女贞子及其成分对体内外氧化应激诱导骨量减少的预防作用及作用机制

Chinese herb Fructus Ligustri lucidi is commonly used on strengthening tendons and bones, and nourishing liver and kidney yin, which is also suitable for long-term use. Modern pharmacological research has proved Fructus Ligustri lucidi and its components some positive functions, such as antioxidation, osteogenesis promotion and immunity improving. They also showed good ability on protecting livestock health and improving production performance at the same time in intensive farming. So Fructus Ligustri lucidi and its components will be the ideal feed additives product. This project is based on animal osteopenia model induced by oxidative stress and osteoblast oxidative damage model induced by H2O2 in vivo and in vitro. After Fructus Ligustri lucidi and its components are added in the animal model and osteoblast model, the bone mineral density and the SOD and MDA content in blood, liver or kidney will be assayed, the model cell proliferation, differentiation and apoptosis will be detected, SOD and MDA at cellular level will be assayed, and expression level of cell OPG and RANKL gene and protein will be tested, so we can explore the protection effect of Fructus Ligustri lucidi and its components when the animal bones and osteoblasts were challenged with oxidative stress. Then through the MAPK signaling pathway inhibitors assays and expression of MAPK and phosphorylated MAPK, upon the basis of cell pharmacodynamics studies, we can explore the anti-oxidative stress effect of MAPK signaling pathway on Fructus Ligustri lucidi and its components on osteoblast injury. This project will provide the basis for further in-depth research on the antioxidative mechanism of Fructus Ligustri lucidi.

中药女贞子强筋健骨，滋肝肾之阴，久服轻身。药理研究表明女贞子及其成分具有抗氧化、促成骨、提高免疫力等作用，在保护集约养殖条件下动物健康的同时可提高其生产性能，是理想的中药添加剂。本项目以体内外氧化应激诱导动物骨量减少模型和成骨细胞氧化损伤模型为基础，将女贞子及其成分分别作用于模型动物和模型细胞后，检测模型小鼠骨密度、血液及肝肾SOD和MDA；检测模型细胞增殖、分化、凋亡，SOD和MDA水平，以及OPG和RANKL基因和蛋白水平；研究氧化应激状态下，中药女贞子及其成分对动物骨骼和成骨细胞的保护作用；并在细胞药效研究的基础上，通过MAPK通路抑制剂以及通路蛋白磷酸化水平的研究，探讨MAPK通路在女贞子及其成分抗成骨细胞氧化损伤中的作用，为进一步深入研究女贞子的抗氧化作用机制提供依据。

中药女贞子强筋健骨，可滋肝肾之阴。药理研究表明女贞子及其成分因其抗氧化、促成骨、提高免疫力等作用，有作为养殖动物营养代谢骨量减少的添加剂的潜力。本项目通过D-半乳糖联合低钙饲喂20d后，灌胃不同浓度的女贞子水提液作用于模型小鼠，检测模型小鼠及中药各组小鼠的股骨系数、股骨干湿比、股骨骨密度以及血清SOD、MDA以及OPG含量，确立小鼠骨量减少模型构建方法，并分析女贞子对小鼠骨量减少模型的影响；通过0.25-0.6mmmol/LH2O2作用于MC3T3-E1成骨细胞系，确立成骨细胞氧化损伤模型，并将一定浓度的女贞子及其成分熊果酸UA和齐墩果酸OA，确定浓度的UA或OA联合p38MAPK、JNK、ERK通路抑制剂作用于模型细胞24h后，通过测定细胞增殖、细胞上清ALP、OPG和RANKL水平，分析女贞子及其成分对细胞氧化损伤模型的影响及MAPK通路机制。研究结果表明，通过10g/Kg·d剂量的D-半乳糖联合0.1%低钙饮食饲养20d后可构建小鼠骨量减少模型，高浓度（7g/Kg·d）和中浓度（3.5g/Kg·d）的女贞子均可通过提升模型小鼠的股骨系数和骨干湿比以及骨密度，表明女贞子具有对氧化应激骨量减少具有保护作用；通过0.25-0.6mmol/L的H2O2可构建成果细胞氧化损伤模型，且1-1E-6mg/ml的女贞子以及50-1μM的UA和OA可以提高氧化损伤细胞活性，降低ALP活性，提高模型细胞的OPG蛋白分泌量，但对RANKL蛋白无影响；通过抑制剂研究发现，女贞子成分UA和OA的抗成骨细胞氧化损伤作用主要是通过p38MAPK和JNK通路起到提高模型细胞活性和OPG分泌功能的作用，且可以与UA和OA联合降低血清ALP分泌，而ERK通路抑制剂仅可抑制UA升氧化应激模型细胞损伤细胞OPG作用。本项目结果为进一步深入研究女贞子的抗氧化作用机制提供依据。