Objective:To screen out the potential key genes of endotoxin tolerance (ET), and to provide theoretical and experimental evidence for treatment and prognosis of sepsis.Methods:①Experiment 1 (gene chip and bioinformatics analysis): ET related data set GSE47783 was downloaded from the Gene Expression Omnibus (GEO). The data set was obtained from lipopolysaccharide (LPS) stimulated mouse macrophages to establish sepsis model (LPS group) and ET model (ET group). IDEP 0.92 software was used to screen differential expressed gene (DEG) between the two groups, analyze gene ontology (GO), and locate the main functions and signaling pathways of differential genes. The protein-protein interaction (PPI) network of DEG was constructed by the Search Tool for the Retrieval of Interacting Genes Database (STRING) to screen core genes hepatitis A virus cell membrane protein receptor 2 (HAVCR2) for following up validation study. ②Experiment 2 (reproduction of mouse macrophage RAW264.7 model): RAW264.7 cells were cultured in vitro, the ET model (ET group, cells were cultured with 10 μg/L LPS for 24 hours and then with 100 μg/L LPS for 4 hours) and sepsis model (LPS group, cells were cultured with 100 μg/L LPS for 4 hours) were reproduced by LPS stimulation. Phosphate buffer saline (PBS) group was given equal volume of solvent PBS for 4 hours. The mRNA and protein expressions of HAVCR2 were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting. ③Experiment 3 (RAW264.7 cells transfected with HAVCR2 lentiviral vector): to further clarify whether HAVCR2 was involved in the formation of ET, after knockdown of HAVCR2 in RAW264.7 cells by lentiviral short hairpin RNA (shRNA) technology, the ET model (HAVCR2 --ET group) was constructed again, and the control group (ET group) without knockdown of HAVCR2 was set up. RT-qPCR method was used to detect the mRNA expressions of macrophage polarization key proteins [arginase 1 (ARG1), CD206, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide synthase 2 (NOS2)] in cells. Results:①Experiment 1: a total of 1 013 DEG were identified, compared with LPS group, 521 genes were up-regulated and 492 genes were down-regulated in ET group. The function of these DEG was to increase biosynthesis and reduce inflammatory reaction. Signal pathways were mainly enriched in Janus kinase/signal transducers and activators of transcription (JAK/STAT) , NOD like receptor, Toll-like receptor (TLR), TNF, hypoxia inducible factor-1 (HIF-1). The first up-regulated HAVCR2 in the ET group was selected as the target of the study. ②Experiment 2: the results of in vitro experiment showed that the mRNA expression of HAVCR2 after high-dose LPS stimulation was down-regulated as compared with PBS group, and the mRNA expression of HAVCR2 in ET group was significantly higher than that in LPS group (2 -ΔΔCT: 1.10±0.10 vs. 0.60±0.10, P < 0.05). The results of Western blotting were consistent with RT-qPCR results. ③Experiment 3: the mRNA expressions of ARG1 and CD206 in HAVCR2 --ET group were significantly lower than those in ET group [ARG1 mRNA (2 -ΔΔCT): 0.50±0.10 vs. 1.00±0.10, CD206 (2 -ΔΔCT): 0.73±0.10 vs. 1.00±0.10], and the mRNA expressions of TNF-α and IL-1β were significantly higher than those in ET group [TNF-α mRNA (2 -ΔΔCT): 2.20±0.10 vs. 1.00±0.10, IL-1β mRNA (2 -ΔΔCT): 9.00±0.10 vs. 1.00±0.10), with significant differences (all P < 0.05). There was no significant difference in the expression of NOS2 mRNA between the two groups. Conclusion:HAVCR2 is involved in the regulation of inflammatory factors downstream of sepsis and the formation of ET, which is expected to become a new therapeutic target of sepsis.

BACKGROUND: Chinese medicines have better effects on preventing and treating diabetic nephropathy at early period, and the effects are caused by the diversity of the effective components of Chinese medicines which acts on the different targets of diabetic nephropathy.OBJECTIVE: To observe the effect of Xiaokening on the proliferation of mesangial cells under high glucose, and investigate the possible mechanism. DESIGN: A controlled observation.SETTING: Department of Endocrinology, the First Affiliated Hospital of Nanjing Medical University.MATERIALS: The experiments were completed in the Research Room of Cell Culture, Research Center of Endocrine Metabolism, the First Affiliated Hospital of Nanjing Medical University from July 2003 to May 2004. The rat mesangial cell lines HBZY-1 were bought from Chinese Center for Typical Culture Collection.METHODS: The mesangial cells were passaged and cultured according to the instructions. ① Grouping according to different concentration of stimulation: In the high glucose+Xiaokening group, Xiaokening of 6 concentrations were used, I.e., 20.0, 40.0, 60.0, 80.0, 120.0, 200.0 g/L. Meanwhile,normal glucose control group and high glucose control group were also set,the glucose concentration in the medium was 5.6 and 30 mmol/L respectively. The proliferations were observed after 72 hours. Grouping according to different time points of stimulation: There were normal glucose control group, high glucose control group and high glucose+Xiaokening group, the glucose concentration in the medium was 5.6, 30 and 30 mmol/L respectively, and the Xiaokening concentration was 60 g/L. The proliferations were observed at 24, 48 and 72 hours respectively in the three groups.② The dispensed cell suspension was placed into the 96-well plate, and 200 μL suspension for each well. The culture plate was placed in the inoculation box containing CO2 (0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant was deserted, cell solutions containing different drugs of different concentrations were added in each group, 200 μL for each well. The 96-well plate was then placed in the culture box containing CO2 (0.05 in volume fraction) and 100% humidity at 37 ℃ for inoculation.In the groups of different concentrations, 20 μL MTT (5 g/L) was added into the wells after 72 hours. In the groups of different time points, 20 μL MTT (5 g/L) was added into the wells at 24, 48 and 72 hours. Then the plates were inoculated at 37 ℃ for 4 hours. Under microscope, it was observed that MTT get into the cells, the supernatant was sucked away, then 150 μL dimathyl sulfoxide was added to dissolve methyl alcohol, and shaken to even with plate rocking bed for 30 s, and the absorbance (A) values were recorded with the microplate reader at the wavelength of 492 nm.MAIN OUTCOME MEASURES: The proliferations of mesangial cells (A value) were observed at different time points and in Xiaokening of different concentrations.RESULTS: ①Proliferation of mesangial cells at different time points: The A values in the high glucose control group at 24, 48 and 72 hours (0.685 ±0.010, 0.750±0.087, 0.659±0.018) were higher than those in the normal glucose control group (0.586±0.054, 0.598±0.040, 0.527±0.047, P ＜ 0.05). In both the high and normal glucose control groups, the A value at 48 hours was higher than that at 24 hours (P ＜ 0.05), but the A value at 72 hours was lower than that at 48 hours (P ＜ 0.05). The A value in the high glucose+Xiaokening group at 72 hours was 0.538±0.023, and it was lower than that in the high glucose control group (P ＜ 0.05). ② Proliferation of mesangial cells in Xiaokening of different concentrations: Xiaokening high er than 60.0 g/L could obviously restrain the excessive stimulation of high glucose to the mesangial cells, and the effect was in a concentration-de pendent manner, but too high concentration (120.0 g/L) would result in the abscission and death of cells, whereas no survived cells could be observed in extremely high concentration (200.0 g/L). CONCLUSION: Xiaokening could inhibit the proliferation of mesan gial cells stimulated by high glucose after 72 hours in a concentration dependent manner, but too high concentration would cause strong cytotoxicity.

Objective To investigate the protective effects of pentoxifylline on gastric mucosal injury in rats with endotoxemia. Methods 30 Wistar rats were randomly divided into 3 groups : sham operated control group (C group) ,LPS group and PTX group. The serum tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) levels in each group were measured. The immunohistochemistry was used to assess the expression of the ICAM-1 in the gastric mucosal tissues. Results The levels of TNF-α and IL-1β in serum in LPS group were higher than those in C group (P <0.01). The level of ICAM-1 in the gastric mucosal tissues in LPS group was higher than those in sham operated control group(P < 0.01). After FIX treatment, the levels of TNF-α and IL-1β in serum in PTX group were lower than those in LPS group(P < 0.01) and the level of ICAM-1 in the hepatic tissues in PTX group was also lower than those in LPS group(P <0.01). Conclusions TNF-α,IL-1β and ICAM-1 take part in the progress of gastric mucosal inju-ry in rats with endotoxemia. PTX can eliminate the gastric mueesa injury and protect the gastric mucosal tissues by downregulating the levels of TNF-α, IL-1β and ICAM-1.

Object To study the optimal conditions and parameters for the enrichment process of scoparone in Yinchenhao Decoction (YCHD) with macroporous resin. Methods With the enrichment degree of scoparone in YCHD as an index, the optimal conditions of the enrichment process were investigated. Results The optimal conditions were that the aqueous extract of YCHD was taken into the macroporous resin column, and kept for 30 min, then the resin was washed by five times of distilled water to get rid of impurity and three — four times of 70% ethanol to elute scoparone. Conclusion The process is feasible to enrich effective ingredient scoparone from YCHD.

Cardiopulmonary resuscitation is the most comonly used method facing cardiac arrest.The 2010 CPR guidelines emphasized high quality chest compressions and recommended continuous compression for 2 minutes after defibrillation to minimize interruptions in compressions.However,starting chest compressions immediately after a defibrillation shock may be harmful,if the heart is providing spontaneous beats and being subjected to external compressions at the same time.So it is very important to recognize ROSC during CPR,the methods of which include touching the pulse,amplitude spectral area,partial pressure end-tidal carbon dioxide,coronary perfusion pressure,central venous oxygen saturation,chest compression fraction,regional cerebral oxygen saturation,photoplethysmography,conjunctival oxygen tension,transthoracic-impedance plethysmography and echocardiography.This paper gives a review of the ROSC prognosis and recognition methods during CPR.

Object To probe into the objectivity and authenticity of four properties (cold, heat, warm, and cool) of raw Coptidis Rhizoma (RCR) and their preparations from biophysics and biochemistry. Methods Microcalorimetry was used to obtain the metabolic growth power-time curve of bacteria affected by the total alkaloid in different preparations of RCR and biothermokinetic parameters. Combined with the records of ancient herbal literatures, the influence of properties by different preparations to RCR were synthetically evaluated. Results All the effective total alkaloid in different preparations of RCR can restrain the Escherichia coli growth and metabolism in different level. Drugs, such as Coptidis Rhizoma Stir-fried with ginger juice (CRGJ), Coptidis Rhizoma stir-fried with wine (CRW), and Coptidis Rhizoma stir-fried with Fructus Evodiae (CRFE), with a little Warm Property can decrease the multiplication rate constants of E. coli index and increase heat output in growth metablism remarkably, while the drugs, such as RCR, Coptidis Rhizoma stir-fried with vinegar (CRV), and Coptidis Rhizoma stir-fried with gallbladder juice (CRGBJ), with a little Cold Property can decrease the multiplication rate constants and increase heat output a little. There was a stable difference between them. Conclusion Microcalorimetry is a new and useful mean for the study of the properties of tradition Chinese medicine.

Through our pre-investigation and literature analysis, it was found that rhubarb could be categorized into two types, chrysophanol-type and rhein-type, based on the proportion of the two constituents in the total content of anthraquinones after acid hydrolysis. In this paper, the antimicrobial activities of chrysophanol-type and rhein-type rhubarbs against Staphylococcus aureus were compared with microcalorimetric analysis, in order to illustrate the bioactive differentiability between the two chemotypes. For the aim to display the distinction of chrysophanol and rhein percentage in total anthraquinones, the sampling volume was regulated to make the total anthraquinones equivalent, thus, the antimicrobial difference was only attributed to the difference of chemotypes. The results indicated that the antimicrobial difference between the two chemotypes was confirmable labeled at the biothermokinetic parameters of S. aureus growth affected by the rhubarb samples. The growth rate constant (k1) of the first exponential phase for the growth of S. aureus affected by the rhein-type rhubarb was significantly lower than that of chrysophanol-type (P<0.01), which suggested stronger antimicrobial activity of rhein-type rhubarb than that of chrysophanol-type. However, the antimicrobial activities of rhein-type rhubarbs were not positively correlated to the contents of rhein. It suggested that the antimicrobial activity of rhubarb might be related to some unknown components which were of same accumulating pattern of rhein. The findings in present study provided some experimental evidence on categorizing rhubarb into two chemotypes through the difference of antimicrobial activity on S. aureus by microcalorimetric analysis and, further, offered references to revision of the commercial specification of rhubarb from chemical view.

To establish a bioassay method and quality standard of Banlangen granula, agglutinated activity assay was used in the analysis of the traditional Chinese medicine, Banlangen granula. It showed that masculined effect could be picked up effectively and the products quality of different pharmaceutical factories and different batch numbers from the same factory could be revealed conveniently, accurately, quickly and directly with this method (valence value was between 2 and 11). The established bioassay method had a good reproducibility with RSD = 2%. The dependablity of the activity of red cell agglutination and restrainting influenza virus NA was conspicuous (r2 = 0.878 3). In conclusion, this bioassay method is suitable to control and evaluate the quality of Banlangen granula. Thus the method may provide a simple and effective technique in supervising and examining the quality of other traditional Chinese medicine.

Object To demonstrate the naked truth of four therapeutic features of TCM (cold, hot, warm and cool) from biophysical and biochemical point of view. Methods Growth thermogenesis curves of Escherichia coli were determined under different conditions by microcalorimetry to give biothermodynamic parameters, such as multiplication constant (k), median inhibitory concentration (IC 50 ), and enthalpy (?H). Differences between folia ginseng and flos ginseng were comprehensively compared with reference to classical Chinese literatures on material medica. Results The bacteriostatic curve of both drugs were basically similar. With increasing concentration of decoctions of both drugs, the lag phase was delayed and the peaks shifted to the right. While the difference of ?H was stable, which decreases with the increasing concentration of Folium ginseng, but increases with increasing concentration of Flos ginseng. Conclusion Microcalorimetry is not only a new effective method to describe the therapeutic features of TCM, but also could be used as a tool for the screening of active TCM.

Objective To establish an objective method for evaluating the intrinsic characteristics between cold and hot nature of Chinese materia medica(CMM)through the different effects of Mahuang decoction(MHD)and Maxing Shigan decoction(MSD)on animal temperature tropism.Methods The equipment with cold/hot pads was used to investigate the variety ofthe temperature tropism between two groups of mice treated by MHD and MSD,respectively.Meanwhile,the activities ofadenosine triphosphatase(ATPase),superoxide dismutase,succinate dehydrogenase,and malondialdehyde were measured.Results After treated by MHD,the macroscopic behavioral index of remaining rate on warm pad(40 ℃)of mice decreasedsignificantly(P < 0.05),suggesting the enhancement of cold tropism,meanwhile,the internal indices of ATPase activity and oxygen consuming volume increased significantly(P < 0.05),suggesting the enhancement of energy metabolism.On theother hand,the above-mentioned indices in MSD group changed on the inverse way.Conclusion The relative drug natureof MHD and MSD revealed in this study is consistent with the theoretical prognostication or definition.It indicates that theinternal cold and hot nature of CMM could be reflected in ethological way on the changes of animal temperature tropismwhich might be internally regulated by body energy metabolism.