ObjectiveTo explore and establish the liver injury risk prediction model of indirect toxicity of Chinese medicinals under the condition of compound formulas， and provide new ideas and methods for the study of evaluation of liver injury of Chinese medicinals based on indirect toxicity. MethodsTaking Buguzhi （Fructus Psoraleae） pre-parations as model drug， the combined Chinese medicinals with Buguzhi （Fructus Psoraleae） of high frequency are screened out， and their components and action targets were obtained through TCMSP， TCMIP and PharmMapper databases. The association strength value and risk value of Chinese medicinals that acted on the nuclear factor κB （NF-κB） pathway were analyzed. For those having greater values than the median association strength value and risk value were regarded as indirect Chinese medicinals of liver injury risk. In this way， a prediction model of liver injury risk of Chinese medicinals was constructed based on immune activation-related indirect liver injury process （taking NF-κB pathway as an example）. And verification of the prediction model was performed using Heshouwu （Radix Polygoni Multiflori） preparations. ResultsThe prediction model of liver injury risk based on important immunoactivated pathway （taking NF-κB pathway as an example） found that Yinyanghuo （Herba Epimedii） （association strength value = 0.18， risk value = 0.25） was a Chinese medicinal with potential risk of indirect liver injury within Buguzhi （Fructus Psoraleae） prepartions， which may increase the risk of liver injury by positively regulating Bruton's tyrosine kinase （Btk） and protein kinase C theta （PKCθ） on NF-κB pathway. Further verification of prediction model by Heshouwu （Radix Polygoni Multiflori） preparations showed that Buguzhi （Fructus Psoraleae） （association strength value = 0.25， risk value = 0.33） and Tusizi （Semen Cuscutae） （Semen Cuscutae， association strength value = 0.34， risk value = 0.33） may increase the liver injury risk of Heshouzu. ConclusionThe liver injury risk prediction model of indirect toxicity of Chinese medicinals has been constructed in this study， providing metho-dological reference for the identification of Chinese medicinals of indirect liver injury risk under the condition of compound formulas.

Objective: To evaluate the effect of 3D-printed oval root canal preparation by using small field-of-view cone beam CT (CBCT) combined with computer-aided technologies. Methods: An extracted tooth with a suitable single oval canal was selected by small field-of-view CBCT scanning. Three-dimensional (3D)-printed resin teeth were obtained based on the CBCT data after data conversion and processing. 50 general dentists were selected to prepare the oval root canal of the resin teeth with X-Gold rotary Ni-Ti instruments. Small field-of-view CBCT was applied to scan the oval root canals before and after preparation. Then, computer-aided technologies were used to calculate and compare these CBCT data, analyzing the effects of oval root canal cleaning as well as the root canal deviation by 3D reconstruction and rendering of the images. Results : Among the 50 cases, the mean unprepared area of the oval root canal wall was（56.20 ± 11.91）% and the mean maximum deviation distance of the root canal was（0.140 ± 0.041）mm. There was no correlation between root canal cleanliness and deviation (r=0.18, P=0.212). Conclusion Combined with small field-of-view CBCT and computer-aided technology, we can effectively quantitatively evaluate the root canal preparation effect.

The main constituents of a typical medicinal herb,Polygonum muMflorum (Heshouwu in Chinese),that induces idiosyncratic liver injury remain unclear.Our previous work has shown that cotreatment with a nontoxic dose of lipopolysaccharide (LPS) and therapeutic dose of Heshouwu can induce liver injury in rats,whereas the solo treatment cannot induce observable injury.In the present work,using the constituent "knock-out" and "knock-in" strategy,we found that the ethyl acetate (EA) extract of Heshouwu displayed comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats.Results indicated a significant elevation of plasma alanine aminotransferase,aspartate aminotransferase,and liver histologic changes,whereas other separated fractions failed to induce liver injury.The mixture of EA extract with other separated fractions induced comparable idiosyncratic hepatotoxicity to the whole extract in LPS-treated rats.Chemical analysis further revealed that 2,3,5,4'-tetrahydroxy trans-stilbene-2-O-β-glucoside (trans-SG) and its cis-isomer were the two major compounds in EA extract.Furthermore,the isolated cis-,and not its trans-isomer,displayed comparable idiosyncratic hepatotoxicity to EA extract in LPS-treated rats.Higher contents of cis-SG were detected in Heshouwu liquor or preparations from actual liver intoxication patients associated with Heshouwu compared with general collected samples.In addition,plasma metabolomics analysis showed that cis-SG-disturbing enriched pathways remarkably differed from trans-SG ones in LPS-treated rats.All these results suggested that cis-SG was closely associated with the idiosyncratic hepatotoxicity of Heshouwu.Considering that the cis-trans isomerization of transSG was mediated by ultraviolet light or sunlight,our findings serve as reference for controlling photoisomerization in drug discovery and for the clinical use of Heshouwu and stilbene-related medications.

Objective Helicobacter pylori ( Hp ) infection is an important risk factor of the gastrointestinal disease, including chronic gastritis, peptic ulcer and gastric cancer. However, many recent experimental and clinical studies have shown that its addition to causing gastrointestinal diseases, but also associated with many diseases, and closely related with the occurrence of liver diseases. Summarized recent advances in the study of the relevant studies,including the relationship between hepatitis and cirrhosis,and the occurrence and development of liver cancer.

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.

To establish kinetic assay method for the analysis of hemolysis and to investigate dynamic hemolytic process of polysorbate 80. The UV-VIS spectrum of heme changes when hemoglobin is released continuously during the hemolytic process. Therefore, dynamic hemolytic curve was determined as a new way to characterize the kinetic process of interaction between polysorbate 80 and red blood cells. The effect of polysorbate 80 on blood cells could be perfectly investigated by the hemolytic dynamics. Dynamic hemolytic parameters of polysorbate 80 were calculated according to the hemolytic curves. The constants of hemolytic rate and maximum hemolytic rate of polysorbate 80 had fine linear relationships at the range of 1-20 mg x mL(-1) and 5-20 mg x mL(-1), respectively. In comparison with the present official method such as macroscopic observation and static spectrophotometric methods, kinetic spectrophotometry has the advantages of real time, on-line determination, sensitive, objective, good reproducibility and 2-dimensional information acquired. Therefore, as a biological technique, kinetic spectrophotometry could be applied to evaluate the quality of polysorbate 80 and to screen other solubilizing excipients.

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.

BACKGROUNDTo clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell.

METHODShTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection.

RESULTSElectrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line.

CONCLUSIONSThe hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.