赤芍801对小鼠巨噬细胞COX-1和COX-2活性、mRNA及蛋白表达的影响

目的研究赤芍801(propyl gallate,PrG)对环氧酶(cy-c looxygenase,COX)活性、mRNA及蛋白表达的影响。方法采用基于小鼠腹腔巨噬细胞的COX-1和COX-2体外筛选模型,用钙离子导入剂(calc ium ionophore A23187)短时刺激小鼠腹腔巨噬细胞,测定培养上清液中的6酮--前列腺素F1α(6-keto-PGFlα)的量反映COX-1活性;用脂多糖(lipopolysac-charide,LPS)长时间刺激细胞,测定培养上清液中的前列腺素E2(PGE2)的量反映COX-2活性。半定量RT-PCR法检测PrG对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2 mRNA表达的影响。W estern b lot法检测PrG对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2蛋白表达的影响。结果PrG体外10,5μmol.L-1浓度下不影响6-keto-PGFαl生成(P>0.05),而在1,0.5,0.1,0.05μmol.L-1浓度下则诱导6-keto-PGF1α生成(P<0.01),并呈较好的剂量依赖性。PrG体外1,10μmol.L-1可抑制PGE2生成(P<0.05)。PrG不同浓度对LPS刺激的Raw小鼠巨噬细胞COX-1、COX-2 mR-NA表达无明显影响。PrG(100,10,1μmol.L-1)浓度下可抑制COX-2蛋白表达,但对COX-1蛋白表达无影响。结论在0.05~10μmol.L-1浓度范围内,PrG低浓度时促进6-keto-PGF1α生成,高浓度时抑制PGE2生成,提示其高浓度时抑制COX-2,低浓度时激活COX-1;不同浓度PrG对COX-1、COX-2 mRNA表达均无明显影响。PrG(1~100μmol.L-1)浓度下可抑制COX-2蛋白表达,但对COX-1蛋白表达无影响。

Effect of propyl gallate on cyclooxygenase-1/-2 activity, mRNA and protein expression in murine macrophages

Aim This study aimed to investigate effects of propyl gallate(PrG) on cyclooxygenase-1 and-2(COX-1/-2)activities,mRNA and protein expression in murine macrophages.Methods A screening model for COX inhibitors in vitro based on murine peritoneal macrophages was used.COX-1 activity was determined by the level of 6-keto-PGF_(1α) in supernatants of cultured macrophages which were stimulated withcalcium ionophore A23187 for 1 h,while COX-2 activity was determined by the level of PGE_2 in supernatants of cultured macrophages which were stimulated with lipopolysaccharide(LPS) for 9 hours.Semi-quantative RT-PCR was used to determine the effect of PrG on mRNA expression of COX-1 and COX-2 in RAW murine macrophage cell line stimulated by LPS.Western blotting was used to determine the effect of PrG on protein expression of COX-1 and COX-2 in RAW cells stimulated by LPS.Results PrG did not affect 6-keto-PGF_(1α) synthesis at concentrations of 5 and 10 μmol·L~(-1)(P>0.05),but enhanced 6-keto-PGF_(1α) synthesis at concentrations of 1,0.5,0.1 and 0.01 μmol·L~(-1)(P<0.01)in vitro.It inhibited PGE_2 synthesis at concentrations of 10 and 1 μmol·L~(-1)(P<0.05).PrG at tested concentrations did not affect mRNA expression of COX-1 and COX-2.PrG at concentrations of 100,10 and 1 μmol·L~(-1) inhibited COX-2 protein expression,while showed no effect on COX-1 protein expression.Conclusion Within the range of 0.05~10 μmol·L~(-1),PrG activated COX-1 at lower concentrations and inhibited COX-2 at higher concentrations in murine peritoneal macrophages.PrG had no effect on COX-1 and COX-2 mRNA expression,while PrG could inhibit COX-2 protein expression.