Objective To observe the influence of N-acetylcysteine(NAC)on the formation of Pseudomonas aeruginosa biofilm(BF),and to study the synergism of bactericidal activities of NAC combined with levofloxacinon to the mature Pseudomonas aeruginosa biofilm.Methods The experiment was done in experiment centre of Xiangya Hospital.Scanning electronic microcopy(SEM)were utilized to demonstrate the effect of NAC on the formation of biofilm.Then biofilm cultured without NAC were detected by optical microscopy after being stained with AgNO3.The minimal inhibitory concentration were detected by double test tube diluted method.The mature biofilm was treated by NAC and/or different concentrations of levofloxacin for 24 hours.The influence of levofloxacin combined with NAC were examined by MTT method on the bacterial quantity in biofilms.Results Biofilm was achieved on the silicon slides in all groups 7 days after the incubation of Pseudomonas aeruginosa.Scanning electronic microcopy showed that the mucoid materials among bacteria was significantly reduced and the thickness of biofilm was decreased in NAC groups.Levofloxacin beyond 1 MIC could significantly decline the viable cells on biofilms(P

RhoA, a small GTPase, is involved in a wide array of cellular functions in the central nervous system, such as cell motility, cytoskeleton rearrangement, transcriptional regulation, phagocytosis and cell growth. It is not known how spinal cord injury (SCI) affects the expression of RhoA in different nerve cells. In the present study, we investigated the changes of RhoA expression in remote areas of the injury at the 3rd, 7th and 30th day after SCI, which was established by T10 contusion method. Moreover, we examine its expression profile in neurons, astrocytes and microglia. RhoA was found to be weakly expressed in these nerve cells in normal spinal cord. Western blotting showed that, after SCI, the total RhoA expression was up-regulated, and the RhoA expression was increased and peaked at the 7th day. Double immunostaining revealed specific and temporal expression patterns of RhoA in different nerve cells. The expression of RhoA in neurons started to increase at day 3, peaked at day 7 and then decreased slightly at day 30. Expression of RhoA in astrocytes increased moderately after SCI and peaked at day 7. There was no obvious change in RhoA expression in microglia after SCI in remote areas. This study demonstrated that, after SCI, RhoA expression exhibited different patterns with different nerve cells of spinal cord. RhoA expression patterns also changed with time after SCI, and among different nerve cells in the injured spinal cord. These findings can help us better understand the roles of RhoA in SCI.

Objective To evaluate the effects of nerve growth factor-beta (NGF-β) pretreatment on cell apoptosis induced by endoplasmic reticulum stress during ischemia-reperfusion in isolated rat hearts.Methods Male Sprague-Dawley rats,weighing 180-220 g,were anesthetized with intraperitoneal 10％ chloral hydrate 300 mg/kg.The hearts were excised and perfused in a Langendorff apparatus with K-H solution aerated with 95％ O2 and 5％ CO2 at 37 ℃.Thirty-two isolated rat hearts were randomly divided into 4 groups (n =8 each) using a random number table:control group (group C),I/R group,NGF-β pretreatment group (group N) and NGF-β combined with K252a (trkA receptor antagonist) pretreatment group (group N + K).In group C,the hearts were continuously perfused with K-H solution for 195 min.The hearts were perfused with K-H solution for 45 min in group I/R.In N and N + K groups,the hearts were perfused with K-H solution for 15 min,and then with K-H solution containing 0.1 μg/ml NGF-β and 0.1 μg/ml NGF-β mixed with 100 nmol/L K252a,respectively,for 30 min.The perfusion was suspended for 30 min followed by 120 min of reperfusion with K-H solution in I/R,N and N + K groups.HR,left ventricular end-diastolic pressure (LVEDP),left ventricular developed pressure (LVDP) and + dp/dtmax were measured at the end of 15 min equilibration (baseline) and at 5,30,60 and 120 min of reperfusion.Myocardial specimens were obtained at 120 min of reperfusion for detection of myocardial apoptosis (by TUNEL) and expression of glucose-related protein 78 (GRP78),CCAAT/enhancer-binding protein homologous protein (CHOP),and caspase-12 (by Western blot analysis).Apoptosis index was calculated.Results Compared with group C,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index and expression of GRP78 and CHOP were increased in I/R and N groups,and the expression of caspase-12 was upregulated in I/R group.Compared with group I/R,HR,LVDP,and + dp/dtmax were significantly increased,and LVEDP,apoptosis index and expression of GRP78,CHOP and caspase-12 were decreased in group N,and the expression of GRP78 was down-regulated in group N + K.There was no significant difference in cardiac function indexes between group I/R and N + K.Compared with group N,HR,LVDP and + dp/dtmax were significantly decreased,and LVEDP,apoptosis index,and expression of GRP78,CHOP and caspase-12 were increased in group N + K.Conclusion NGF-β pretreatment can protect the isolated rat hearts against ischemia-reperfusion injury,and inhibition of the endoplasmic reticulum stress-triggered cell apoptosis after activating trkA receptors is involved in the mechanism.

AIM To establish a quantitative analysis of multi-components by single marker(QAMS) method for the content determination of six catechins in Xinnaojian Capsules (Tablets) (tea extract).METHODS The analysis of 50％ methanol extract of this drug was performed on a 35 ℃ thermostatic Shimadzu Wonda Cract ODS-2 column (4.6 mm ×250 mm,5 μm),with the mobile phase comprising of 0.5％ acetic acid (A)-acetonitrile (B) flowing at 1.0 mL/min in a gradient elution manner,and the detection wavelength was set at 280 nm.With epigallocatechin gallate as an internal standard,the relative correction factors of epigallocatechin,catechin,epicatechin,gallocatechin gallate and epicatechin gallate were calculated,from which the content determination was made.RESULTS Six constituents showed good linear relationships within their own ranges (r ≥ 0.999 8),whose average recoveries were 96.00％-98.47％ with the RSDs of 2.09％-2.91％.The results obtained by QAMS method approximated those obtained by external standard method.CONCLUSION This simple and reliable method can be used for the quality control of Xinnaojian Capsules (Tablets).

Arsenic trioxide albumin microspheres (As2O3-BSA-NS) were prepared by using methods of chemical cross-linking. The desirability function (DF), calculated according to the size (<1 microm) distribution, drug loading and drug trapping efficiency, was introduced as a total index for the microspheres formulation. Four factors, inculding W/O ratio, decentralization speed, BSA concentration and stirring stabilization time, were selected and arranged in an orthogonal experimental table. The release characteristic was studied by the drug release experiment in vitro. The four factors affected DF differently. Decentralization speed behaved as the maximum (P<0.01), followed by BSA concentration (P<0.05) and the W/O ratio dose (P<0.05). Stirring stabilization time did not influence DF (P>0.05). The release experiment in vitro showed that As2O3 in As2O3-BSA-NS was released more slower than pure As2O3. It was concluded that regular As2O3-BSA-NS may be prepared by the methods of chemical cross-linking, which was optimized by orthogonal experimental analysis of different factors, and the microspheres can release As2O3 slowly.
Arsenicals/*chemistry ; Cross-Linking Reagents/pharmacology ; Delayed-Action Preparations ; Drug Carriers/*chemistry ; Drug Delivery Systems ; Microspheres ; Oxides/*chemistry ; Serum Albumin, Bovine/*chemistry ; Technology, Pharmaceutical

Objective To determine the interference effect of H. hepaticus infection on the functional characteris-tics of dendritic cell ( DC) surface molecules and immune response in mice. Methods Male BALB/c mice were inocula-ted with H. hepaticus (ATCC 51450). Murine bone marrow-derived dendritic cells (DC) were isolated and co-cultured which were stimulated by GM-CSF and IL-4 at the fifth month after the last inoculation. Then the DCs were subjected to FACS analysis for surface markers (CD11c, CD40, CD80 and MHCII) detection. On this basis, virus suspension of New-castle disease virus( NDV) ZJ1 strain was inoculated into the mice. Serum was collected for detection of the NDV antibody titer in serum weekly to explore the difference of antibody titer between the two groups. Results The expression rates of CD40 and MHCII on the mouse DCs in experimental group were higher than that in the control group. The NDV antibody ti-ter of experimental group was slightly lower than that in the control group in the first week. During the 2nd to 5th weeks, the titer was higher than that in the control group, with a very significant difference. In the 6th week, the titer of both the two groups tended to fall. Conclusions H. hepaticus infection can promote bone marrow DC maturation in mice, stimulate the expression rates of MHC II and CD40, and enhance the NDV antibody levels.

Objective To compare the efficiency of bacteria culture and PCR assays for detection of Staphylococcus aureus ( S.aureus) , Pseudomonas aeruginosa ( P.aeruginosa) and Klebsiella pneumoniae ( K.pneumoniae) in laboratory rats and mice.Methods Bacteria culture combined with biochemical identification and PCR assay were used to detect 78 SPF rats and 422 SPF mice and the results of the two methods were compared .Results All the 78 rats were negative .Of the 422 mice, the positive rate by culture was 7.11%(30/422), of which, 10 were S.aureus, 22 were P.aeruginosa, and 2 were K.pneumoniae.The positive rate by PCR was 7.58%(32/422), of which, 10 were S.aureus, 25 were P. aeruginosa, and 2 were K.pneumoniae.Conclusions The high sensitivity , rapid procedure and easy to operate of PCR assay makes it valuable for rapid bacteria diagnosis and large-scale screening in laboratory animals .

Objective To evaluate the role of 1-phosphatidylinositol 3-kinase (PI3K) signaling pathway in nerve growth factor-beta (NGF-β)-produced mitigation of cell apoptosis induced by endoplasmic reticulum stress during hypoxia-reoxygenation (H/R) in rat cardiomyocytes.Methods H9c2 cells were seeded in 96-well plates at a density of 5× 105 cells/ml (100 μl/well).The wells were randomly divided into 5 groups (n =15 each) using a random number table: control group (group C);group H/R;NGF-β group (group N);NGF-β+NGF receptor trkA antagonist K252a group (group N+K);NGF-β+ PI3K inhibitor LY294002 group (group N+L).The cells were exposed to 95％ N2-5％ CO2 for 4 h in an anaerobic incubator, followed by reoxygenation in a standard incubator for 4 h in H/R, N, N+K and N+L groups.In addition, the cells in N, N + K and N + L groups were incubated in a standard incubator containing NGF-β, the mixture of NGF-β and K252a and the mixture of NGF-β and LY294002, respectively, during reoxygenation, and the final concentrations of NGF-β , K252a and LY294002 were 50 ng/ml, 100 nmol/L and 50 μmol/L, respectively.The cell viability was detected by using CCK-8 assay, and the cell survival rate was calculated.The cell apoptosis was examined by flow cytometry, and apoptosis rate was calculated.The expression of glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), caspase-12, Akt and phosphorylated Akt (p-Akt) was detected by Western blot.The ratio of p-Akt to Akt was calculated.Results Compared with group C, the cell survival rate was significantly decreased, and the apoptosis rate was increased in H/R and N groups, and the expression of GRP78, CHOP and caspase-12 was significantly up-regulated in group H/R, and p-Akt/Akt was significantly increased in group N (P＜0.05).Compared with group H/R, the cell survival rate was significantly increased, the apoptosis rate was decreased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was increased in group N (P＜0.05), and no significant change was found in the parameters mentioned above in N+K and N+L groups (P＞0.05).Compared with group N, the cell survival rate was significantly decreased, the apoptosis rate was increased, the expression of GRP78, CHOP and caspase-12 was up-regulated, and p-Akt/Akt was decreased in N+K and N+L groups (P＜0.05).Conclusion NGF-β can mitigate the cell apoptosis induced by endoplasmic reticulum stress during H/R, and activation of PI3K signaling pathway is involved in the mechanism.

Objective To analyze the effect of mesporous calcium silicate (m-CS)/calcium sulfate cement (CSC),m-CSC for short,in bone defect repair.Methods Setting time and compressive strength of the m-CSC (15 m-CSC as group Ⅰ and 30 m-CSC as group Ⅱ) were tested.CSC was used as the control.Cement samples were immersed in Tris-HCl solution,andin vitro degradation of the m-CSC was measured.Cell morphology and cell proliferation as well as differentiation on the samples were assessed.The cements were implanted into the traumatic femoral defects in rabbits,and the in vivo degradability and osteogenesis of the cements were investigated by histological evaluation after implantation for 4,8 and 12 weeks.Results Addition of m-CS into CSC prolonged the setting time (7.8 min in group Ⅰ and 10.5 min in group Ⅱ),obviously longer than 3.7 min in control group and did not have obvious effect on compressive strength of the cements.Weight loss of m-CSC solution was obviously lower (61.8 wt％ in group Ⅰ and50.3 wt％ in group Ⅱ),compared to70.4 wt％ in control group,pH value in group Ⅱ decreased from 7.40 to 7.26,while decreased from 7.40 to 6.86 in control group,m-CSC could promote cell proliferation and differentiation compared to CSC.At postoperative 12 weeks,histological sections showed massive new bony tissue (55.2％) in group Ⅱ,obviously higher than 25.6％ in control group.Conclusion m-CSC exhibits good biocompatibility,degradability and osteogenesis,and can promote bone regeneration in bone defect repair.

Objective To more intuitively understand the quality control for laboratory animals and further achie-ving a more scientific and reasonable management of laboratory animals, the infection index as evaluation criteria was intro-duced. Then the best way to calculate infection index was explored in order to more scientifically reflect the infection status of laboratory animals. Methods Infection index, also called the degree of infection, is a qualitative indicator of monito-ring laboratory animal quality. After arranging, analyzing, processing and gathering the data from laboratory animal quality monitoring, the index reflects synthetically the pathogen infection status or trend of a particularly investigated experimental animal population or the development of certain experimental animals. Results In general, the pathogen infection index of mice was slightly decreased, while the pathogen infection index of rats roughly increased year by year. In comparing infec-tion index by different pathogens, the parasite infection index of mice was found to be higher than bacteria and virus infec-tion indexes, while the bacteria infection index of rats was higher than parasite infection index and virus ones. Conclusions The infection index model intuitively reflects the quality control status of laboratory animals. The analysis also reveals that the parasite monitoring of the mice and the bacteria detection of rat needs to be reinforcement. In addition, the index of infection reveals that the pathogen infection of mice is well under control, while that of rats tends to be more serious year by year.