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Development of a complex scintillation proximity assay for highthroughput screening of PPAR7 modulators

Acta Pharmacologica Sinica 2005年第3期26卷 339-344页

作者： Bin WU Jie GAO Ming-wei WANG~2 The National Center for Drug Screening,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of sciences graduate school of chinese academy of sciences,shanghai 201203,china The National Center for Drug Screening Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences graduate school of chinese academy of sciences Shanghai China

Aim:To develop a complex high-throughput screening （HTS） assay based onscintillation proximity assay （SPA） technology for identification of novel peroxi-some proliferator-activated receptor gamma （PPARγ） ***:Full-length PPARγ and retinoid X receptor alpha （RXRα）,biotinylated PPAR responseelement （PPRE）,[3H]BRL49653 and streptavidin-coated FlashPlate or microbeadwere used to develop an HTS assay based on SPA ***‘ABCDE’method was validated against conventional hydroxyapatite （HA） assay and ap-plied to large-scale screening of 16 000 synthetic compounds and natural ***:（1） IC50values of positive control compounds （BRL49653 andtroglitazone） obtained from the‘ABCDE’method and HA assay were compa-rable and consistent with those reported elsewhere;（2） Approximately 178compounds,showing more than 70% competitive inhibition on BRL49653 bind-ing to PPARγ,were identified initially by the‘ABCDE’method （microbead）;（3）Secondary screening using FlashPlate and cross-reactivity studies with RARα,β,γ and RXRα,β,γ confirmed that 12 compounds possessed specific PPARγ bind-ing properties including 2 with IC50values less than 0.5 μmol/L and novel chemi-cal ***:The‘ABCDE’method using either FlashPlate ormicrobead,is a highly efficient,automatable,and robust tool to screen potentialPPARγ modulators in HTS *** application may be expanded to othernuclear receptors that form heterodimers upon activation.

关键词： peroxisome proliferator-activated receptor gamma retinoid X receptor alpha scintillation proximity assay high-throughput screening

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Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes

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Acta Pharmacologica Sinica 2005年第8期26卷 969-975页

作者： Yan CAI~(1,2), Li-kun GONG~(1,2), Xin-ming QI~(1,2), Xiang-hong LI~1, Jin REN~(1,3) ~1State Key Laboratory of Drug Researeh, shanghai Institute of Materia Medica, shanghai Institutes for Biological sciences, chinese academy of sciences, shanghai 201203, china ~2graduate school of the chinese academy of sciences, shanghai 201203, china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim: To investigate the mitochondria-initiated apoptosis pathway involved in Carbon tetrachloride （CCl4） hepatotoxicity in vitro. Methods: Several cytotoxic-ity endpoints, including WST-8 metabolism, lactate dehydrogenase leakage and morphological changes, were examined. The 5,5'-dithio-bis（2-nitrobenzoic acid） reaction was used to measure reduced glutathione level, and the malondialdehyde level was determined using the thiobarbituric acid assay. The release of cyto-chrome c and Bcl-XLwas detected by Western blot. Caspase-3 activity was mea sured using the fluorogenic substrate Ac-DEVD-AMC. DNA fragmentation wasused to evaluate cell apoptosis. Results: A time-and dose-dependent decrease in cellular glutathione content was observed, along with a concomitant increase in malondialdehyde levels following the application of CCl4. Caspase 3 activity was stimulated at all doses of CCl4, with the most significant activation at 3 mmol/L. Cytochrome c was released obviously after CCl4treatment. A time-dependent decrease in BcI-XLexpression was observed. DNA fragmentation results revealed apoptosis and necrosis following CCl4treatment. Conclusion: Oxidative damage is one of the essential mechanisms of CCl4hepatotoxicity, which triggers apoptosis via the mitochondria-initiated pathway.

关键词： carbon tetrachloride hepatocytes mitochondria caspase 3 cytochrome c

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Prevention of lipopolysaccharide-induced injury by 3,5-dicaffeoylquinic acid in endothelial cells

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Acta Pharmacologica Sinica 2007年第8期28卷 1143-1148页

作者： Ruo-peng ZHA~(1,2) Wei XU~(1,2) Wen-yi WANG~(1,2) Li DONG~1 Yi-ping WANG~(1,3) ~1State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of Seiences,shanghai 201203,china ~2graduate school of the chinese academy of sciences,shanghai 201203,china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim:To investigate the effect of 3,5-dicaffeoylquinic acid（3,5-diCQA）onlipopolysaccharide（LPS）-induced injury in human dermal microvascular endothe-lial cells（HMEC-I）.Methods:The anti-oxidant effect was detected using themalondialdehyde（MDA）assay in a rat liver microsome model of lipid *** viability was analyzed using the 3-（4,5-dimethylthiazol-2-yl）-2,5diphenyltetrazolium bromide *** lipid peroxide injury was measured bylactate dehydrogenase（LDH）*** cells were detected by flowcytometry,and confirmed by DNA fragmentation ***-3 activitywas measured using a specific assay *** level of intracellular reactive oxygenspecies（ROS）was determined by flow cytometry with a 2,7-dichlorodihydro-fluorescein diacetate fluorescence ***:The exposure of microsomesto ascorbate-Fe2+resulted in lipoperoxidation according to an increase in the levelof *** formation decreased in a dose-dependent manner on treatmentwith 5,10,or 50μmol/L 3,*** with LPS for 16 h resulted in a 60%decrease in cell viability and an increase in LDH release from 47.6% to 61.5%.DNA laddering was observed by agarose gel *** level ofapoptotic cells peaked at 27% after treatment with LPS for 12 *** treat-ment with LPS for 12 h,intracellular ROS and caspase-3 activity ***-treatment with 3,5-diCQA at 5,10,or 50μmol/L for 1 h attenuated LPS-mediatedendothelial cell *** anti-apoptotic action of 3,5-diCQA was partiallydependent on its capacity for anti-oxidation and the suppression of ***:3,5-diCQA displays anti-oxidative and anti-apoptotic activ-ity in HMEC-1 due to scavenging of intracellular ROS induced by LPS,and thesuppression of caspase-3 activity.

关键词： 3 5-dicaffeoylquinic acid lipopolysaccharide human dermal microvascular endo-thelial cells reactive oxygen species apoptosis caspase-3

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Neuroprotective effects of cyclooxygenase-2 inhibitor celecoxib against toxicity of LPS-stimulated macrophages toward motor neurons

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Acta Pharmacologica Sinica 2005年第8期26卷 952-958页

作者： Yong HUANG, Jing LIU, Li-zhen WANG, Wei-yu ZHANG, Xing-zu ZHU~1 Department of Pharmacology, shanghai Institute of Materia Medica, shanghai Institutes for Biological sciences, chinese academy of sciences, graduate school of chinese academy of sciences, shanghai 201203, china Department of Pharmacology Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of Chinese Academy of Sciences Shanghai China

Aim: To establish an in vitro injured motor neuronal model and investigate the neuroprotective effects and possible mechanism of celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, on this model. Methods: After macrophages were stimulated with lipopolysaccharide (LPS)+interferon-γ(IFN-γ) in the pre-sence or absence of celecoxib for 24 h, the cell-free supernatant of LPS-stimulated macrophages was transferred to the culture of NSC34 cells. Viability of NSC34 cells was assessed by MTT assay after a further 24 h and 72 h incubation. After macrophages were stimulated by LPS+IFN-γ for 12 h or 24h, the release of pros-taglandin E2(PGE2), nitric oxide (NO), reactive oxygen species (ROS), tumor necro-sis factor α(TNF-α) and interleukin-1β (IL-1β) from macrophages was measured by radioimmunoassay, Griess assay, fluorescence assay and enzyme-linked immunosorbent assay, respectively. The mRNA levels of COX-2, inducible nitric oxide synthase (iNOS), TNF-α and IL-1β in macrophages were determined by reverse transcription-polymerase chain reaction after macrophages were stimu-lated for 6 h and 12 h. Results: The supernatant of LPS-stimulated mouse mac-rophages induced the death of NSC34 cells and celecoxib protected the NSC34cells against this toxicity. The LPS-induced increases in the release of PGE2, NO,TNF-α and IL-1β from macrophages were attenuated by pre-treatment with celecoxib. However, celecoxib showed no effect on the ROS levels upregulated by LPS+IFN-γin the macrophage supernatant. The mRNA levels of COX-2, iNOS, TNF-α and IL-1β were increased in LPS-activated macrophages and, except COX-2, reduced by pre-treatment with celecoxib. Conclusion: An in vitro injured motor neuronal model was established by using the toxicity of LPS-stimulated mouse macrophages toward motor neuronal NSC34 cells. In this model, celecoxib exerted neuroprotective effects on motor neurons via an inhibition of the neurotoxic secretions from activated macrophages.

关键词： amyotrophic lateral sclerosis celecoxib macrophage NSC34 cell lipopolysaccharide cyclooxygenase-2 prostaglandin E2 nitric oxide reactive oxygen species

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Altered expression of cytochrome P450 and possible correlation with preneoplastic changes in early stage of rat hepatocarcinogenesis

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Acta Pharmacologica Sinica 2005年第6期26卷 737-744页

作者： Lin-lin LIU~(2,3), Li-kun GONG~(2,3), Xin-ming QI~(2,3),Yan CAI~(2,3), Hui WANG~2, Xiong-fei WU~(2,3),Ying XIAO~(2,3), Jin REN~(2,4) ~2 State Key Laboratory of Drug Research, shanghai Institute of Materia Medica, shanghai Institutes for Biological sciences, chinese academy of sciences, shanghai 201203, china ~3 graduate school of the chinese academy of sciences, shanghai 201203, china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China graduate school of the chinese academy of sciences Shanghai China

Aim: Correlation of cytochrome P450 (CYPs) with preneoplastic changes in the early stage of hepatocarcinogenesis is still unclear. To detect the expression of carcinogen-metabolizing related microsomal P450 enzymes, namely the CYP1 Al, CYP1A2, CYP2B1/2, CYP2E1, and CYP3A, we performed the medium-term bioas-say of Ito's model in Sprague-Dawley rats. Methods: The amount and activity of CYP were assessed by biochemical and immunohistochemical methods in week 8. The correlation between CYP expression and microsomal oxidative stress was investigated by comparing the generation of microsomal lipid peroxidation in the presence or absence of specific CYP inhibitor. Results: In the DEN-2-AAF and 2-AAF alone groups, the expression of CYP1 Al and CYP2E1 were up-regulated and the expression of CYP2B1/2 and CYP1A2 were quite the contrary. Strong staining of CYP2E1 and CYP2B1/2 was found around the centrolobular vein and weak staining in the altered hepatic foci revealed by immunohistochemical procedure. There was no significant change in the activity of CYP3A among the 4 groups. Altered hepatic tissue bore more microsomal NADPH (nicotinamide adenine dimucleotide phosphate,reduced form)-dependent lipid peroxidation than normal tissue. And the difference among the 4 groups disappeared when CYP2E1 was inhibited. More microsomal lipid peroxidation was generated when incubated with CYP1A inhibitor a-naphthoflavone. Conclusion: CYP altered their expression levels and these alterations can play important roles in the alteration of cell redox status of preneoplastic tissue in the early stage of hepatocarcinogenesis.

关键词： cytochrome P450 preneoplastic changes hepatocarcinogensis

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Design,synthesis,and anti-inflammatory evaluation of a series of novel amino acid-binding 1,5-diarylpyrazole derivatives

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Acta Pharmacologica Sinica 2005年第7期26卷 865-872页

作者： Ming-hui LI Lin-lin YIN Mao-jun CAI Wei-yu ZHANG Yue HUANG Xin WANG Xing-zu ZHU~1 Jing-kang SHEN~1 State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of sciences,graduate school of the chinese academy of sciences,shanghai 201203,china State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Graduate School of the Chinese Academy of Sciences Shanghai China

Aim:To design and synthesize a series of novel amino acid-binding 1,5-diarylpyrazole derivatives,which are intended to act as prodrugs with better aque-ous solubility than celecoxib,and which will exert potent anti-inflammatory activi-ties after being converted to their parent compounds in ***:To intro-duce an amino acid,celecoxib analogs containing amino or methylamino groupwere synthesized first through multi-step chemical *** the synthesizedcompounds were screened in an intact cell-based assay in vitro and in carrag-eenan-induced mouse paw edema in *** active compounds were selectedfor further evaluation in a carrageenan-induced rat paw edema *** prelimi-nary pharmacokinetics experiments were conducted using high performance liq-uid chromatography/mass spectrometry(HPLC/MS).Results:Celecoxib,6 of the1,5-diarylpyrazole class of celecoxib analogs,and their amino acid derivatives(hydrochloride salts)were *** vitro screening,the hydrochloridesalts showed decreased inhibitory effects on cyclooxygenase(COX)-1 and COX-2 compared with their parent compounds,but some exhibited potent anti-inflam-matory activity in *** 4a was selected for further evaluation,and itsanti-inflammatory effect was equivalent to that of celecoxib after oral administra-tion in the carrageenan-induced rat paw edema *** three doses(25 mg/kg,50 mg/kg,and 100 mg/kg)the percentage inhibition on edema was 20.7%,52.6%,and 62.6%(for compound 4a)and 27.8%,38.4%,and 40.1%(for celecoxib),*** pharmacokinetic evaluations support the hypothesisthat compound 4a was actually converted to its parent compound,compound ***:The compound bound with amino acid acts like prodrug,which canexert anti-inflammatory effect similar to celecoxib after being converted to its par-ent *** finding will be of great benefit in carrying out structuralmodifications of prodrug-like selective COX-2 inhibitors.

关键词： nonsteroidal anti-inflammatory agents cyclo oxygenase inhibitors celecoxib pyrazole sulfonamide prodrugs

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Insulin-sensitizing effects of a novel α-methyl-α-phenoxylpropionate derivative in vitro

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Acta Pharmacologica Sinica 2007年第3期28卷 417-422页

作者： Hao-shu WU~(2,4) Juan-hong YU~(3,4) Ying-yin LI~2 Yu-she YANG~3 Qiao-jun HE~2 Yi-jia LOU~(2,5) Ru-yun JI~3 ~2Department of Pharmacology and Toxicology,College of Pharmaceutical sciences,Zhejiang University,Hangzhou 310058,china ~3State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,graduate school of the chinese academy of sciences,shanghai 201203,china Department of Pharmacology and Toxicology College of Pharmaceutical Sciences Zhejiang University Hangzhou China Department of State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Graduate School of the Chinese Academy of Sciences Shanghai China

Aim:To examine the insulin sensitizing effects of a novel α-methyl-α-phenoxylpropionate derivative YY20 in insulin-sensitive cell ***:Theperoxisome proliferator-activated receptor γ（PPARγ）agonist bioactivities of YY20were detected by a preadipocyte differentiation ***-PCR and Westernblotting analysis were used to detect the expression of the target gene or *** effects of YY20 on insulin-mediated glucose consumption were determined inthe HepG2 human hepatocellular carcinoma ***:YY20 could enhancethe differentiation of preadipocytes to adipocytes and upregulate the gene ex-pression of PPARγ2,as well as the protein expression of insulin receptor sub-strate-1（IRS-1）,glucose transporter-4（GLUT4）,and adiponectin（ACRP30）.Theeffects on GLUT4 and ACRP30 could be reversed by the PPARγ inhibitor ***,YY20 efficiently reduced glucose consumptions in HepG2 cells after24 h culture,and the effects were related to insulin and YY20 ***:YY20,a potential insulin-sensitizing agent like rosiglitazone,couldenhance glucose consumption in HepG2 cells in a concentration-and insulin-dependent *** may improve the insulin resistance associated with type 2diabetes.

关键词： YY20 peroxisome proliferator-activated receptor y insulin sensitivity glucose consumption

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Design,synthesis,antitumor evaluations and molecular modeling studies of novel 3,5-substituted indolin-2-one derivatives

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Acta Pharmacologica Sinica 2007年第1期28卷 140-152页

作者： Hai-hong LI~2 Xiu-hua ZHANG~3 Jin-zhi TAN~2 Li-li CHEN~2 Hong LIU~(2,5) Xiao-min LUO~(2,5) Xu SHEN~(2,4) Li-ping LIN~(3,5) Kai-xian CHEN~2 Jian DING~3 Hua-liang JIANG~(2,4)2 Center for Drug Discovery and Design,State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,and graduate school,chinese academy of sciences,shanghai 201203,china 3 Division of Anti-tumor Pharmacology,shanghai Institute of Materia Medica,shanghai Institutes for Biological sciences,chinese academy of sciences,shanghai 201203,china 4 school of Pharmacy,East china University of Science and Technology,shanghai 200237,china Center for Drug Discovery and Design State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences and Graduate School Chinese Academy of Sciences Shanghai China Division of Anti-tumor Pharmacology Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China school of Pharmacy East China University of Science and Technology Shanghai China

Aim:To design and synthesize a novel class of antitumor agents,featuring the 3,5-substituted indolin-2-one ***:Based on enzyme binding fea-tures of（Z）-SU5402,introducing a β-pyrrole group at the 3-position of the indolin-2-one core,a series of novel 3,5-substituted indolin-2-ones were designed *** human carcinoma cell lines of A-43l,A-549,MDA-MB-468,and Autosomal Dominant Polycystic Kidney disease were chosen for the cellproliferation ***:Twenty new compounds（1a-t）with E configurationhave been designed,synthesized and *** structural features weredetermined by nuclear magnetic resonance（NMR）spectra,low-and high-resolu-tion mass spectra,and confirmed by X-ray *** the enzymeassay showed a weak inhibition effect against the epidermal growth factor receptor,vascular endothelial growth factor receptor,fibroblast growth factor receptor andplatelet-derived growth factor receptor tyrosine kinases,the cell-based antitumoractivity was *** 1 g and lh showed higher inhibitory activitytoward the A-549 and MDA-MB-468 cell lines with IC50of 0.065-9.4 μmol/***:This study provides a new template for further development ofpotent antitumor drugs.

关键词： protein-tyrosine kinase indolin-2-one antitumor drug screening assays drug design molecular models

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In vitro Potentiation of Antimalarial Activities by Daphnetin Derivatives Against Plasmodium falciparum

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Biomedical and Environmental sciences 2006年第5期19卷 367-370页

作者： FANG HUANG LIN-HUA TANG LIN-QIAN YU YI-CHANG NI QIN-MEI WANG FA-JUN NAN National Institute of Parasite Diseases Chinese Center for Disease Control and Prevention Shanghai 200025 China chinese National Center for Drug Screening Shanghai Institute of Materia Medica Shanghai Institute for Biological Sciences Graduate School of the Chinese Academy of Sciences Shanghai 201203 China

Objective To screen the antimalarial compounds of daphnetin derivatives against Plasmodium falciparum in vitro. Method Plasmodium faciparum （FCC1） was cultured in vitro by a modified method of Trager and Jensen. Antimalarial compounds were screened by microscopy-based assay and microfluorimetric method. Results DA79 and DA78 showed potent antimalarial activity against Plasmodiumfalciparum cultured in vitro. Conclusion Though the relationship between the structures of daphnetin derivatives and their antimalarial activities has not been clarified yet, this study may provide a new direction for discovery of more potential antimalarial compounds.

关键词： Daphnetin Antimalarial Drug screening

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2023 chinese national clinical practice guideline on diagnosis and management of Crohn’s disease

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中华医学杂志(英文版) 2024年第14期137卷 2024 Jul 2页

作者： Inflammatory Bowel Disease Group, chinese Society of Gastroenterology, chinese Medical Association Inflammatory Bowel Disease Quality Control Center of china Xijing Hospital Air Force Medical University China Peking Union Medical College Hospital China West china Hospital Sichuan University China The First Affiliated Hospital Dalian Medical University China University of Nottingham Ningbo China GRADE Centre United Kingdom The First Affiliated Hospital Sun Yat-sen University China Sir Run Run Shaw Hospital Zhejiang University School of Medicine China Peking University People's Hospital China The Second Affiliated Hospital Zhejiang University School of Medicine China Renmin Hospital Wuhan University China chinese PLA General Hospital China The First Affiliated Hospital Naval Medical University China The Sixth Affiliated Hospital Sun Yat-sen University China The First Affiliated Hospital Anhui Medical University China Zhongshan Hospital Xiamen University China Zhongnan Hospital Wuhan University China The First Hospital of Shanxi Medical University China The Eighth Affiliated Hospital Sun Yat-sen University China Peking University Third Hospital China Peking University First Hospital China Qilu Hospital Shandong University China General Hospital of Eastern Theater Command China Xiangya Hospital Central South University China The Tenth People's Hospital Tongji University China The First Affiliated Hospital Kunming Medical University China shanghai Pudong New District Zhoupu Hospital China Ruijin Hospital Shanghai Jiao Tong University School of Medicine China Tongji Medical College Huazhong University of Science and Technology China General Hospital of Nanjing Military Command China The Third Xiangya Hospital Central South University China Daping Hospital Chongqing China The Second Xiangya Hospital Central South University China Jiangsu Provincial People's Hospital China The Second Hospital of Hebei Medical University China Nanjing Drum Tower Hospital China Nanfan Hospital Southern Medical University China GRADE Center University of Nottingham Ningbo China The Fifth Affiliated Hospital Zhengzhou University

The role of chinese expert consensus in standardization and improvement of the clinical diagnosis and treatment of inflammatory bowel disease (IBD) is self-evident. As clinical and basic research in china becomes standardized under a better understanding of IBD, more data on chinese patients have become available to develop a consensus and guideline. This guideline, collaboratively developed by the IBD Group, chinese Society of Gastroenterology, incorporates the latest international consensus statements,[1-7] domestic research findings, and practical considerations, as an update based on the 2018 chinese consensus on IBD management.[8] The IBD guideline consists of two parts: ulcerative colitis (UC) and Crohn's disease, and this manuscript presented the UC part. The evidence used in this guideline was collected and analyzed by the standard of guideline, and the contents were organized as problem statements, for clarity. The formulation of this guideline aimed to reflect the latest progress in IBD, providing comprehensive and valuable guidance for the clinical management of IBD.

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