辛伐他汀抑制人外周血单核巨噬细胞脂蛋白相关磷脂酶A2表达

Simvastatin suppress lipopolysaccharides induced upregulation of lipoprotein associated phospholipase A2 expression in macrohages via inactivation of p38MAPK pathway

目的 观察辛伐他汀对人外周血单核巨噬细胞脂蛋白相关磷脂酶A2（Lp-PLA2）表达的影响,并探讨其调控机制.方法 分离培养人外周血单核巨噬细胞,实验分为脂多糖（LPS）组、辛伐他汀组和丝裂原活化蛋白激酶（MAPK）干预组.LPS组：分别用不同浓度（0、1、10、102、103和104ng/ml）的LPS与细胞共同孵育6 h,观察不同浓度的辛伐他汀对LPS诱导的Lp-PLA2 mRNA和蛋白表达的影响 并用1μg/ml的LPS与细胞孵育不同时间（0、6、12、24和48 h）,观察辛伐他汀作用不同时间对LPS诱导的Lp-PLA2 mRNA和蛋白表达的影响.辛伐他汀组：1 μg/ml的LPS＋不同浓度的辛伐他汀（10-2～10-7mmol/L）与单核巨噬细胞共同孵育24 h,1 μg/ml LPS＋10-3mmol/L的辛伐他汀与单核巨噬细胞孵育不同时间（0、6、24、24和48 h）,观察辛伐他汀对LPS诱导的Lp-PLA2mRNA和蛋白表达及酶活性的影响.MAPK组：分别用10 μmol/L的p38抑制剂SB203580、20 μmol/L的ERK抑制剂U0126和20 μmol/L的JNK抑制剂SP600125预处理30 min后,将单核巨噬细胞与1μg/ml的LPS共同孵育24 h,观察MAPK信号通路在LPS介导的Lp-PLA2表达中的作用.逆转录-多聚酶链反应（RT-PCR）方法 检测Lp-PLA2 mRNA表达,比色法测定酶活性,Western blot方法 检测Lp-PLA2蛋白表达以及p38-MAPK蛋白及磷酸化水平.结果 （1）0.1μg/ml的LPS刺激6 h即可显著增加单核巨噬细胞Lp-PLA2 mRNA和蛋白的表达及其酶活性,并且随LPS浓度的增加和刺激时间的延长,该作用增强.（2）辛伐他汀可以明显抑制LPS诱导的Lp-PLA2的表达增加,并且降低酶活性,该作用呈浓度及时间依赖性.（3）辛伐他汀抑制LPS诱导的p38MAPK蛋白活化,p38MAPK的抑制剂SB203580可以完全阻断LPS介导的Lp-PLA2蛋白表达增加,与辛伐他汀作用相似.而MEK1/2的抑制剂U0126和JNK的抑制剂SP600125对LPS介导的Lp-PLA2蛋白表达的增加没有影响.结论 在培养的人外周血单核巨噬细胞中,辛伐他汀可以明显抑制LPS诱导的Lp-PLA2 mRNA和蛋白表达,降低Lp-PLA2酶活性,该作用至少部分由抑制p38MAPK信号转导通路介导.

Objective To investigate the effects of simvastatin on lipopolysaccharides （LPS）induced upregulation of Lp-PLA2 in human peripheral blood monocytes-macrophages and the related mechanisms. Methods Peripheral blood monocytes of healthy volunteer were isolated and incubated for 2-3 days. Monocytes were incubated with various concentrations of LPS for 6 h or with 1 μg/ml of LPS for different times in LPS group. In simvastatin group and MAPK inhibitors groups, cells were pre-treated with simvastatin （ 10-2 - 10-7mmol/L）or various MAPK inhibitors （ 10 μmol/L SB203580, 20 μmol/L U0126,and 20 μmol/L SP600125） before LPS co-incubation. Lp-PLA2 activity was measured by chromometry, LpPLA2 mRNA expression was detected by RT-PCR. Protein expressions of Lp-PLA2 and p38MAPK and phosphorylated p38MAPK were examined by Western blot. Results （ 1 ） LPS significantly upregulated LpPLA2 mRNA and protein expression, as well as the enzyme activity in a time and concentration dependentmanner, which could be significantly attenuated by simvastatin in a time and concentration dependent manner. （2） Simvastatin significantly reduced LPS-induced p38MAPK phosphorylation. The p38 MAPK inhibitor SB203580, but not MEK1/2 inhibitor U0126 and JNK inhibitor SP600125, completely prevented LPS-mediated up-regulation of Lp-PLA2 at protein level. Conclusion This study demonstrated that LPS significantly upregulated Lp-PLA2 mRNA and protein expression, as well as the enzyme activity in a time and concentration dependent manner via Rho-p38MAPK pathway, which could be significantly suppressed by simvastatin.