温肾通络止痛方干预脂肪细胞外泌体对骨质疏松小鼠模型骨脂平衡的影响  

Effects of Wenshen Tonglong Zhitong Recipe on Bone-fat Balance in Osteoporosis Mouse Model by Adipocyte-derived Exosomes

作　　者：员浬 王礼宁 潘娅岚 孙杰[1,4] 刘孟敏 李沐哲 张天驰 吾米提·塔西 郭杨[1,4] 马勇[1,2,5] YUN Li;WANG Li-ning;PANG Ya-lan;SUN Jie;LIU Meng-min;LI Mu-zhe;ZHANG Tian-chi;WUMITI Ta-xi;GUO Yang;MA Yong(Laboratory for Novel Repairing and Reconstructive Technology of Orthopedics and Traumatology,Nanjing University of Traditional and Western Medicine,Nanjing 210023;School of Integrated Chinese and Western Medicine,School of TCM,Nanjing University of Chinese Medicine,Nanjing 210023;School of Nursing,Nanjing University of Chinese Medicine,Nanjing 210023;School of Medicine and Holistic Integrative Medicine,Nanjing University of Chinese Medicine,Nanjing 210023;Wuxi Traditional Chinese Medicine Hospital Affiliated to Nanjing University of Chinese Medicine,Jiangsu Province Clinical Medical Innovation Center for Degenerative Osteoarthropathy of Traditional Chinese Medicine,Jiangsu 214071)

机构地区：[1]南京中医药大学骨伤修复与重建新技术实验室,南京210023 [2]南京中医药大学中医学院中西医结合学院,南京210023 [3]南京中医药大学护理学院,南京210023 [4]南京中医药大学医学院整合医学院,南京210023 [5]南京中医药大学无锡附属医院,江苏省中医退行性骨关节病临床医学创新中心,江苏214071

出　　处：《中国中西医结合杂志》2025年第3期303-312,共10页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金面上项目(No.82074458);江苏省自然科学基金面上项目(No.BK20221351);江苏省自然科学基金青年项目(No.BK20220470);江苏省高等学校自然科学研究面上项目(No.22KJB360012);江苏省研究生科研创新计划(No.KYCX22-1883);江苏省中医退行性骨关节病临床医学创新中心资助项目(No.苏中医科教[2021]4号)。

摘　　要：目的观察温肾通络止痛方干预脂肪细胞外泌体对骨质疏松模型小鼠骨脂平衡的影响。方法80只Balb/c小鼠随机分为假手术组、模型组、外泌体组、中药干预外泌体组。构建去卵巢骨质疏松小鼠模型,假手术组及模型组分别按照200μL尾静脉注射生理盐水,外泌体组及中药干预外泌体组按照30μg(200μL)尾静脉注射外泌体。每周1次,维持12周。采用酶联免疫法测血清中骨组织特异性碱性磷酸酶(B-ALP)、骨钙素(OCN)、抗酒石酸酸性磷酸酶(TRAP)、白介素-6(IL-6)含量,使用全自动生化分析仪检测血清中甘油三酯(TG)、总胆固醇(TC)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)含量;光学显微镜下观察股骨HE染色、TRAP染色组织病理改变并计算破骨细胞数量(N.Oc/BS)及破骨细胞表面积与骨表面比值(Oc.S/BS);Micro-CT检测小鼠股骨骨微结构[骨小梁分离度(Tb.Sp)、骨密度(BMD)、骨体积分数(BV/TV)]及周围脂肪组织分布[诱导型骨髓脂肪组织(rMAT)及组成型骨髓脂肪组织(cMAT)]并进行三维重建图像分析;免疫组化法及qPCR法分别检测骨组织小鼠Runt相关转录因子2(RUNX2)、成骨相关转录因子(Osterix)、CCAT增强子结合蛋白α(CEBP-α)及过氧化物酶体增殖激活受体γ2(PPARγ2)蛋白及mRNA的表达。结果与假手术组比较,模型组B-ALP、OCN、TRAP、IL-6、TC、TG、LDL-C水平、rMAT、cMAT占比升高,RUNX2、Osterix mRNA及蛋白水平升高(P<0.05,P<0.01),HDL-C含量下降(P<0.05),CEBP-α、PPARγ2m RNA及蛋白表达降低(P<0.01);同时模型组骨小梁被破坏,网状结构断裂,骨小梁数量减少,骨质流失,骨微结构遭到破坏,Tb.Sp、BMD、BV/TV降低(P<0.01);与模型组比较,外泌体组和中药干预外泌体组血清中B-ALP、OCN水平、HDL-C含量升高,Tb.Sp、BMD、BV/TV、CEBP-α、PPARγ2m RNA及蛋白升高(P<0.05,P<0.01),CHOL、TG、LDL-C含量、r MAT、c MAT占比、RUNX2、Osterix m RNA及蛋白表达降低(P<0.Objective To observe the effect of Wenshen Tongluo Zhitong Recipe(WTZR)on adipocyte exosomes on bone-fat balance in osteoporosis model mice.Methods Totally 80 Balb/c mice were randomly divided into sham operation group(Sham),model group(OVX),exosome group(EXO),and WTZR intervention exosome group(EXO+WTZR).An ovariectomized osteoporosis mouse model was constructed.The Sham and OVX group were injected physiological saline into the caudal vein at a dose of 200μL respectively,and EXO and EXO+WTZR group were injected exosomes into the caudal vein at a dose of 30μg(200μL),once a week for 12 weeks.The contents of bone tissue-specific alkaline phosphatase(B-ALP),osteocalcin(OCN),tartrate-resistant acid phosphatase(TRAP),and interleukin-6(IL-6)in serum were detected by ELISA,and the levels of triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),and high density lipoprotein cholesterol(HDL-C)in serum were detected by a fully automatic biochemical analyzer.The histopathological changes of femur were observed by HE staining and TRAP staining under optical microscope,and the number of osteoclasts(N.Oc/BS)and the ratio of osteoclasts surface area to bone surface(Oc.S/BS)were calculated.Micro-CT was used to detect the microstructure of mouse femur bone[trabecular separation(Tb.Sp),bone mineral density(BMD),bone volume fraction(BV/TV)]and the distribution of surrounding adipose tissue[regulated bone marrow adipose tissue(rMAT)and constitutive bone marrow adipose tissue(cMAT)],and the three-dimensional reconstruction image analysis was performed.IHC and qPCR methods were used to detect the expression of mouse Runt-related transcription factor 2(RUNX2),osteogenesis-related transcription factor(Osterix),CCAT enhancer binding proteinα(CEBP-α)and peroxisome proliferation-activated receptorγ2(PPARγ2)protein and mRNA in bone tissue,respectively.Results Compared with the Sham group,the levels of B-ALP,OCN,TRAP,IL-6,TC,TG,LDL-C,rMAT,and cMAT increased,the mRNA and protein levels of RUNX2 and Osterix in

关 键 词：温肾通络止痛方 骨质疏松症 外泌体 脂肪细胞 骨脂平衡 中药复方 

分 类 号：R285.5[医药卫生—中药学]