Ugonin K enhances maturation and protects from reactive oxygen species-induced cell death in osteoblasts

• Main Authors: Chia-Hsin Lee, 李家昕
• Other Authors: Jyh-Fei Liao
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/42085565943797181053

博士 === 國立陽明大學 === 藥理學研究所 === 100 === Osteoporosis usually occurs in women after menopause and also develops in men during aging, which is due to an increased bone resorption (mediated by osteoclasts) and/or a decreased new bone formation (mediated by osteoblasts). Drug that represses bone resorption or accelerates bone formation therefore has the potential to ameliorate bone lose. Several lines of recent evidence also strongly suggest that the oxidative stress represents a principal cause of osteoblastic injury and death in the pathological conditions of osteoporosis. Thus, the strategy to increase bone mass includes stimulating the differentiation/maturation of osteoblasts and protecting from reactive oxygen species (ROS)-induced cell death. Ugonin K is a flavonoid isolated from the roots of Helminthostachys zeylanica (L.) Hook, a folk medicine used to strengthen bone mass and cure bone fracture. The present study showed that ugonin K significantly induced the increase of alkaline phosphatase (ALP) activity, expressions of bone sialoprotein (BSP) and osteocalcin (OCN), and subsequent mineralization in MC3T3-E1 cells. Western blot showed that ugonin K increased the nuclear level of estrogen receptor (ER)-α protein and the phosphorylated levels of Src, Akt, p38 and ERK1/2. Ugonin K-induced mRNA expressions of the transcription factors runt-related transcription factor 2 (Runx2) and osterix were inhibited by an ER antagonist ICI 182,780, Src inhibitor PP2, PI3K inhibitor wortmannin, p38 inhibitor SB203580 and MEK inhibitor PD98059. Except PD98059, pretreatment with ICI 182,780, PP2, wortmannin and SB203580 significantly repressed ugonin K induced ALP activity, mRNA expression of BSP and mineralization. Except SB203580, pretreatment with ICI 182,780, PP2, wortmannin and PD98059 repressed ugonin K-induced mRNA expression of OCN. These results suggested that ER, Src, Akt, p38 and ERK participated in regulating ugonin K evoked osteogenesis with different roles. Oxidative stress has recently been linked with osteoporosis. Therefore, the present study also investigated the protective effects of ugonin K against H2O2-induced cytotoxicity in osteoblasts. The results showed that ugonin K attenuated H2O2-induced cell death and this effect was better than ascorbic acid, α-tocopherol and probucol treatments. Within 2 hr challenge of H2O2, ugonin K posttreatment also significantly reversed H2O2-induced cell death. Annexin V-FITC/propidium iodide and ssDNA apoptosis ELISA assay confirmed that H2O2-induced cell death was due to cell apoptosis. H2O2 significantly induced ROS production, cytochrome c release, caspase-9, caspase-3 and poly(ADP) ribose polymerase activations, while ugonin K significantly repressed these H2O2-induced effects. This cytoprotective effect of ugonin K was abolished by ICI 182,780, PP2, wortmannin, SB203580 and PD98059. These results suggested that ugonin K protects osteoblasts from H2O2-induced apoptosis through ER mediated activations of Src, Akt, p38 and ERK1/2. In conclusion, this study found that ugonin K promotes ER into nucleus to bind estrogen response elements and stimulates transcriptional activity to increase expressions of bone-related growth factors via classical pathway in MC3T3-E1 cells. On the other hand, this study demonstrated that ugonin K-induced expressions of transcription factors (Runx2 and osterix) and stimulates expressions of osteoblast-related factors and mineralization may act through an ER dependent activation of a non-classical pathway, resulting in activations of Src, Akt, p38 and ERK1/2. Furthermore, ugonin K protects MC3T3-E1 cells against H2O2-induced apoptosis also through ER-dependent pathway. These results indicate that ugonin K may have the potential to be a therapeutic agent for the treatment of osteoporosis and it is worth performing further animal study.