Department of Trauma Surgery, Technical University Munich, MRI, Munich, Germany.
Department of experimental Trauma Surgery, Technical University Munich, MRI, Munich, Germany.
J Inflamm (Lond). 2014 May 18;11:15. doi: 10.1186/1476-9255-11-15. eCollection 2014.
Oxidative stress is involved in the pathogenesis of bone diseases such as osteoporosis, which has a high coincidence with fractures in elderly. Several studies showed positive effects of herbal bioactive substances on oxidative stress. This study analyses the effect of green tea extract (GTE) Sunphenon 90LB on primary human osteoblasts differentiation and viability during H2O2-induced oxidative stress. Moreover, it was analyzed, whether GTE acts during the HO-1 signaling pathway.
Human osteoblasts were isolated from femoral heads of patients undergoing total hip replacement. Beneficial effects of GTE on osteoblasts were examined in a dose- and time-dependent manner. Furthermore, GTE was given before, simultaneous with and after induction of oxidative stress with 1 mM H2O2 to simulate prophylactic, acute and therapeutic use, respectively. Cell damage was measured by LDH leakage and cell viability by MTT assay. Flow cytometry was applied to measure formation of Reactive Oxygen Species by using 27-dichlorofluorescein-diacetate. The formation of Extracellular Matrix after differentiation with GTE supplementation during oxidative stress was visualized with von Kossa and Alizarin Red staining. Last one was additionally photometrically quantified. To assess the effects of H2O2 and GTE on the osteogenic genes, RT-PCR was performed. To evaluate the intramolecular influence of GTE after the stimulation the protein levels of HO-1 were analyzed.
Stimulation of primary human osteoblasts with low doses of GTE during oxidative stress over 21 days improved mineralization. Furthermore, GTE supplementation in combination with H2O2 leads to a higher gene expression of osteocalcin and collagen1α1 during osteoblasts differentiation. Both are important for bone quality. Pre-incubation, co-incubation and post-incubation of osteoblasts with high doses of GTE protect the osteoblasts against acute oxidative stress as shown by increased cell viability, decreased LDH leakage, and reduced production of intracellular free radicals. Functional analysis revealed an increased HO-1 protein synthesis after stimulation with GTE.
Incubation of human primary osteoblasts with GTE significantly reduces oxidative stress and improves cell viability. GTE also has a beneficial effect on ECM production which might improve the bone quality. Our findings suggest that dietary supplementation of GTE might reduce inflammatory events in bone-associated diseases such as osteoporosis.
氧化应激与骨质疏松等骨骼疾病的发病机制有关，而骨质疏松症在老年人中与骨折的发生率非常高。一些研究表明，草药生物活性物质对氧化应激有积极的影响。本研究分析了绿茶提取物（GTE）Sunphenon 90LB 对过氧化氢诱导的氧化应激下人原代成骨细胞分化和活力的影响。此外，还分析了 GTE 是否作用于 HO-1 信号通路。
从接受全髋关节置换术的患者的股骨头中分离出人原代成骨细胞。以剂量和时间依赖性的方式研究 GTE 对成骨细胞的有益作用。此外，GTE 分别在诱导氧化应激前、同时和之后给予 1mM H2O2，分别模拟预防、急性和治疗用途。通过 LDH 漏出和 MTT 测定来测量细胞损伤。使用 2'7'-二氯荧光素二乙酸酯通过流式细胞术测量活性氧的形成。在用 GTE 补充物进行分化后形成细胞外基质，通过 von Kossa 和茜素红染色进行可视化。最后通过比色法进行定量。为了评估 H2O2 和 GTE 对成骨基因的影响，进行了 RT-PCR。为了评估 GTE 在刺激后的分子内影响，分析了 HO-1 的蛋白水平。
在氧化应激期间用低剂量 GTE 刺激原代人成骨细胞可改善矿物质化。此外，GTE 补充与 H2O2 联合使用可导致成骨细胞分化过程中骨钙素和胶原 1α1 的基因表达更高。两者对骨质量都很重要。用高剂量 GTE 对成骨细胞进行预孵育、共孵育和后孵育可保护成骨细胞免受急性氧化应激，表现为细胞活力增加、LDH 漏出减少和细胞内自由基产生减少。功能分析显示 GTE 刺激后 HO-1 蛋白合成增加。
用 GTE 孵育人原代成骨细胞可显著减轻氧化应激并提高细胞活力。GTE 对细胞外基质的产生也有有益的影响，这可能会提高骨质量。我们的研究结果表明，膳食补充 GTE 可能会减少与骨质疏松症等骨骼疾病相关的炎症事件。
