电针刺激后大鼠血清对成骨细胞OPG、RANKL mRNA及其蛋白表达的影响

Effect of Rat Serum after Electroacupuncture Stimulation on the Expressions of Osteoblastic OPG and RANKL mRNAs and Their Proteins

目的观察电针刺激去卵巢骨质疏松症模型大鼠的血清对体外培养成骨细胞的骨形成因子骨保护素(osteoprotegerin,OPG)和骨吸收因子(receptor activator for nuclear factor-κB ligand,RANKL)mRNA表达及其蛋白浓度的影响。方法将45只雌性SD大鼠,随机分为空白组(A组)、模型组(B组)、电针刺激组(C组),每组15只。除A组外,B组、C组均切除双侧卵巢。造模成功后,C组给予电针刺激,B组正常饲养,A组不做任何处理,常规正常饲养。12周后,采用水合氯醛麻醉各组大鼠,心脏采血,经过处理后加到体外培养的大鼠成骨细胞培养液中。采用碱性磷酸酶(ALP)检测成骨细胞活性,RT-qPCR法检测大鼠血清对OPG、RANKL mRNA表达的影响,ELISA法检测大鼠血清对OPG、RANKL蛋白浓度的影响。结果各组大鼠血清处理成骨细胞后,与A组比较,B组ALP的活性明显降低(P<0.05),C组电刺激后成骨细胞内ALP的活性明显增加(P<0.05)。与B组比较,A组及C组OPG mRNA表达及其蛋白浓度表达明显降低(P<0.05);与A组比较,C组OPG mRNA表达及其蛋白浓度均明显降低(P<0.05)。与B组比较,C组及A组RANKL mRNA表达及其蛋白浓度表达明显降低(P<0.05);与A组比较,C组RANKL mRNA及蛋白浓度明显降低(P<0.05)。C组大鼠骨密度明显高于B组(P<0.05)。结论电刺激后的大鼠血清可以提升ALP、OPG水平,降低RANKL水平,其治疗骨质疏松症的机制可能与此有关。

Objective To investigate the effect of ovariectomized osteoporosis rat serum after electroacupuncture stimulation on the expressions of in vitro cultured osteoblastic bone formation factor osteoprotegerin(OPG)and bone resorption factor receptor activator of nuclear factor-κB ligand(RANKL)mRNAs and the concentrations of their proteins.Method Forty-five female SD rats were randomized to a blank group(group A),a model group(group B)and an electroacupuncture stimulation group(group C),15 rats each.Bilateral ovaries were excised in groups B and C except group A.After successful model making,group C was given electroacupuncture stimulation and group B,fed regularly.Group A was fed regularly without any treatment.After 12 weeks,each group of rats was anesthetized with chloral hydrate and blood was taken from the heart.The blood was processed and put in culture fluid for in vitro cultured rat osteoblasts.Osteoblast activity was measured using alkaline phosphatase(ALP).The effect of rat serum on the expressions of osteoblastic OPG and RANKL mRNAs was assessed using RT-qPCR.The effect of rat serum on the concentrations of OPG and RANKL proteins was determined using ELISA.Result After osteoblasts were treated with rat serumin in each group of rats,ALP activity decreased significantly in group B(P<0.05),and osteoblastic ALP activity increased significantly in group C after electroacupuncture stimulation(P<0.05),in comparison with group A.OPG mRNA expression and its protein concentration decreased significantly in groups A and C compared with group B(P<0.05)and in group C compared with group A(P<0.05).RANKL mRNA expression and its protein concentration decreased significantly in groups C and A compared with group B(P<0.05)and in group C compared with group A(P<0.05).Bone mineral density was significantly higher in group C of rats than in group B(P<0.05).Conclusion Rat serum after electroacupuncture stimulation can increase ALP and OPG levels and decrease RANKL levels,which may be related to the mechanism by which electroacupuncture treats osteoporosis.