AIM: To investigate the signaling mechanisms involved in the priming effects of lipopolysaccharide(LPS) on heat killed Staphylococcus aureus(HKSa)-induced nitric oxide(NO) production in macrophages.METHODS: Murine macrophage RAW264.7 was used in the experiment.Griess reagent was used to measure the content of nitrite in culture medium.Real-time PCR and Western blot was utilized to examine the mRNA and protein levels of toll-like receptor 2(TLR2),respectively.Dual luciferase reporter assay was used to assess the transcriptional activity of nuclear factor of activated T cells(NF-AT).RESULTS: The RAW264.7 cells pretreated with LPS for 24 h significantly enhanced NO production induced by HKSa,suggesting that LPS primed the macrophages and increased the reactivity of the cells to HKSa.LPS increased the mRNA and protein levels of TLR2 in a dose-dependent manner in RAW264.7 cells.The RAW264.7 cells pretreated with LPS enhanced NO production induced by peptidoglycan,one of the specific ligand of TLR2.The priming effect of LPS on HKSa-induced NO production was partly blocked by TLR2 neutralizing antibody.LPS significantly enhanced the transcriptional activity of NF-AT,which was inhibited by BAPTA/AM(a cell-permeable cytosolic calcium chelator) and cyclosporine A(CsA,an inhibitor for calcineurin).Both BAPTA/AM and CsA inhibited the priming effect of LPS on HKSa-induced NO production in RAW264.7 cells.CONCLUSION: The present study confirms the priming effect of LPS on the reactivity of RAW264.7 cells to HKSa.Pattern recognition receptor TLR2 and calcium/calcineurin/NF-AT signaling pathway may be involved in the priming process initiated by LPS.

OBJECTIVETo study plasma protein binding rate of bufalin.

METHODHPLC was employed to determine the concentration of bufalin. Plasma protein binding rate studies were conducted by equilibrium dialysis method. The influence of drug concentration and plasma in different species on plasma protein binding rate were studied.

RESULTThere was no significant difference in the plasma protein binding rates at low, middle and high bufalin concentrations in dilution medium. The protein binding rate of bufalin in human plasma was higher than in the rat plasma.

CONCLUSIONBufalin has higher protein binding extent with both rat plasma and human plasma.

Animals ; Blood Proteins ; chemistry ; Bufanolides ; chemistry ; Humans ; Kinetics ; Male ; Protein Binding ; Rats ; Rats, Sprague-Dawley

BACKGROUND:A novel biodegradable Mg-Zn alloy has been designed,in which the density and the Young's modulus are proximal to human bone,at the same time,it depletes the toxicity of aluminium and rare earth element in commercial magnesium alloys.OBJECTIVE:To evaluate the cytocompatibility of biodegradable Mg-Zn alloy.DESIGN,TIME AND SETTING:Contrast study was performed in the central laboratory of the Sixth People's Hospital of Shanghai Jiao Tong University between November 2007 and March 2008.MATERIALS:The Mg-6wt%Zn was prepared by School of Materials Science and Engineering of Shanghai Jiao Tong University,with the density was 1.82 g/cm3 and the Young's modulus was 44 GPa.L-929 cells for cytocompatibility test were provided by Chinese Academy of Science Type Culture Collection.Ten male New Zealand rabbits were employed in the hemolysis test.METHODS:The Mg-Zn alloy extraction medium was prepared by serial dilutions with fresh medium to 10%,50% and 100%.The experiments were carried out in a 96-well tissue-culture plate.Simple DMEM culture solution was taken as negative controls,while DMEM culture solution supplemented with 0.64% phenol served as positive controls.MAIN OUTCOME MEASURES:Relative proliferation rate of L-929 cells was determined at 2,4 and 7 days with MTT assay.The cytotoxicity of Mg-Zn alloy was evaluated according to ISO 10993-5:1999.The L-929 cell morphology and growth at 2,4 and 7 days were determined under inverted microscope.Based on ISO 10993-4:2002,hemolysis in vitro was evaluated through measuring erythrocyte lysis and ferrohemoglobin freeing degree with indirect contact method.RESULTS:The number of L-929 cells increased significantly and the morphology was not changed.The growth and morphology of cells in different Mg-Zn extraction medium had no difference from negative control group.Cytotoxicity test showed that biodegradable Mg-Zn alloy did not have obvious toxicity on L-929 cells,and the cytotoxicity of these extracts was in grade 0-1.Hemolysis test suggested that the Mg-Zn alloys did not have obvious hemolysis reaction,and the hemolysis index was 3.4%,which was less than the national standard (5%).CONCLUSION:The Mg-Zn alloys do not have obvious cytotoxicity and hemolysis reaction,which demonstrate that Mg-Zn alloys have good cytocompatibility.

Objective The aim of this study was to investigate the mechanism of sanguinarine(SANG)on the inhibitory pro-liferation in ovarian cancer SKOV3 cells. Methods CCK-8 assay was used to detect proliferation of SKOV3 cells. Flow cytometry was used to detect the effect of SANG on apoptosis in SKOV3 cells. The spectrophotometer was used to detect the production of reac-tive oxygen species(ROS)by SANG. The mouse ovarian cancer xenograft model was used to detect the inhibitory effect of SANG on tumor growth. Results SANG promoted apoptosis in SKOV3 cells in a dose-and time-dependent manner. The SANG-induced ap-optosis was associated with the production of ROS,Activated the c-Jun-N-terminal kinase( JNK) and nuclear factor-κB( NF-κB)signaling pathways. In mouse model of ovarian cancer xenografts,after intravenous injection of mice with SANG,SANG was signifi-cantly inhibited the growth of ovarian cancer xenografts when compared to the control group. SANG also significantly induced apoptosis in ovarian cancer xenografts. Conclusion SANG can significantly inhibit the proliferation of ovarian cancer SKOV3 cells,induce ap-optosis,increase the production of ROS,and inhibit the growth of ovarian cancer.

Objective:To analyze the correlation between cognitive impairment and cortical atrophy in elderly patients with asymptomatic carotid artery stenosis (ACAS).Methods:In this cross-sectional study, 40 consecutive elderly patients with ACAS treated in the Department of Neurology, Northern Jiangsu People′s Hospital from July 1, 2020 to June 30, 2021 (ACAS group), and 40 elderly healthy controls who accepted physical examination during the same period (control group) were included. Cognitive assessment was performed using the Mental State Examination Scale (MMSE) and the Montreal Cognitive Assessment Scale (MoCA), and brain magnetic resonance imaging scanning was performed in the ACAS group. The artificial intelligence technique was applied for brain lobe segmentation and cortical volume calculation. The χ2-test, independent sample t-test and Wilcoxon non-parametric test were used to analyze the difference of clinical data and cognitive scores between the two groups. In the ACAS group, the cortical volumes of the side with carotid stenosis was compared with that of the normal side, and Spearman′s correlation analysis was used to assess the correlation between cognitive scores and cortical atrophy. Results:Compared with the control group, the ACAS group got significantly lower scores of MMSE and MoCA, as well as lower scores of visuospatial executive function, attention and calculation, language function, abstraction ability and delayed recall [(25.60±2.49) vs (27.18±1.01), (22.05±3.59) vs (25.60±1.43), (2.73±1.04) vs (4.08±0.62), (4.53±0.93) vs (5.03±0.66), 2.00 (0.00) vs 3.00 (0.00), 1.00 (1.00) vs 2.00 (0.00), and (2.95±0.96) vs (3.35±0.62)] (all P<0.05). There was not significant differences in naming and orientation ability between the two groups (both P>0.05). The volume of cortical, temporal lobe, frontal lobe, parietal lobe and insular lobe on the side with carotid stenosis in the ACAS group were significantly smaller than those on the normal side [186.23 (177.97, 202.53) vs 194.67 (185.65, 204.82) cm 3, 54.74 (50.66, 56.95) vs 55.61 (51.24, 58.49) cm 3, 72.98 (70.76, 78.34) vs 75.27 (72.34, 80.66) cm 3, 53.66 (51.11, 57.86) vs 56.59 (52.80, 60.09) cm 3, 6.57 (6.35, 7.07) vs 6.72 (6.46, 7.34) cm 3] (all P<0.05). The MoCA score in the ACAS group was positively related to the cortical volume ratio of the two sides ( r=0.427, P<0.01). The attention ( r=0.353) and abstraction ( r=0.226) ability scores were positively correlated with the temporal lobe volume ratios of the two sides (both P<0.05). The visuospatial executive ( r=0.187) and language ( r=0.373) ability scores were positively correlated with frontal lobe volume ratios of the two sides (both P<0.05), and visuospatial executive ( r=0.386), naming ( r=0.344), language ( r=0.517), abstraction ( r=0.335) and delayed recall ( r=0.333) ability scores were positively correlated with parietal lobe volume ratios of the two sides (all P<0.05). Conclusion:In elderly patients with ACAS, the cognitive impairment and cortical atrophy on the sides with carotid stenosis are significant and a positive correlation is detected between them.