葛根素对废用性骨质疏松大鼠模型的防治作用及机制研究
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摘要
探讨葛根素(puerarin,Pur)对废用性骨质疏松(disuse osteoporosis,DOP)大鼠模型的防治作用及可能机制。2月龄健康Wistar雌性大鼠30只,随机数字表法分组为对照组(Control)、尾吊组(HLS)、尾吊时服用葛根素组(HLS+Pur),每组10只。通过尾巴悬吊法建立废用性骨质疏松模型,HLS+Pur组每天给予15.4 mg·kg~(-1)葛根素混悬液,Control组和HLS组分别灌服等体积蒸馏水。28 d后,腹主动脉采血处死大鼠,摘取大鼠主要脏器,剥离大鼠骨组织,计算大鼠各器官系数并镜下观察脏器组织病理学情况,进行骨密度检测和骨生物力学实验,骨组织切片后观察双荧光标记和VG染色结果,测定血清骨代谢生化指标。各组大鼠器官系数无统计学意义,脏器病理学检测未见明显异常;骨密度结果显示,葛根素能大幅度提升尾吊引起的胫骨和椎骨的骨密度下降;骨生物力学参数结果表明,葛根素能有效增加胫骨和椎骨的最大载荷和弹性模量;荧光标记观察发现,葛根素用药期间,荧光间距变大,骨形成增强;VG染色结果证明,与HLS组相比,葛根素组骨小梁数目增多、骨小梁厚度变宽但骨分离度减小,极大地改善了尾吊之后的骨微结构;此外血清生化指标显示,葛根素能促使骨形成指标骨钙素(osteocalcin,OC)含量明显增加,同时显著抑制骨吸收指标抗酒石酸酸性磷酸酶5b(tartrate-resistant acid phosphatase 5b,TRACP 5b)的生成。葛根素对废用性骨质疏松大鼠模型具有防治作用且效果良好,其作用机制可能与促进骨形成和抑制骨吸收有关。
To investigate the preventive effect and possible mechanism of puerarin(Pur) in rat model of disuse osteoporosis(DOP),thirty healthy Wistar female rats of 2 months old were randomly divided into control group(Control), hindlimb suspension group(HLS), and puerarin group(HLS+Pur) in hindlimb suspension, with 10 rats in each group. A disuse osteoporosis model was established by tail suspension method, and 15.4 mg·kg~(-1) puerarin suspension was administered to HLS+Pur group every day, and the same volume of distilled water was administered to Control group and HLS group respectively. After 28 days, the rats were sacrificed by abdominal aorta blood collection, the main organs of the rats were removed, and the bone tissues of the rats were dissected. The organ index of the rats was calculated and the histopathology of the organs was observed under microscope. Bone mineral density test and bone biomechanical experiment were performed. Bone histomorphometry results were observed after bone tissue sectioning, and serum biochemical markers of bone metabolism were determined. There was no significant difference in organ index between the groups. There was no obvious abnormality in the pathological examination of the organs. The results of bone mineral density showed that puerarin could significantly increase the bone density of the tibia and vertebrae caused by hindlimb suspension. The mechanical parameters experiments showed that puerarin could effectively increase the maximum load and elastic modulus of the tibia and vertebrae. Fluorescence labeling showed that the fluorosis interval increased and the bone formation increased during puerarin treatment. The VG staining results showed that compared with the HLS group, in the puerarin group, the number of trabecular bone increased, the thickness of the trabecular bone became thicker, and the bone separation became smaller, which greatly improved the bone microstructure after hindlinb suspension. In addition, serum biochemical indicators showed that puerarin could promote bone formation index bone calcium. The content of osteocalcin(OC) increased and inhibited the formation of tartrate-resistant acid phosphatase 5 b(TRACP 5 b). Puerarin has a preventive effect in the rat model of disuse osteoporosis and its effect is good, and its mechanism may be related to promoting bone formation and inhibiting bone resorption.
To investigate the preventive effect and possible mechanism of puerarin(Pur) in rat model of disuse osteoporosis(DOP),thirty healthy Wistar female rats of 2 months old were randomly divided into control group(Control), hindlimb suspension group(HLS), and puerarin group(HLS+Pur) in hindlimb suspension, with 10 rats in each group. A disuse osteoporosis model was established by tail suspension method, and 15.4 mg·kg~(-1) puerarin suspension was administered to HLS+Pur group every day, and the same volume of distilled water was administered to Control group and HLS group respectively. After 28 days, the rats were sacrificed by abdominal aorta blood collection, the main organs of the rats were removed, and the bone tissues of the rats were dissected. The organ index of the rats was calculated and the histopathology of the organs was observed under microscope. Bone mineral density test and bone biomechanical experiment were performed. Bone histomorphometry results were observed after bone tissue sectioning, and serum biochemical markers of bone metabolism were determined. There was no significant difference in organ index between the groups. There was no obvious abnormality in the pathological examination of the organs. The results of bone mineral density showed that puerarin could significantly increase the bone density of the tibia and vertebrae caused by hindlimb suspension. The mechanical parameters experiments showed that puerarin could effectively increase the maximum load and elastic modulus of the tibia and vertebrae. Fluorescence labeling showed that the fluorosis interval increased and the bone formation increased during puerarin treatment. The VG staining results showed that compared with the HLS group, in the puerarin group, the number of trabecular bone increased, the thickness of the trabecular bone became thicker, and the bone separation became smaller, which greatly improved the bone microstructure after hindlinb suspension. In addition, serum biochemical indicators showed that puerarin could promote bone formation index bone calcium. The content of osteocalcin(OC) increased and inhibited the formation of tartrate-resistant acid phosphatase 5 b(TRACP 5 b). Puerarin has a preventive effect in the rat model of disuse osteoporosis and its effect is good, and its mechanism may be related to promoting bone formation and inhibiting bone resorption.
引文
[1] Qi W, Yan Y B, Lei W, et al. Prevention of disuse osteoporosis in rats by Cordyceps sinensis extract[J]. Osteoporosis Int, 2012, 23(9):2347.
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[3] Smith E M, Comiskey C M, Carroll A M. A study of bone mineral density in adults with disability[J]. Arch Phys Med Rehab, 2009, 90(7):1127.
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[5] Chang K, Chang W H, Huang S, et al. Pulsed electromagnetic fields stimulation affects osteoclast formation by modulation of osteoprotegerin, RANK ligand and macrophage colony-stimulating factor[J]. J Orthop Res, 2005, 23(6):1308.
[6] Kr?lner B, Toft B. Vertebral bone loss: an unheeded side effect of therapeutic bed rest[J]. Clin Sci, 1983, 64(5):537.
[7] Lang T, Leblanc A, Evans H, et al. Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight[J]. J Bone Miner Res, 2004, 19(6):1006.
[8] Li Y. Protective roles of puerarin and danshensu on acute ischemic myocardial injury in rats[J]. Phytomedicine, 2007, 14(10):652.
[9] 郑高利,张信岳,郑经伟,等.葛根素和葛根总异黄酮的雌激素样活性[J].中药材,2002,25(8):566.
[10] Suthon S, Jaroenporn S, Charoenphandhu N, et al. Anti-osteoporotic effects of Pueraria candollei var. mirifica on bone mineral density and histomorphometry in estrogen-deficient rats[J]. J Nat Med, 2016, 70(2):225.
[11] Dongdong Y U, Shuai M U, Zhao D, et al. Puerarin attenuates glucocorticoid-induced apoptosis of hFOB1.19 cells through the JNK-and Akt-mediated mitochondrial apoptotic pathways[J]. Int J Mol Med, 2015, 36(2):345.
[12] 葸慧荣, 李文苑, 杨芳芳,等. 中药葛根素对青年大鼠峰值骨量的影响研究[J]. 中国骨质疏松杂志, 2018, 31(4):635.
[13] Zhang P, Kazunori H, Hiroki Y. A brief review of bone adaptation to unloading[J]. Genom Proteom Bioinf, 2008, 6(1):4.
[14] Jing D, Cai J, Wu Y, et al. Pulsed electromagnetic fields partially preserve bone mass, microarchitecture, and strength by promoting bone formation in hindlimb-suspended rats[J]. J Bone Miner Res, 2014, 29(10):2250.
[15] Shirazi-Fard Y, Anthony R A, Kwaczala A T, et al. Previous exposure to simulated microgravity does not exacerbate bone loss during subsequent exposure in the proximal tibia of adult rats[J]. Bone, 2013, 56(2):461.
[16] Ji Z, Shi C, Huang S, et al. Elcatonin attenuates disuse osteoporosis after fracture fixation of tubular bone in rats[J]. J Orthop Surg Res, 2015, 10(1):103.
[17] 罗新新, 徐国良, 黎宇,等. 葛根调节脂肪细胞糖脂代谢改善胰岛素抵抗的研究[J]. 中国中药杂志, 2016, 41(14):2687.
[18] 刘东吉, 余智奎, 刘春生,等. 葛根种质资源的分子地理标识研究[J]. 中国中药杂志, 2011, 36(3):299.
[19] 梁丽谊,王汝上.葛根总黄酮提取工艺优化研究[J].今日药学,2014,24(12):866.
[20] 裴莉昕,纪宝玉,陈随清,等.葛根多糖提取工艺的优化[J].中国现代中药,2017,19(4):553.
[21] 李波, 余金钟, 党中勤. 风药在久泻治疗中应用的理论探讨[J]. 中国中医基础医学杂志, 2018,24(5):713.
[22] 李珊珊, 刘逢芹, 郑世存. 异黄酮调控机体骨代谢现代研究[J]. 辽宁中医药大学学报, 2015(11):131.
[23] Dhainaut A, Hoff M, Syversen U, et al. Technologies for assessment of bone reflecting bone strength and bone mineral density in elderly women: an update[J]. Womens Health, 2016, 12(2):209.
[24] Oftadeh R, Perezviloria M, Villacamacho J C, et al. Biomechanics and mechanobiology of trabecular bone: a review[J]. J Biomech Eng, 2015, doi: 10.1115/1.4029176.
[25] Vidal B, Pinto A, Galv?o M J, et al. Bone histomorphometry revisited[J]. Acta Reumatológica Portuguesa, 2012, 37(4):294.
[26] Guo L, Zhang J P, Zhang K Y, et al. Effects of 1.8 GHz radiofrequency field on microstructureand bone metabolism of femur inmice[J].Bioelectromagnetics, 2018, 39(5):386.
[2] Shinchuk L M, Morse L, Huancahuari N, et al. Vitamin D deficiency and osteoporosis in rehabilitation inpatients[J]. Arch Phys Med Rehab, 2006, 87(7):904.
[3] Smith E M, Comiskey C M, Carroll A M. A study of bone mineral density in adults with disability[J]. Arch Phys Med Rehab, 2009, 90(7):1127.
[4] Kasturi G C, Cifu D X, Adler R A. A review of osteoporosis: part Ⅰ. Impact, pathophysiology, diagnosis and unique role of the physiatrist[J]. PM R, 2009, 1(3):254.
[5] Chang K, Chang W H, Huang S, et al. Pulsed electromagnetic fields stimulation affects osteoclast formation by modulation of osteoprotegerin, RANK ligand and macrophage colony-stimulating factor[J]. J Orthop Res, 2005, 23(6):1308.
[6] Kr?lner B, Toft B. Vertebral bone loss: an unheeded side effect of therapeutic bed rest[J]. Clin Sci, 1983, 64(5):537.
[7] Lang T, Leblanc A, Evans H, et al. Cortical and trabecular bone mineral loss from the spine and hip in long-duration spaceflight[J]. J Bone Miner Res, 2004, 19(6):1006.
[8] Li Y. Protective roles of puerarin and danshensu on acute ischemic myocardial injury in rats[J]. Phytomedicine, 2007, 14(10):652.
[9] 郑高利,张信岳,郑经伟,等.葛根素和葛根总异黄酮的雌激素样活性[J].中药材,2002,25(8):566.
[10] Suthon S, Jaroenporn S, Charoenphandhu N, et al. Anti-osteoporotic effects of Pueraria candollei var. mirifica on bone mineral density and histomorphometry in estrogen-deficient rats[J]. J Nat Med, 2016, 70(2):225.
[11] Dongdong Y U, Shuai M U, Zhao D, et al. Puerarin attenuates glucocorticoid-induced apoptosis of hFOB1.19 cells through the JNK-and Akt-mediated mitochondrial apoptotic pathways[J]. Int J Mol Med, 2015, 36(2):345.
[12] 葸慧荣, 李文苑, 杨芳芳,等. 中药葛根素对青年大鼠峰值骨量的影响研究[J]. 中国骨质疏松杂志, 2018, 31(4):635.
[13] Zhang P, Kazunori H, Hiroki Y. A brief review of bone adaptation to unloading[J]. Genom Proteom Bioinf, 2008, 6(1):4.
[14] Jing D, Cai J, Wu Y, et al. Pulsed electromagnetic fields partially preserve bone mass, microarchitecture, and strength by promoting bone formation in hindlimb-suspended rats[J]. J Bone Miner Res, 2014, 29(10):2250.
[15] Shirazi-Fard Y, Anthony R A, Kwaczala A T, et al. Previous exposure to simulated microgravity does not exacerbate bone loss during subsequent exposure in the proximal tibia of adult rats[J]. Bone, 2013, 56(2):461.
[16] Ji Z, Shi C, Huang S, et al. Elcatonin attenuates disuse osteoporosis after fracture fixation of tubular bone in rats[J]. J Orthop Surg Res, 2015, 10(1):103.
[17] 罗新新, 徐国良, 黎宇,等. 葛根调节脂肪细胞糖脂代谢改善胰岛素抵抗的研究[J]. 中国中药杂志, 2016, 41(14):2687.
[18] 刘东吉, 余智奎, 刘春生,等. 葛根种质资源的分子地理标识研究[J]. 中国中药杂志, 2011, 36(3):299.
[19] 梁丽谊,王汝上.葛根总黄酮提取工艺优化研究[J].今日药学,2014,24(12):866.
[20] 裴莉昕,纪宝玉,陈随清,等.葛根多糖提取工艺的优化[J].中国现代中药,2017,19(4):553.
[21] 李波, 余金钟, 党中勤. 风药在久泻治疗中应用的理论探讨[J]. 中国中医基础医学杂志, 2018,24(5):713.
[22] 李珊珊, 刘逢芹, 郑世存. 异黄酮调控机体骨代谢现代研究[J]. 辽宁中医药大学学报, 2015(11):131.
[23] Dhainaut A, Hoff M, Syversen U, et al. Technologies for assessment of bone reflecting bone strength and bone mineral density in elderly women: an update[J]. Womens Health, 2016, 12(2):209.
[24] Oftadeh R, Perezviloria M, Villacamacho J C, et al. Biomechanics and mechanobiology of trabecular bone: a review[J]. J Biomech Eng, 2015, doi: 10.1115/1.4029176.
[25] Vidal B, Pinto A, Galv?o M J, et al. Bone histomorphometry revisited[J]. Acta Reumatológica Portuguesa, 2012, 37(4):294.
[26] Guo L, Zhang J P, Zhang K Y, et al. Effects of 1.8 GHz radiofrequency field on microstructureand bone metabolism of femur inmice[J].Bioelectromagnetics, 2018, 39(5):386.