文献类型：期刊文献

中文题名：针刺疗法干预RANKL/OPG信号通路调控骨质疏松模型大鼠骨代谢平衡机制研究

英文题名：Mechanism of Acupuncture Therapy on RANKL/OPG Signaling Pathway to Regulate Bone Metabolism Balance in Osteoporosis Model Rats

作者：尹秦[1];潘虹[1];丁小娟[1];秦晓光[1];郭小荣[1]

第一作者：尹秦

机构：[1]甘肃中医药大学附属医院,甘肃兰州730020

第一机构：甘肃中医药大学第二附属医院

年份：2021

卷号：31

期号：4

起止页码：292

中文期刊名：康复学报

外文期刊名：Rehabilitation Medicine

收录：CSTPCD;;CSCD:【CSCD_E2021_2022】；

基金：国家自然科学基金项目(81660802);甘肃省自然科学基金项目(17JR5RA056);甘肃省科技计划项目(1304FKCA096)。

语种：中文

中文关键词：骨质疏松;针刺疗法;RANKL/OPG;信号通路;骨代谢

外文关键词：osteoporosis;acupuncture therapy;RANKL/OPG;signal pathway;bone metabolism

摘要：目的:探讨针刺疗法对去卵巢大鼠核因子Kappa B受体活化因子配体(RANKL)/骨保护素(OPG)信号通路的影响,并分析其调控骨代谢平衡的机制。方法:选择SPF级雌性Wistar大鼠60只,按照随机数字表法分为空白组、模型组、雌二醇组和针刺组,每组15只。除空白组外,其他3组大鼠均采用去卵巢法建立骨质疏松模型。造模成功后进行为期12周的干预,空白组、模型组大鼠给予0.9%Na Cl溶液50μg/(kg·d)灌胃,雌二醇组给予戊酸雌二醇50μg/(kg·d)灌胃,针刺组给予电针刺激肾俞、三阴交、关元、足三里等穴位,刺激强度2 mA,频率2 Hz,15 min/次,1次/d。采用酶联免疫法(ELISA)分析4组大鼠血清中骨特异性碱性磷酸酶(BALP)、I型胶原羧基末端肽(CTX-I)、核结合因子-α1(CBF-α1)、I型前胶原氨基端前肽(PINP)、骨钙素(OC)等骨代谢标志物含量;采用Micro-CT分析股骨远端骨密度(BMD)、相对骨体积(BV/TV)、骨小梁厚度(Tb.Th)、连接密度(Conn.D)、骨小梁分离度(Tb.Sp)、骨小梁数量(Tb.N)、结构模型指数(SMI)等;采用TUNEL法检测大鼠股骨远端细胞凋亡情况;采用蛋白免疫印迹法(Western blotting)和实时荧光定量聚合酶链式反应(RT-PCR)检测大鼠股骨和下丘脑组织中RANKL、OPG蛋白和mRNA表达水平。结果:(1)骨密度及骨代谢标志物:与空白组比较,模型组BMD、BALP、CBF-α1、CTX-I、PINP、OC均明显降低(P<0.05)。与模型组比较,针刺组BMD、BALP、CBF-α1、CTX-I、PINP、OC均明显升高(P<0.05)。(2)骨小梁微结构:与空白组比较,模型组BV/TV、Tb.N、Tb.Th、Conn.D均明显降低,Tb.Sp、SMI均明显升高(P<0.05)。与模型组比较,针刺组BV/TV、Tb.N、Tb.Th、Conn.D均明显升高,Tb.Sp、SMI均明显降低(P<0.05)。(3)骨远端细胞凋亡情况:与空白组比较,模型组凋亡细胞数量明显增加;与模型组比较,雌二醇组、针刺组干预后大鼠股骨远端细胞凋亡数量均明显减少。(4)下丘脑及股骨组织OPG、RANKL mRNA和蛋白表达:与空白组比较,模型组下丘脑、股骨中OPG mRNA和蛋白表达均明显降低,RANKL mRNA和蛋白表达均明显升高(P<0.05)。与模型组比较,针刺组下丘脑、股骨中OPG mRNA和蛋白表达均明显上升,RANKL mRNA和蛋白表达均明显下降(P<0.05)。结论:针刺疗法能够改善OP模型大鼠骨密度,调节骨代谢水平,改善骨小梁微结构,其机制可能与RANKL/OPG信号通路调控有关。
Objective:To explore the effect of acupuncture therapy on receptor of activator of nuclear factor-k B ligand(RANKL)/osteoprotegerin(OPG)signaling pathway in ovariectomized rats,and to analyze its mechanism of regulating bone metabolism balance.Methods:A total of 60 SPF female Wistar rats were randomly divided into the blank group,the model group,the estradiol group and the acupuncture group,with 15 rats in each group.Except the blank group,the other three groups were ovariectomized to establish osteoporosis model.The rats in the blank group and the model group were given 0.9%NaCl solution for 50μg/(kg·d)by gavage,the estradiol group was given estradiol valerate 50μg/(kg·d)by gavage,the acupuncture group was given electroacupuncture at Shenshu,Sanyinjiao,Guanyuan,Zusanli acupoints with stimulation intensity of 2 mA,frequency of 2 Hz,15 min/time,once a day.Enzyme linked immunosorbent assay(ELISA)was used to analyze bone specific alkaline phosphatase(BALP),cterminal type I collagen telopeptide(CTX-I),core-binding factor-α1(CBF-α1),procollagen type I amino-terminal peptide(PINP),osteocalcin(OC)of the four groups;The relative bone volume fraction(BV/TV),trabecular number(Tb.N),trabecular bone thickness(Tb.Th),connective density(Conn.D),trabecular bone separation(Tb.Sp),structural model index(SMI)were analyzed by Micro-CT;TUNEL method was used to detect apoptosis of distal femur cells in four groups;Western blot and RT-PCR method were used to detect the protein expression levels of RANKL,OPG mRNA in femur and hypothalamus in four groups.Results:①BMD and bone metabolism markers:compared with the blank group,BMD,BALP,CBF-α1,CTX-I,PINP and OC in the model group were significantly decreased(P<0.05).Compared with the model group,BMD,BALP,CBF-α1,CTX-I,PINP and OC in the acupuncture group were significantly increased(P<0.05).②Trabecular microstructure:compared with the blank group,BV/TV,Tb.N,Tb.Th,Conn.D in the model group were significantly decreased,while Tb.Sp,SMI were significantly increased(P<0.05).Compared with the model group,BV/TV,Tb.N,Tb.Th and Conn.D in the acupuncture group were significantly increased,while Tb.Sp and SMI were significantly decreased(P<0.05).③Apoptosis of distal bone cells:compared with the blank group,the number of apoptotic cells in the model group increased significantly;compared with the model group,the number of apoptotic cells in the distal femur of the estradiol group and the acupuncture group decreased significantly.④OPG and RANKL mRNA and protein expression in hypothalamus and femur:compared with the blank group,OPG mRNA and protein expression in hypothalamus and femur of the model group were significantly decreased,while RANKL mRNA and protein expression were significantly increased(P<0.05);compared with the model group,the expression of OPG mRNA and protein in hypothalamus and femur of the acupuncture group were significantly increased,while the expression of RANKL mRNA and protein were significantly decreased(P<0.05).Conclusion:Acupuncture therapy can improve bone density,regulate bone metabolism level and improve bone trabecular microstructure in OP model rats,its mechanism may be related to the regulation of RANKL/OPG signal pathway.