自噬调控Keap1/Nrf2信号通路对成骨细胞氧化应激损伤的保护作用及其与骨质疏松的关系研究

Oxidative stress can damage various cellular components of osteoblasts, and is regarded as a pivotal pathogenic factor for osteoporosis. Recently, we found that autophagy could protect against oxidative stress damage to osteoblasts, but the mechanism is largely unknown. Furthermore, we discovered that autophagy could up-regulate the Keap1/Nrf2 pathway. Keap1/Nrf2 pathway plays an important role in promoting bone reconstruction as well as suppressing oxidative stress. Our present project is designed to investigate the role of autophagy mediated Keap1/Nrf2 pathway in oxidative stress damage to osteoblasts and its contribution against osteoporosis. At first, we will measure the change of expressions of autophagy related factors and factors in the Keap1/Nrf2 pathway in the histological samples from osteoporotic patients and from an osteoporotic animal model. Then, these factors will be analyzed in the osteoblasts under oxidative stress in vitro. Meanwhile, the effect of autophagy on the Keap1/Nrf2 pathway will be evaluated by using gene transfection and RNA interference techniques. Finally, the in vivo function of BECN-1 on the proliferation, apoptosis and differentiation of osteoblasts will be investigated in the LC3-GFP transfected mice. Our results should be helpful to improve the understanding of the role of autophagy in oxidative stress damage to osteoblasts and provide a new antioxidant target for the management of osteoporosis.

氧化应激诱导成骨细胞凋亡是骨质疏松始动因素之一，申请者前期研究表明自噬抑制成骨细胞氧化应激，但具体机制尚不清楚。预实验发现自噬上调Keap1/Nrf2通路，而其在抗氧化应激和促进骨重建中起重要作用。故我们提出假说：自噬激活Keap1/Nrf2通路，抑制氧化应激，促进骨重建。为验证这一假说，本课题拟利用人体标本和骨质疏松动物模型在组织学水平检测自噬和Keap1/Nrf2通路相关基因表达；通过体外诱导成骨细胞氧化应激损伤，检测自噬和Keap1/Nrf2信号分子表达；利用基因转染和阻断技术，明确自噬调控Keap1/Nrf2信号通路关键信号分子；最后应用GFP-LC3转基因小鼠建立骨质疏松模型以验证BECN-1对成骨细胞Keap1/Nrf2信号通路和骨代谢影响；系统分析自噬调控Keap1/Nrf2信号通路在氧化应激诱导成骨细胞凋亡过程中的作用机制，为骨质疏松的抗氧化治疗开辟新思路。

目的：BECN-1是自噬关键调控基因，研究证实BECN-1在衰老，骨端关节炎等退变性疾病过程中的发挥重要作用。然而，BECN-1在骨质疏松和骨形成中的作用尚不。本项研究的目的在于观察BECN-1基因对成骨细胞Keap1/Nrf2信号通路的影响。方法：应用慢病毒 (pGC-FU-3FLAG-BECN-1)和Talen技术分别构建过表达和干扰BECN-1基因载体，建立BECN-1过表达和沉默细胞系；CCK-8检测成骨细胞增殖率；流式检测BECN-1基因过表达和沉默对细胞周期影响；成骨诱导分化4周，茜素红染色检测成骨细胞矿化能力；成骨诱导分化48hrs，qRT-PCR和western blot检测自噬相关keap1、p-Nrf2、Nrf2、SOD1、SOD2基因和蛋白表达表达水平；最后，Annexin V-PE/7AAD染色流式细胞检测BECN-1基因对成骨细胞凋亡的影响。结果：成功构建稳定BECN-1过表达和沉默的Mc3T3-E1细胞系；CCK-8检测结果显示： BECN-1基因沉默导致细胞增殖受抑制，但是BECN-1过表达细胞增殖能力无明显改变； BECN-1基因沉默细胞矿化结果数量明显减少，而BECN-1过表达不影响细胞矿化能力；成骨诱导分化48 hrs，qRT-PCR和western blot检测BECN-1基因沉默抑制keap1、p-Nrf2、Nrf2、SOD1、SOD2基因和蛋白表达下降，而BECN-1过表达对Keap1/Nrf2信号通路相关蛋白表达影响不明显。过氧化氢处理12 hrs，BECN-1基因沉默导致细胞更易受过氧化氢损伤，成骨细胞凋亡率升高，而BECN-1过表达则抑制成骨细胞凋亡。结论：自噬基因BECN-1缺失抑制成骨细胞增殖和分化，诱导成骨细胞凋亡。BECN-1基因沉默导致细胞更易受氧化应激损害，而BECN-1表达则抑制成骨细胞氧化应激损伤。BECN-1可能是骨质疏松抗氧化治疗的有效靶点。