阿托伐他汀对TNF-α和IL-1β诱导的大鼠主动脉内皮细胞PAPP-A表达的影响

Effects of atorvastatin on expression of PAPP-A induced by TNF-α and IL-1β in rat aortic endothelial cells

目的:研究阿托伐他汀对炎症因子肿瘤坏死因子α(TNF-α)和白细胞介素-1β(IL-1β)诱导大鼠主动脉内皮细胞表达妊娠相关血浆蛋白A(PAPP-A)的影响。方法:原代培养大鼠主动脉内皮细胞,选择生长良好的第3-4代细胞用于实验。实验分组:(1)空白对照组:不加干预措施原培养液继续培养;(2)阿托伐他汀浓度组:加入浓度分别为0.1、1、10μmol/L的阿托伐他汀作用24 h;(3)阿托伐他汀时间组:加入浓度为10μmol/L的阿托伐他汀分别作用6、12、24 h;(4)阿托伐他汀干预组:加入TNF-α(60μg/L)或者IL-1β(20μg/L)作用1 h后,再加入不同浓度的阿托伐他汀(0.1、1、10μmol/L)进行培养,分别于作用6、12、24 h终止实验。MTT法观察药物对细胞增殖的影响,RT-PCR技术测定细胞中PAPP-A mRNA的表达,ELISA方法检测上清液中PAPP-A蛋白水平。结果:(1)外源性加入阿托伐他汀及TNF-α或者IL-1β后,分别作用3、6、12、24、48 h,同对照组相比,细胞增殖能力无明显变化(P<0.05),说明干预药物本身对细胞没有毒性作用。(2)单独加入阿托伐他汀,细胞PAPP-A表达水平与空白对照组比较没有明显差异(P<0.05)。(3)炎症因子作用一段时间后再加入阿托伐他汀,PAPP-A mRNA和蛋白表达水平随着阿托伐他汀浓度和作用时间的增加而逐渐降低,有一定的浓度-时间依赖关系,与TNF-α组和IL-1β组比较明显下降(P<0.05)。结论:阿托伐他汀本身对细胞表达PAPP-A没有影响,但在一定程度上抑制TNF-α和IL-1β诱导的内皮细胞PAPP-A的表达,并有浓度-时间依赖关系。

AIM： To investigate the effects of atorvastatin on the expression of pregnancy-associated plasma protein A（PAPP-A）induced by TNF-α and IL-1β in endothelial cells.METHODS： The rat aortic endothelial cells were isolated from thoracic aortas and cultured by the tissue explant method.The cells in passage 3-4 were used in the experiment and were randomly divided into 4 groups： blank control group： the cells were treated without any intervention;atorvastatin concentration groups： the cells were incubated with atorvastatin at the concentrations of 0.1,1 and 10 μmol/L for 24 h;atorvastatin time groups： the cells were incubated with atorvastatin at the concentration of 10 μmol/L for 6 h,12 h and 24 h;atorvastatin＋inflammatory factors groups： the cells were pre-incubated with 60 μg/L TNF-α or 20 μg/L IL-1β for 1 h,then different concentrations of atorvastatin（0.1,1.0,10 μmol/L） were added for 6 h,12 h and 24 h.MTT reduction assay was used to observe the cell proliferation.The mRNA expression of PAPP-A was detected by RT-PCR.The protein level of PAPP-A in the supernatants of cultured cells was measured by ELISA.RESULTS： Compared with blank control group,no significant change of cell proliferation was observed after the intervention of atorvastatin and TNF-α/IL-1β for 3 h,6 h,12 h,24 h and 48 h,indicating that the drugs had no toxic effects on the cells.No significant difference of PAPP-A expression between atorvastatin groups and blank control groups was found.Compared with TNF-α groups and IL-1β groups,PAPP-A expressions in atorvastatin intervention groups significantly decreased.The protein level of PAPP-A was gradually decreased with the raised concentration of atorvastatin and the prolonged time in a concentration-and time-dependent manner.CONCLUSION： Atorvastatin doesn＇t influence the PAPP-A expression,but inhibits the expression of PAPP-A activated by inflammatory factors in a concentration-and time-dependent manner in primary cultured rat aortic endothelial cells.