Systematization of evaluation method for the antiinflammatory agent.

1.本项目的目的是建立一种合理的方法来评价药物的抗炎效果，因为可以根据炎症反应的病理生理方面的分析和炎症部位的化学介质的分析来进行评价。结论：1.利用本实验室建立的气囊型炎症实验模型，可以很好地说明该方法的原理。方法和结果：(1)采用氢氧化钠消化、乙醇澄清的方法，从大鼠真皮中几乎完全提取荧光素标记的牛血清白蛋白。用荧光分光光度法测上清液中白三烯的含量，以确定真皮炎症反应中的血浆渗出量。(2)建立了测定炎症囊液中白三烯的高效液相色谱方法。SEP-PAK C-18色谱柱被证明能有效地从囊液中提取白三烯。从色谱柱中分离出来的白三烯首先被装载到反相柱上。收集的每个白三烯组分再次加载到直相柱上进行最终测定。该方法成功地分离和测定了白三烯B4，而多肽三烯的回收率并不令人满意。因此，我们建立了测定单个气囊炎大鼠白三烯B4的测定方法。(3)我们的PGE高效液相测定试验没有成功，因为放射免疫法被证明是更方便地测定气囊液中的PGE。(4)目前建议合理评价抗炎药的方法是结合渗出液中的PGE水平来测量血浆渗出量，并结合渗出液中的趋化活性来测量白细胞的渗透。

1. Aim of the project: It is to develop a rational method for evaluating antiinflammatory efficacy of a drug in that the evaluation could be done based on the assay of pathophysiological aspects of inflammatory reaction together with the assay of chemical mediators in the site of inflammation. The principle of the assay may be well materialized by using experimental models of the inflammation of air pouch type which has been developed in this laboratory.2. Methods and the results:(1) Almost complete extraction of fluorescein-labeled bovine serum albumin from rat dermis was attempted with successful results by digesting the tissue with NaOH followed by clearing it with the aid of ethanol. The supernatant can be assayed in a fluorospectrometer to determine plasma exudation in dermal inflammatory response.(2) An HPLC method was investigated to determine leukotrienes in inflammatory pouch fluids. Sep-pak C-18 cartridge was proved to be effective in the extraction of leukotrienes from the pouch fluids. The leukotriene faction from the cartridge was loaded on a reverse phase column first. Each leukotriene fraction collected was again loaded on a straight phase column for the final determination. This was successful to separate and measure leukotriene B4, while recovery rates of peptidoleukotrienes were unsatisfactory. The assay method has been thus established for the measurement of leukotriene B4 for individual rats with the air pouch inflammation.(3) Our trial for HPLC measurement of PGE was unsuccessful, because radioimmunoassay has been shown to be more convenient to measure PGE in the pouch fluid.(4) The present proposal for the rational evaluation of antiinflammatory agents is to mesure plasma exudation in combination with the PGE level in the exudate and to measure leukocyte infiltration in combination with the chemotactic acitivity of the exudate.