摘要
目的:观察骨碎补总黄酮对骨质疏松模型大鼠磷酸钙骨水泥强化椎体骨质量和生物力学性能的影响,并探讨其可能的作用机制。方法:将60只12周龄雌性SD大鼠随机分为4组。模型组(10只)、空白组(20只)、骨碎补总黄酮组(20只)大鼠均采用切除双侧卵巢的方法进行骨质疏松造模,假手术组(10只)大鼠仅从双侧卵巢附近切除少量脂肪组织。造模手术后4周,处死假手术组和模型组大鼠,采用双能X线吸收法测量第4腰椎骨密度,验证造模是否成功。确认造模成功后,空白组和骨碎补总黄酮组进行第4腰椎磷酸钙骨水泥椎体强化;术后第1天开始骨碎补总黄酮组以2 mL骨碎补总黄酮溶液(骨碎补总黄酮用量为0.22 g·kg^(-1))灌胃,空白组以等量蒸馏水灌胃,药物干预每天1次,连续干预8周。药物干预结束后,处死空白组和骨碎补总黄酮组大鼠,取经磷酸钙骨水泥强化的第4腰椎,采用双能X线吸收法测定骨密度;采用显微CT检测骨微结构,测定骨体积分数(bone volume/tissue volume,BV/TV)、骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数量(trabecular number,Tb.N);采用压缩实验进行生物力学性能检测,测定椎体最大负荷和刚度;采用实时荧光定量PCR技术检测碱性磷酸酶(alkaline phosphatase,ALP)、骨钙素(osteocalcin,OCN)、骨桥蛋白(osteopontin,OPN)等骨形成标志基因的mRNA表达水平;采用Western Blot技术检测ALP、OCN、OPN等骨形成标志基因,以及Wnt1、Wnt3a、β联蛋白等Wnt信号通路相关基因的蛋白表达水平。结果:①造模结果。模型组第4腰椎骨密度低于假手术组(t=11.311,P=0.000)。②强化椎体的骨密度及骨微结构检测结果。骨碎补总黄酮组强化椎体的骨密度、BV/TV、Tb.Th、Tb.N均高于空白组(t=3.328,P=0.004;t=3.795,P=0.001;t=2.340,P=0.031;t=2.256,P=0.037)。③强化椎体的生物力学性能检测结果。骨碎补总黄酮组强化椎体的最大负荷和刚度均大于空白组(t=3.689,P=0.002;t=2.335,P=0.031)。④强化椎体组织中骨形成标志基因的mRNA和蛋白表达水平检测结果。骨碎补总黄酮组强化椎体组织中ALP、OCN、OPN的mRNA和蛋白表达水平均高于空白组(mRNA:t=9.738,P=0.000;t=7.576,P=0.000;t=9.880,P=0.000;蛋白:t=10.923,P=0.000;t=17.896,P=0.000;t=16.751,P=0.000)。⑤强化椎体组织中Wnt信号通路相关基因的蛋白表达水平检测结果。骨碎补总黄酮组强化椎体组织总蛋白中Wnt1、Wnt3a蛋白表达水平,以及核蛋白中β联蛋白的蛋白表达水平均高于空白组(t=13.252,P=0.000;t=13.379,P=0.000;t=9.765,P=0.000);2组强化椎体组织总蛋白中β联蛋白的蛋白表达水平的差异无统计学意义。结论:骨碎补总黄酮可改善骨质疏松模型大鼠磷酸钙骨水泥强化椎体的骨质量和生物力学性能,其机制可能是通过激活Wnt信号通路,促进β联蛋白进入细胞核,增加ALP、OCN、OPN表达,从而促进成骨分化。
Objective:To investigate the effects of total flavonoids of rhizoma drynariae(TFRD)on bone quality and biomechanical properties of calcium phosphate cement(CPC)-augmented vertebrae in osteoporotic(OP)model rats,and to explore its underlying mechanism.Methods:Sixty 12-week-old female SD rats were randomly assigned into model group(10 ones),blank group(20 ones),TFRD group(20 ones),and sham-operated group(10 ones).All rats but the ones in sham-operated group were treated by bilateral ovariectomy for inducing OP model,while the ones in sham-operated group merely underwent removal of peri-ovarian adipose tissues,with ovaries retained.Four weeks after the modeling surgery,the rats in sham-operated group and model group were sacrificed,and the L_(4) lumbar vertebra was dissected for measuring the bone mineral density(BMD)via dual-energy X-ray absorptiometry(DEXA)to evaluate whether the OP models were built successfully.After successful modeling,the rats in blank group and TFRD group were subjected to CPC augmentation at the L_(4) lumbar vertebra.From the postoperative day 1,the rats in TFRD group were intervened by intragastric administration with 2 mL TFRD solution(TFRD dosage:0.22 g/kg),while the ones in blank group with the same dose of distilled water,once a day for consecutive 8 weeks.After the end of drug intervention,the rats in blank group and TFRD group were executed,and the CPC-augmented L_(4) lumbar vertebra were harvested to measure the BMD via DEXA,meanwhile,the bone microstructure was assessed,and the bone volume/tissue volume(BV/TV),trabecular thickness(Tb.Th),as well as trabecular number(Tb.N)were detected by micro-CT.Moreover,the biomechanical properties were tested by compression testing to determine the maximum load and stiffness of the CPC-augmented L_(4) lumbar vertebra.Furthermore,the mRNA expression levels of bone formation marker genes,including alkaline phosphatase(ALP),osteocalcin(OCN),and osteopontin(OPN)were detected by real-time fluorescence quantitative PCR technology,and the protein expression levels of bone formation marker genes such as ALP,OCN,and OPN,as well as Wnt signaling pathway-related genes such as Wnt1,Wnt3a,andβ-catenin,were detected by Western Blot.Results:①Modeling results.The BMD of the L_(4) lumbar vertebra was lower in model group compared to sham-operated group(t=11.311,P=0.000).②BMD and bone microstructure of the augmented vertebra.The BMD,BV/TV,Tb.Th,and Tb.N of the augmented L 4 lumbar vertebra were all higher in TFRD group compared to blank group(t=3.328,P=0.004;t=3.795,P=0.001;t=2.340,P=0.031;t=2.256,P=0.037).③Biomechanical properties of the augmented vertebra.The maximum load and stiffness of the augmented L_(4) lumbar vertebra were greater in TFRD group compared to blank group(t=3.689,P=0.002;t=2.335,P=0.031).④The mRNA and protein expression levels of bone formation marker genes in the augmented vertebra tissues.The mRNA and protein expression levels of ALP,OCN,and OPN in the augmented L_(4) vertebra tissues were all higher in TFRD group compared to blank group(mRNA:t=9.738,P=0.000;t=7.576,P=0.000;t=9.880,P=0.000;protein:t=10.923,P=0.000;t=17.896,P=0.000;t=16.751,P=0.000).⑤The protein expression levels of Wnt signaling pathway-related genes in the augmented vertebra tissues.The protein expression levels of Wnt1 and Wnt3a in the total protein extracts,as well as that ofβ-catenin in the nuclear protein extracts of the augmented L_(4) vertebra tissues,were all higher in TFRD group compared to blank group(t=13.252,P=0.000;t=13.379,P=0.000;t=9.765,P=0.000);while the comparison of the protein expression level ofβ-catenin in the total protein extracts between the 2 groups revealed no significant differences.Conclusion:TFRD can improve the bone quality and biomechanical properties of CPC-augmented vertebrae in OP model rats.It may work by stimulating osteoblastic differentiation through activating the Wnt signaling pathway,promoting nuclear translocation ofβ-catenin,and up-regulating the expression of ALP,OCN,and OPN.
作者
张鑫
马朋朋
刘肃
李伟
张麦粒
赵立春
张春玲
ZHANG Xin;MA Pengpeng;LIU Su;LI Wei;ZHANG Maili;ZHAO Lichun;ZHANG Chunling(The First Affiliated Hospital of Hebei North University,Zhangjiakou 075061,Hebei,China)
出处
《中医正骨》
2025年第7期15-20,共6页
The Journal of Traditional Chinese Orthopedics and Traumatology
基金
2022年度河北省医学科学研究课题(20220612)。
