补肾活血复方藤黄健骨胶囊藤黄健骨胶囊通过激活Sirt1/FoxO3a的抗骨质疏松研究

Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule Attenuates Postmenopausal Osteoporosis via Activating Sirt1/FoxO3a

关键词:  补肾活血复方藤黄健骨胶囊  绝经后骨质疏松  Sirt1/FoxO3a信号轴  自噬

Key words:  Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule  postmenopausal osteoporosis  Sirt1/FoxO3a signal axis  autophagy

摘要:

目的 观察补肾活血复方藤黄健骨胶囊对绝经后骨质疏松模型鼠Sirt1、FoxO3a表达及自噬分子P62、Beclin1、LC3-Ⅱ表达水平变化的影响，旨在探讨补肾活血复方藤黄健骨胶囊治疗绝经后骨质疏松的作用机制。方法 建立去卵巢诱导的大鼠绝经后骨质疏松模型，观察一般状况；显微CT观察股骨骨微结构变化及骨体积分数（BV/TV）、骨小梁厚度（Tb.Th）；ELISA法检测ALP和CTX-I含量；实时荧光定量PCR（qPCR）检测Sirt1、FoxO3a、P62的基因水平；蛋白印迹法检测Sirt1、FoxO3a、P62、Beclin1、LC3-Ⅱ的表达。结果 1.一般状况和体重结果显示补肾活血复方藤黄健骨胶囊高剂量组能显著改善大鼠的一般状况，显著减轻大鼠的体重（p＜0.05）。2.显微CT结果显示补肾活血复方藤黄健骨胶囊能显著减轻股骨骨微结构损伤程度，补肾活血复方藤黄健骨胶囊中、高剂量组能显著升高BV/TV，高剂量组能显著升高Tb.Th（p＜0.05或p＜0.01）。3.ELISA结果显示补肾活血复方藤黄健骨胶囊各剂量组显著升高ALP含量，补肾活血复方藤黄健骨胶囊高剂量组能显著降低CTX-Ⅰ含量（p＜0.01）。4.Sirt1/FoxO3信号轴关键分子基因和蛋白检测结果显示补肾活血复方藤黄健骨胶囊各剂量组能显著升高Sirt1/FoxO3a信号轴关键分子Sirt1、FoxO3a基因表达和蛋白表达（p＜0.05或p＜0.01）。5.自噬分子基因和蛋白检测结果显示补肾活血复方藤黄健骨胶囊各剂量组能显著降低自噬分子P62基因表达和蛋白表达，显著升高自噬分子Beclin1蛋白表达，中、高剂量组能显著升高自噬分子LC3-Ⅱ蛋白表达（p＜0.05或p＜0.01）。结论 补肾活血复方藤黄健骨胶囊能够减轻股骨骨微结构损伤，治疗绝经后骨质疏松，可能是通过激活Sirt1/FoxO3a信号轴关键分子Sirt1、FoxO3a表达，进一步通过下调自噬分子P62和上调自噬分子Beclin1、LC3-Ⅱ来实现的。

Abstract:

OBJECTIVE To observe the effects of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsuleon the expression levels of Sirt1, FoxO3a, and the expression levels of autophagy molecules P62, Beclin1, and LC3-Ⅱ, its aim was to investigate the mechanism of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsulein postmenopausal osteoporosis (PMOP) rats. METHODS To A rats postmenopausal osteoporosis model was established using bilateral ovariectomy method, the general situation was observed. The microstructure changes were observed, and the bone volume fraction (BV/TV) , the trabecular thickness (Tb.Th) of femoral bone was checked using micro CT. The ALP and CTX-I content were detected by ELISA method. The gene levels of Sirt1, FoxO3a, and P62 were detected using real time fluorescence quantitative PCR (qPCR). The expression of Sirt1, FoxO3a, P62, Beclin1, LC3-Ⅱ was analyzed using Western blotting. RESULTS 1. The general situation and weight results showed that the general situation significantly improved, the weight significantly reduced in the high-dose group of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule (p<0.05). 2.The micro CT results showed that the Bushen Huoxue Fu Fang Tenghuang Jiangu Capsulecould significantly reduce the degree of femoral bone microstructure damage, the medium-dose and high-dose groups of the Bushen Huoxue Fu Fang Tenghuang Jiangu Capsulecould significantly increase BV/TV, while the high-dose group could significantly increase Tb.Th (p<0.05 or p<0.01). 3.The ELISA results showed that the ALP contents significantly increased in the high-dose group of Bushen Huoxue Recipe, the CTX-Ⅰ contents significantly decreased in the each-dose group of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule (p＜0.01). 4. The detection results of gene and protein of key molecules in signal axis showed that the gene and protein expressions of key molecule Sirt1, FoxO3a in the Sirt1/FoxO3a signal axis significantly raised in the each-dose group of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsulecould (p<0.05 or p<0.01). 5. The detection results of gene and protein of autophagy molecule showed that each-dose group of Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule could significantly reduce the gene and protein expression of autophagy molecule P62, and significantly increase the protein expression of autophagy molecule Beclin1 , the medium-dose and high-dose groups could significantly increase the protein expression of autophagy molecule LC3-Ⅱ (p<0.05 or p<0.01). CONCLUSION Bushen Huoxue Fu Fang Tenghuang Jiangu Capsule can alleviate microstructural damage to femoral bones and and treat PMO, possibly through activating the Sirt1 and FoxO3a expression of key molecules in Sirt1/FoxO3a signal axis, and further down- regulating the expression level of autophagy molecule P62 and upregulating the expression levels of autophagy molecules Beclin1 and LC3-Ⅱ.