of cytosol, 2 mM GTP-c-S, and 100 mM sodium dodecyl-sulfate. Drugs 
were incubated for 2 min before NADPH oxidase assembly by SDS. 4. Assay of Superoxide Anion Production The assay of the generation of superoxide anion was based on the superoxide dismutase -inhibitable reduction of ferricytochrome c. In brief, after supplementation with 0.5 mg/ml ferricytochrome c and 1 mM Ca2+, neutrophils were equilibrated at 37uC PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19718258 for 2 min and incubated with ursolic acid for 5 min. Cells were activated with 100 nM N-formylmethionyl-leucyl-phenylalanine in the pre-priming with 1 mg/ml cytochalasin B for 3 min. Changes in absorbance with the reduction of ferricytochrome c at 550 nm were continuously monitored with a double-beam, six-cell positioner spectrophotometer with constant stirring. 5 min. Cells were activated by FMLP in the presence of CB, and changes in absorbance at 405 nm were continually monitored for 15 min. The results are expressed as a percentage of elastase release in the FMLP/CB-activated, drugfree control system. 6. Superoxide Anion-Scavenging Activity The superoxide anion-scavenging ability of ursolic acid was determined using xanthine/xanthine oxidase in a cell-free system. After 0.1 mM xanthine was added to the assay buffer for 15 min at 30uC, the absorbance associated with the superoxide anion-mediated WST-1 reduction was measured at 450 nm. 5. Assay of Elastase Release The assay of the release of elastase was performed using MeOSuc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate. Briefly, after supplementation with MeO-Suc-Ala-AlaPro-Val-p-nitroanilide, neutrophils were equilibrated at 37uC for 2 min and incubated with ursolic acid for 7. Rats Adult male Sprague-Dawley rats were used in this study. All animal experiments were performed according to the guidelines of the Animal Welfare Act and The Guide for Care and Use of Laboratory Animals from the National Institutes of Health. All procedures and protocols were approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital. The rats were obtained from the National Science Council Experimental Animal Center. 8. Rat Trauma-Hemorrhagic Shock Model A trauma-hemorrhagic shock rat model was used in the study. Twenty-four male Sprague-Dawley rats were divided into 4 groups according to a table of random numbers. All rats were caged individually in the animal house with controlled humidity 7075%, temperature and lighting. The animals were provided with water and basal diet and at least PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19718802 1 week was allowed for the animals to adapt to the environment. Animals were fasted overnight but allowed free water access before the experiment. Trauma-hemorrhagic shock and resuscitation was then performed as described previously. In brief, rats were anesthetized with isoflurane inhalation, and soft tissue trauma was performed with midline laparotomy. The polyethylene catheters were placed in the right femoral vein and Effect of Ursolic Acid following Trauma-Hemorrhagic Shock or the vehicle at 30 minutes from the beginning of the resuscitation period. Sham animals underwent all procedures; purchase 518303-20-3 however, neither resuscitation nor hemorrhage was performed. Ursolic acid or vehicle was administered in sham rats. The rats were humanely sacrificed at 24 hours after sham operation or resuscitation. 9. Assay of Hepatic Injury At 24 hours after trauma-hemorrhagic shock or sham operation, blood samples were drawn. Hepatic injury was measured with both femoral arteries. To reduce