodium tetraborate, 60 mmol x L(-1) sodium cholate (CA) and 20% acetonitrile under the applied voltage of 20 kV at 25 degrees C. The detection wavelenth was 203 nm, the sampling time is 5 sec (hydrostatic injection).</p><p><b>RESULT AND CONCLUSION</b>The liner range was 0.38 - 1.65 mg x ml(-1) for Re and 0.42 - 1.76 g x L(-1) for Rb1. The average recovery for Re was 97.2%, RSD = 1.6% and that for Rb1 was 97.7%, RSD = 1.9% (n = 5). The preparation of sample is easy and the chromatogram has much information.</p>