ERK信号转导途径在小檗碱抑制脂多糖诱导的COX-2表达中的作用

The effect of ERK signaling cascade pathways on the inhibition of Berberine on lipopolysaccharide induced COX-2 expression

目的探讨小檗碱是否抑制脂多糖诱导的COX-2mRNA及蛋白表达,以及小檗碱是否通过ERK信号转导途径抑制COX-2的表达。方法取健康志愿者外周血,分离及培养单核细胞,分为五组,分别为对照组;脂多糖(Lipopolysaccharide,LPS)组;LPS+小檗碱25μmol/L组;LPS+小檗碱50μmol/L组;LPS+小檗碱100μmol/L组。分别在培养后30min,6h,12h,24h提取细胞,行RT-PCR法测定COX-2mRNA水平,行westernblot法测定ERK、p-ERK及COX-2蛋白水平。同时加入选择性ERK抑制剂,分别测定COX-2mRNA及蛋白水平。结果与对照组相比,LPS组COX-2mRNA及蛋白表达明显增强(P<0.01)。与LPS组相比,小檗碱组COX-2mRNA及蛋白表达明显抑制(P<0.05),且随着浓度增加,抑制作用更明显,在给药后12h,小檗碱对COX-2抑制作用最强。与LPS组相比,小檗碱组ERK活性水平有明显统计学差异(P<0.05)。加入ERK抑制剂之后,COX-2mRNA及蛋白水平降低明显(P<0.05)。结论小檗碱能抑制人外周血单核细胞COX-2mRNA及蛋白水平,其作用程度呈浓度依赖性,小檗碱对ERK活性蛋白表达有明显抑制作用。小檗碱可能通过ERK信号转导途径抑制人外周血单核细胞COX-2mRNA及蛋白表达。

Objective To investigate the inhibition of berberine （BBR） on lipepolysaccharide （LPS） induced COX-2 expression via ERK signaling cascade pathways in human peripheral blood monocytes （HPMC）. Methods HPMC were isolated and cultured from whole blood and divided into 5 groups treated with null, LPS, LPS ＋BBR 25 μmol/L, LPS ＋BBR 50 μmol/L, LPS ＋ BBR 100 μmol/L respectively for 30 minutes, 6 hours, 12 hours and 24 hours. Then monocytes were extracted for RT-PCR and westernblot analysis to examine COX-2 mRNA and protein activated expression of ERK signaling pathway. Meanwhile, PD98059 （ ERK inhibitor） was incubated together with LPS stimulation to examine COX-2 mRNA and protein expression. Results At the four time points after treatment, the COX-2 mRNA and protein expression decreased at a low ebb at 12 hours after BBR treatment （P 〈 0. 05 ）. On the other hand, with the increasing concentration of BBR, the COX-2 expression decreased progressively （ P 〈 0. 01 ）. In the group of BBR treatment at various time points （at 6 h, 12 h, 24 h after treatment） and three levels of dosage, ERK protein expression was inhibited significantly. At 12 hours after BBR administration the ERK protein expression reduced more prominently than that of other three time points （ P 〈 0. 05 ）, and ERK protein expression decreased progressively as the dosage increased. Human monocytes COX-2 mRNA and protein expression was inhibited significantly while incubated with ERK pathway inhibitor, PD98059 （ P 〈 0. 01 ）. Conclusion It is concluded that BBR inhibits COX-2 mRNA and protein expression in a dose-dependent manner and ERK expression can be suppressed by BBR. BBR inhibits COX-2 expression via ERK signaling pathway in HPMC.