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目的:肥大细胞(MCs)类胰蛋白酶是一种丝氨酸蛋白酶,目前未发现其体内内生的抑制剂。本研究用水蛭源性类胰蛋白酶抑制剂(LDTI)研究MCs颗粒释放液抑制THP-1巨噬细胞源性泡沫细胞胆固醇流出的机制。方法:用Compound 48/80刺激大鼠腹腔MCs活化,制备颗粒释放液。荧光分光光度仪检测MCs组胺释放率。采用丙酮抽提LDTI并用反相高效液相纯化。酶法检测MCs颗粒释放液中类胰蛋白酶的活性。用15%非还原聚丙烯酰胺凝胶电泳检测HDL3的改变。用高效液相色谱检测细胞内总胆固醇(TC)、游离胆固醇(FC)和胆固醇酯(CE)含量,液体闪烁计数器检测细胞内胆固醇流出。结果:MCs颗粒释放液使泡沫细胞内胆固醇流出减少,总、游离胆固醇蓄积增加,HDL3的载脂蛋白A-Ⅰ降解;而用LDTI抑制MCs颗粒释放液中类胰蛋白酶的活性后,可取消MCs颗粒释放液的这些作用。结论:LDTI能抑制MCs颗粒释放液的作用,表明类胰蛋白酶可以抑制胆固醇外流,而且与HDL3的apoA-Ⅰ的降解有关。
AIM: Mast cell (MCs) tryptase is a serine protease and no endogenous inhibitor has been found in vivo. In this study, the leech-derived tryptase inhibitor (LDTI) was used to study the mechanism of MCs particle release inhibiting the cholesterol efflux from THP-1 macrophage-derived foam cells. Methods: Compound 48/80 was used to stimulate rat peritoneal MCs activation to prepare particle release solution. Fluorescence spectrophotometer was used to detect the histamine release rate of MCs. LDTI was extracted with acetone and purified by reverse-phase high performance liquid chromatography. Enzymatic detection of MCs particle release tryptase activity. HDL3 changes were detected by 15% non-reducing polyacrylamide gel electrophoresis. The contents of total cholesterol (TC), free cholesterol (FC) and cholesterol ester (CE) in the cells were detected by HPLC, and the efflux of intracellular cholesterol was detected by liquid scintillation counter. Results: The release of MCs particles reduced the efflux of cholesterol in foam cells, increased the total and free cholesterol accumulation, and degraded apolipoprotein A-Ⅰ in HDL3. However, the inhibitory effect of LDTI on the activity of tryptase in MCs particles release MCs These effects of particle release fluid. Conclusion: LDTI can inhibit the release of MCs particles, indicating that tryptase can inhibit cholesterol efflux, but also with HDL3 apoA-Ⅰ degradation.