脂多糖诱导大鼠主动脉内皮细胞蛋白C受体和蛋白酶活化受体1的表达及血必净注射液的干预作用

摘要

目的 观察脂多糖(LPS)诱导的大鼠主动脉内皮细胞蛋白C受体(EPCR)和蛋白酶活化受体1(PAR1)的表达,并探讨血必净注射液对其的干预作用.方法 采用组织贴块法培养Wistar大鼠主动脉内皮细胞,培养1周后用流式细胞仪鉴定内皮细胞纯度.按1∶3传代至3～4代时,随机将细胞分为空白对照组、LPS刺激组(1 mg/L)、血必净干预组(LPS 1 mg/L,血必净注射液10 g/L),活化蛋白C(APC)干预组(LPS 1 mg/L,APC 0.1 mg/L).分别在12、24、48、72 h采用逆转录-聚合酶链反应(RT-PCR)测定内皮细胞EPCR和PAR1的mRNA表达;用流式细胞仪检测EPCR和PAR1的蛋白表达.结果 12 h时各组间EPCR和PAR1蛋白表达水平比较差异均无统计学意义(P均>0.05);LPS刺激组EPCR和PAR1 mRNA表达则均降低,APC和血必净干预后两值均升高(P<0.05或P<0.01).24～72 h LPS刺激组EPCR的mRNA和蛋白表达水平均明显低于空白对照组(P<0.05或P<0.01),EPCR的表达随LPS作用时间延长而减少,呈明显的负相关;而血必净干预组EPCR表达明显高于LPS刺激组(P均<0.01).LPS刺激组PAR1的mRNA和蛋白表达水平较空白对照组有所降低(P<0.05或P<0.01),用血必净干预后,PAR1的mRNA和蛋白表达水平有所增加(P均<0.01).与APC干预组比较,血必净干预组EPCR和PAR1的蛋白表达增加更为明显.结论 血必净注射液能从基因和蛋白水平增加由LPS诱导大鼠主动脉EPCR和PAR1的表达作用,可能与其保护内皮细胞的功能抑制其凋亡有关.%Objective To investigate the effect of Xuebijing injection (血必净注射液) on expression of endothelial protein C receptor (EPCR) and protease activated receptor 1 (PAR1) mRNA and protein in rat aortic endothelial cells (RAECs) after lipopolysaccharide (LPS) challenge. Methods RAECs were cultured for one week, and the purity was determined with flow cytometry. ARECs were randomly divided into four groups: control group, LPS stimulation group (1 mg/L LPS), Xuebijing injection treatment group (LPS: 1 mg/L, Xuebijing injection: 10 g/L) and activated protein C (APC) treatment group (LPS: 1 mg/L, APC: 0.1 mg/L). The RAECs were collected at 12, 24, 48, and 72 hours after being stimulated by LPS. EPCR and PAR1 mRNA of RAECs were assessed by reverse transcription-polymerase chain reaction (RT-PCR). EPCR and PAR1 protein were assessed by flow cytometry. Results At 12 hours, EPCR and PAR1 protein expressions were not significantly different among groups (all P>0.05). The level of EPCR and PAR1 mRNA were decreased in LPS stimulation group, but they were elevated in both APC and Xuebijing injection treatment groups (P<0.05 or P<0.01). From 24 to 72 hours, compared to control group, the levels of EPCR mRNA and protein expression were significantly decreased after LPS stimulation (P<0.05 or P<0.01). The levels of EPCR expression were decreased and negatively correlated with the time of LPS treatment. Also, compared to LPS stimulation group, treatment with Xuebijing markedly elevated the levels of EPCR (P<0.01). The levels of PAR1 expression were significantly decreased by LPS stimulation compared with those of control group (P<0.05 or P<0.01). After the treatment with Xuebijng, the expression of PAR1 was gradually increased (all P<0.01). Compared with APC treatment group, Xuebijing could increase the PAR1 expression better. Conclusion Xuebijing could raise EPCR and PAR1 mRNA and protein expression of RAECs after LPS challenge, and it may be related to its protection of endothelial cell from undergoing apoptosis.