六味地黄汤对PMOP模型大鼠股骨MAPK通路动态干预研究

Dynamic intervention of Liuwei Dihuang decoction on MAPK pathway in femur of PMOP model rats

目的研究六味地黄汤对PMOP模型大鼠股骨MAPK信号通路中p38、ERK1/2磷酸化蛋白及mRNA表达的动态干预作用及其机理。方法将6~8月龄未孕雌性SD大鼠(SPF级)编号、称重后随机分为5组,除空白组外,其余各组均采用去卵巢法造模。造模后第5天开始,连续给药90 d。分别在术后30、60、90 d,双能X线骨密度仪检测各组大鼠股骨骨密度;蛋白免疫印迹法(Western blot)法检测各组大鼠股骨中p-p38及p-ERK1/2表达;实时荧光定量PCR(RT-qPCR)法检测各组大鼠股骨中p38、ERK1/2 mRNA表达。结果与空白组比较,模型组大鼠的股骨骨密度值在60、90 d时下降(P<0.05);与模型组比较,六(酒)可上调PMOP术后30、60、90 d模型大鼠股骨中p-p38、p-ERK1/2及ERK1/2 mRNA表达(P<0.05、P<0.01)以及术后60、90 d模型大鼠股骨p38 mRNA表达(P<0.01),而六(生)仅能调整术后90 d模型大鼠股骨中p-p38及p-ERK1/2及mRNA表达(P<0.05、P<0.01)及术后30、60 d模型大鼠股骨中p-ERK1/2表达(P<0.05)。结论六(酒)、六(生)均可干预绝经后骨质疏松症,其机理可能与该汤通过调控MAPK通路中p38及ERK1/2两个蛋白,并使其发生磷酸化修饰,激活成骨相关因子,升高BMD,改善PMOP有关;其中六(酒)的干预作用从绝经早期、中期开始。

Objective To study the dynamic intervention of Liuwei Dihuang Decoction on the expression of p38,ERK1/2 phosphorylated protein and mRNA in the femoral MAPK signal pathway of PMOP model rats and its mechanism.Methods Pregnant female SD rats(SPF grade)aged from 6 months to 8 months were randomly divided into 5 groups after numbering and weighing.Except the blank group,all the other groups were made by ovariectomy.The drug was administered continuously for 90 days from the 5th day after the model was established.Bone mineral density(BMD)of femur of rats in each group was measured by dual-energy X-ray absorptiometry at 30 days,60 days and 90 days after operation.Western Blot was used to detect the expression of p-p38 and p-ERK1/2 in femur of rats in each group.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression of p38 and ERK1/2 mRNA in femur of rats in each group.Results Compared with the blank group,the femoral bone mineral density in the model group decreased at 60 days and 90 days(P<0.05).Compared with the model group,Liu(jiu)can up-regulate the expression of p-p38,p-ERK1/2 and ERK1/2 mRNA in the femur of the model rats on the 30th,60th and 90th day after PMOP(P<0.05,P<0.01)and the expression of p38 mRNA in the femur of the model rats on the 60th and 90th day after PMOP(P<0.01).However,Liu(sheng)could only regulate the expression of p-p38,p-ERK1/2 and mRNA in the femur of 90 days after operation(P<0.05,P<0.01)and the expression of p-ERK1/2 in the femur of 30 days and 60 days after operation(P<0.05).Conclusion Liu(jiu)and Liu(sheng)can both interfere with postmenopausal osteoporosis,and its mechanism may be related to the fact that the decoction can regulate p38 and ERK1/2 proteins in MAPK pathway,make them undergo protein phosphorylation modification,activate osteogenesis-related factors,increase BMD and improve PMOP.Among them,the intervention of Liu(jiu)began in the early and middle stages of menopause.