银杏苦内酯B对脂多糖致MS1细胞炎症反应中P38丝裂原活化蛋白激酶的活化作用

The activation of ginkgolide B on P38 MAPK in inflammatory reaction of MS1 cells induced by lipopolysaccharide

【目的】探讨脂多糖(lipopolysaccharide,LPS)刺激的胰腺微血管内皮细胞(MS1细胞)中P38丝裂原活化蛋白激酶(P38mitogen activated protein kinase,P38 MAPK)基因表达量变化和蛋白的磷酸化激活及BN52021对其的干预,阐明BN52021的作用机制。【方法】MTT法探索LPS导致MS1细胞炎症反应的最佳浓度和作用时间点,以该浓度时间点为前提,确定BN52021对MS1细胞炎症反应的最佳抑制浓度。通过Real-time PCR、Western blotting技术检测P38 MAPK基因在不同组之间表达量的变化及蛋白磷酸化激活情况。【结果】MTT法显示当LPS浓度达到10μg/ml,作用时间点为24 h时,MS1细胞株活性(OD490:0.25±0.01)与未作处理对照组细胞活性(OD490:0.63±0.01)相比,前者对细胞产生明显损伤(P<0.05);BN52021在浓度达到50 mmol/L时细胞活性(OD490:0.20±0.03)与LPS阳性对照组细胞活性(OD490:0.14±0.02)相比,前者的细胞活性明显提高(P<0.05)。Realtime PCR结果显示LPS诱导的MS1细胞株炎症状态下P38 MAPK(F=3.87±0.07)基因表达明显上调(P<0.05),在BN52021干预下上调的P38 MAPK基因表达量相比LPS诱导组明显下调(F=1.49±0.18)(P<0.05)。Western blotting蛋白磷酸化激活符合基因表达情况。【结论】BN52021能通过下调P38 MAPK的基因表达和蛋白磷酸化激活有效地抑制LPS所诱导的MS1细胞株的炎症。

【Objective】To explore the effect of ginkgolide B（BN52021） on gene expression and protein phosphorylation of P38 mitogen activated protein kinase（P38 MAPK）in lipopolysaccharide（LPS） stimulated pancreatic microvascular endothelial cells（MS1 cells） and illuminate the mechanism of BN52021.【Methods】The optimal concentration and best time point of LPS and BN52021 were determined by MTT in LPS stimulated MS1 cells. The gene expression and protein phosphorylation of P38 MAPK in different groups were determined by real-time PCR and Western blotting.【Results】The MTT results showed that 10 μg/ml LPS for 24 h led to the significant damage to MS1 cells（OD4900.25±0.01,P〈0.05）compared with the control group（OD4900.63±0.01,P〈0.05）. The MS1 cells activity in50 mmol/L BN52021 group increased obviously（OD490 0.20±0.03,P〈0.05） compared with LPS treated group（OD4900.14±0.02,P〈0.05）. Real-time PCR results showed that the gene expression of P38 MAPK increased significantly in LPS treated MS1 cells（F=3.87±0.07, P〈0.05）. The gene expression of P38 MAPK was markedly down-regulated after the administration of BN52021, compared to LPS treated group（F=1.49 ± 0.18）. The protein phosphorylation results by Western blotting were in accordance with real-time PCR results.【Conclusion】BN52021 could inhibit the inflammation in LPS induced MS1 cells through down-regulating the gene expression and protein phosphorylation of p38-MAPK.