| Background:Large-scale and multidirectional researches for osteoporosis have made great progress at home and abroad, but the study of Chinese medicine in the process of synthesis of b one collagen formation is still in its infancy, especially the research on the role of the molecular mechanism and targets for exploration is less The Guiluerxianjiao is a classic prescription in Chinese medicine. previous studies have confirmed that it can fill jing an d Tonify Qi in the kidney, and has a role in promoting the collagen formation on osteop orosis model. However the studies are not yet deeply, through what way, what pathways and targets are still unknown. According to the literature study, we hypothesized that Gui luerxianjiao play a role of the biology to stimulate osteoblast by regulating the TGF-β/S mads signal transduction, to promote the synthesis of bone collagen type I, which has th e effect on treatment of osteoporosis.Purpose:Discuss the influence of the Guiluerxianjiao on osteoblast synthesis of type I collagen and the G1OP zebrafish model scales form and the organizational structure. Explore the impact of the Guiluerxianjiao on osteoblasts TGF-β/Smad signaling pathway, and to analyse its promotion of type I collagen synthesis and its possible mechanism.Methods:TO culture osteoblast cell in vitro, and apply the drug-containing serum of the Guiluerxianjiao to cultivate the osteoblast. By Western blot method to detect the osteoblast type I collagen, TGF-β1> TGF-β2, Smad2 and Smad3 and Smad4, Smad7 protein synthesis; to detect the expression of osteoblast type I collagen, TGF-β1、TGF-β2, Smad2 and Smad3 and Smad4, Smad7 mRNA through RT-PCR. To explore the influence of the Guiluerxianjiao cultureing osteoblast in vitro by TGF-β/Smads signal pathways to composite the type I collagen.We used adult zebrafish as a model animal and use glucocorticoid dexamethasone as a model drug to establish a rapid and efficient new model of osteoporosis. Using the Guiluerxianjiao to the model, and detect scales Ca, P, ALP, and Hyp content of the zebrafish, using electron microscope scanning to observate the collagen ultrastructure of the zebrafish. By Western blot method to detect the osteoblast type I collagen, TGF-β1、TGF-β2, Smad2 and Smad3 and Smad4, Smad7 gene expression in zebrafish; And to detect By Western blot method the influence in zebrafish type Ⅰ collagen, TGF-β1, Smad3, Smad7 and Smad4 protein expression. To analyse the mechanism of the Guiluerxianjiao to compound osteoporosis zebrafish model of type I collagen through the TGF-β/Smad signaling pathways.Results:1, The result of Western blot method test for Osteoblast shows that:at 24 hours, high dose group of the Guiluerxianjiao can make the synthesis of osteoblast COL1A1, COL1A2 to increasing, other groups were no significant difference compared with normal group. At 48 hours, calcitriol group, low, medium and high dose groups of the Guiluerxinjiao can increase the expression of osteogenesis COL1A1 and COL1A2 protein, and the medium and high dose group of COL1A1 and COL1A2 protein expression were approaching, which were better than that of calcitriol group. At 24 hours and 48 hours, calcitriol group of TGF-β1, TGF-β2 protein expression increased without significantly; but the high dose group of the Guiluerxianjiao about osteoblasts TGF-β1, TGF-P2 expression significantly promoted; At 48 hours, low, medium and high dose group of the Guiluerxianjiao can both make the osteoblast TGF-β1, TGF-β2 protein expression increasing, medium and high dose group of the Guiluerxianjiao has no statistically significant difference. At 24 hours, high dose group of the Guiluerxianjiao could significantly increase the amount of Smad3 protein expression; At 48 hours, low, medium and high dose groups of the Guiluerxianjiao can increase osteoblast Smad3 protein expression. At 24 hours and 48 hours, compared with normal group, the quantity of Smad2 and Smad4, Smad7 protein expression in each dose group was no statistically significant difference.2, Osteoblast for RT-PCR method tests showed:calcitriol, low, medium and high dose groups of the Guiluerxianjiao can make the osteoblast COL1A1 and COL1A2 mRNA level increasing; COL1A1 rubber group, and COL1A2 mRNA level of the Medium and high dose groups of the Guiluerxianjiao increased obviously; TGF-β1 and TGF-β2 mRNA expression of the Calcitriol group had no significant effect; Low, medium and high dose groups of the Guiluerxianjiao can make the osteoblast TGF-β1, TGF-β2 mRNA level increasing;Calcitriol group, low, medium and high doses groups of guiluerxinjiao on osteoblast Smad2 and Smad4 and Smad7 mRNA level had no obvious effect. Smad3 mRNA expression level of Calcitriol group had no obvious effect; the increase of smad3 mRNA level was close in medium and high dose group.3, After establishing the GIOP zebrafish model, we observed the changes of osteoporosis zebra fish scales after dexamethasone induced, using methylene blue staining observation, we found zebrafish scales in blank group compact structure of organization and clear transition. We also found low dose dexamethasone group structure is loose, gap widened, blurred orelapse, changes were most obvious in high dose group. The calcium levels of each building groups’ fish scales began to drop. Among them, the medium dose dexamethasone group dropped obviously, the content of ALP of the medium and high dose dexamethasone began to rise.4, After using the the Guiluerxianjiao, the calcium content of each dose group were obviously higher than that the model group. But the ALP content was lower than the model group.5, We use Western blot method to detect the GIOP zebrafish protein protein expression: calcitriol group, low, medium and high doses groups of the Guiluerxianjiao also can make the GIOP zebrafish type Ⅰ collagen protein expression levels increasing; Among them, the medium and high dose groups of the Guiluexianjiao maked zebrafish type Ⅰ collagen protein expression level to increase significantly; the differences of Calcitriol group of TGF-β1 protein expression level had no statistical significance (P> 0.05); Low, medium and high dose groups of the Guiluerxianjiao can make the GIOP zebrafish TGF-β1 protein expression levels increasing (P< 0.01). The difference of Calcitriol group Smad3 protein expression level differences had no statistical significance (P> 0.05); Low, medium and high dose groups of the Guiluerxianjiao both can make the GIOP zebrafish Smad3 protein expression levels increasing, (P< 0.01). But the Smad4, Smad7 protein expression levels between every group in mutual comparison, there was no statistically significant difference.6, Using RT-PCR method to detect the GIOP zebrafish mRNA expression:calcitriol group, low, medium and high doses group of the Guiluerxianjiao both can make the GIOP zebrafish type Ⅰ collagen mRNA levels increasing (P< 0.01); and the medium,high dose of guiluerxianjiao for GIOP zebrafish is better than that of calcitriol group. There was no obvious effect to the Calcitriol group of zebrafish TGF-β1 Smad3 and Smad4, Smad7mRNA level (P> 0.05); Low, medium and high dose groups of the Guiluerxianjiao can make the GIOP zebrafish TGF-β1 and Smad3 mRNA levels rising, and Smad4, Smad7 mRNA level had no obvious influnence. |
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