小檗碱对脂多糖诱导的COX-2表达的影响

Berberine on LPS-induced COX-2 expression: p38 mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathway comparison

目的:研究中药小檗碱对脂多糖(LPS)诱导的COX-2表达的影响。方法:取人外周静脉血分离及培养单个核细胞,分为3组:空白对照组、LPS组、LPS+小檗碱组。分别在培养后30 min,6 h,12 h,24 h提取细胞,行RT-PCR法测定COX-2 mRNA水平,Western blot法测定p38MAPK,p-p38MAPK,ERK,p-ERK及COX-2蛋白水平。同时加入选择性p38MAPK抑制剂(SB203580),以及特异性ERK抑制剂(PD098059)分别测定COX-2 mRNA及蛋白水平。结果:与空白对照组相比,LPS组COX-2 mRNA及蛋白表达明显增强(P<0.01)。与LPS组相比,LPS+小檗碱组COX-2 mRNA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12 h,小檗碱对COX-2抑制作用最强。与LPS组相比,LPS+小檗碱组P38MAPK活性水平差异无统计学意义(P>0.05)。但是与LPS组相比,LPS+小檗碱组ERK活性水平差异有统计学意义(P<0.05)。加入p38MAPK抑制剂以及ERK抑制剂之后,COX-2 mRNA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个核细胞COX-2 mRNA及蛋白水平,并呈浓度依赖性,但对p38MAPK活性蛋白表达无明显抑制作用;小檗碱对ERK活性蛋白表达有明显抑制作用,可能通过ERK信号转导途径抑制COX-2 mRNA及蛋白表达。

Objective：To investigate the effect of Berberine（BBR） on lipopolysaccharide（LPS） induced cyclooxygenase-2（COX-2） expression via the p38 mitogen activated protein kinase（p38MAPK） and ERK signalling cascade pathways in human peripheral blood monocytes（PBMC）.Method：PBMC were isolated and cultured from whole blood and divided into 5 groups treated with null control,LPS,LPS＋BBR 25 μmol/L,LPS＋BBR 50 μmol/L,LPS＋BBR 100 μmol/L respectively for 30min,6 h,12 h and 24 h.Then monocytes were extracted for RT-PCR and Westernblot analysis to examine COX-2 mRNA and protein activated expression of p38MAPK and ERK signaling pathway.Meanwhile,p38MAPK and ERK inhibitor were incubated along with LPS stimulation to examine COX-2 mRNA and protein expression.Result：COX-2 mRNA and protein expression decreased at a low ebb at 12 h after BBR treatment（P0.05）.On the other hand,with the increasing concentration of BBR treatment,the COX-2 expression decreased progressively（P0.01）.With BBR treatment at different time points（at 6 h,12 h,24 h after treatment） and different levels of dosage,no difference was observed between BBR group and LPS group about p38MAPK protein expression（P0.05）.In BBR group 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 h after BBR administration the ERK protein expression decreased more prominently than that of the other three time points（P0.05）,and ERK protein expression decreased progressively with the dosage increased.Human monocytes COX-2 mRNA and protein expression was inhibited statistically significant when incubated with p38MAPK and ERK inhibitor（P0.05）.Conclusion：BBR inhibited COX-2 mRNA and protein expression at a dose-dependent manner and ERK expression could be inhibited by BBR.BBR inhibits COX-2 expression via ERK signaling pathway in PBMCs.COX-2 expression can be also inhibited by p38MAPK signaling pathway inhibitors.p38 MAPK may have no prominent relationship with the effect of BBR in PBMC.