基于ROS调控BK通道研究成骨细胞骨形成减少及二仙汤干预的分子机制

It may be an important topic for prevention and cure of osteoporosis, which to study the biological basis of bone formation decrease and the effect of Chinese Medicine Herbs. It was established that the apoptosis and its function decrease of osteoblast induced by ROS. The large conductance calcium-activated-potassium channels (BK channel) is expressed in osteoblast, associated with bone mineralization. It was established that the ovarian rat bone tissue BK channel protein expression was significantly increased. The Erxian decoction, a prevention and treatment of osteoporosis prescription of traditional Chinese medicine, can decrease ROS and reduce the BK channel protein expression, promote osteoblast bone formation, however, the expression of the BK channel, current characteristics and regulation factors are not clear. In this study, that the effect of Erxian decoction on ROS, the BK channel expression, BK channel current characteristics, bone formation related gene and protein were examined. The differential protein among ovarian rats tissues or H2O2-stimulated osteoblast and administer of Erxian decoction were discovered by qantitative proteomic techniques. The graph of relationship of protein-protein was established. The expression protein of hyper-expression and shRNA plasmid also constructed. And the BK channel current change detected by Patch Clamp, the concentration of ROS also detected by laser scanning confocal microscope, the expression of BK channel protein, and bone formation related gene and protein were also measured by Realtime PCR or Western blot. The implementation of the project will clarify the protein and protein network relationship in ROS regulated BK channel procession, and discover the molecular mechanism about bone formation reduction of osteoblast and treatment with Erxian decoction.

探讨骨形成减少的生物学基础及中药的干预作用已成为骨质疏松症研究的重要方向。ROS诱导成骨细胞凋亡和功能下降；成骨细胞表达BK通道蛋白，直接影响骨矿化。前期研究发现，去卵巢大鼠骨组织BK通道表达增加，而防治骨质疏松症的二仙汤降低ROS浓度，减少BK通道表达，促进成骨细胞骨形成。但ROS对成骨细胞BK通道表达和功能的调节并不清楚。本项目拟考察二仙汤对成骨细胞ROS、BK通道和骨形成的影响，以定量蛋白质组学检测分析去卵巢大鼠骨组织（H2O2刺激成骨细胞）及二仙汤干预前后的差异表达蛋白，构建蛋白网络关系图；建立差异蛋白过表达、shRNA体系，以膜片钳检测BK通道电流变化；激光共聚焦显微镜检测ROS浓度；Real time-PCR和Western Blot检测BK通道、与骨形成相关基因、蛋白的表达。项目实施将阐明ROS调控BK通道的蛋白相互作用网络关系，揭示成骨细胞骨形成减少和二仙汤干预的分子机制。

骨质疏松症是常见的老年性疾病，严重影响生活质量。探讨骨形成减少的生物学基础及中药的干预作用是骨质疏松症研究的重要方向。ROS诱导成骨细胞凋亡和功能下降，成骨细胞表达BK通道蛋白，直接影响骨矿化。本研究通过大鼠去卵巢复制骨质疏松模型，考察了二仙汤的干预研究，明确了二仙汤改善去卵巢大鼠骨质疏松的作用。以定量蛋白组学分析了差异蛋白变化，明确了二仙汤防治骨质疏松症的作用靶点。通过应用BK通道阻断剂TEA，研究二仙汤对成骨细胞BK通道和骨形成间的作用，发现二仙汤抑制BK通道蛋白表达，能促进成骨细胞骨形成，与PTEN/AKT/FoXo1信号通路的密切关联。通过H2O2诱导成骨细胞骨形成减少模型，考察了二仙汤的干预作用，分析了二仙汤对BK通道表达、骨形成相关基因、蛋白表达的作用。确定BK通道在二仙汤促进成骨细胞骨形成中占重要地位。二仙汤可促进成骨细胞骨形成，与提高AKT和Foxo1的磷酸化，降低PTEN的表达有关，但TEA可逆转二仙汤的这些作用。应用液质联用技术发现二仙汤含602个化合物，包括生物碱65个、黄酮类109个等。网络药理学分析显示槲皮素，木犀草素，芹菜素、芦丁、芒果苷等可能是主要活性成分，构建了活性成分-交集靶点网络图和分子对接图，与BK通道、PTEN、AKT、Foxo1信号通路具有结合活性。研究了二仙汤主要成分芒果苷对成骨细胞的自噬和凋亡的作用，发现芒果苷能降低H2O2诱导的成骨细胞凋亡率，减少ROS和钙离子含量，促进细胞自噬，抑制凋亡。研究了二仙汤对骨髓间充质干细胞（BMSCs）外泌体的影响，分析了血清外泌体，发现二仙汤可下调BMSCs外泌体中miR-26a-5p和miR-143-3p的表达。还研究二仙汤含药血清外泌体纤连蛋白（Fn）对成骨细胞骨形成的作用等。项目实施阐明了ROS调控BK通道的蛋白相互作用网络关系，揭示了成骨细胞骨形成减少和二仙汤干预的分子机制，对深刻认识骨质疏松形成和推动二仙汤的临床应用具有重要科学意义。