相同摩尔浓度下淫羊藿苷及朝藿定C单体在斑马鱼骨质疏松模型中的活性研究

Activity Study of Icariin and Epimedin C Monomer under the Same Molarity in Osteoporosis Zebrafish Model

目的用泼尼松龙诱导的斑马鱼骨质疏松模型评价相同摩尔浓度下淫羊藿苷和朝藿定C单体的抗斑马鱼骨质疏松作用。方法将受精后4日的斑马鱼胚胎分为S组(0.5%二甲亚砜DMSO)、A组(泼尼松龙25μmol/L,0.5%DMSO)、B组(2IU/L鲑降钙素,泼尼松龙25μmol/L,0.5%DMSO)、C组(淫羊藿苷1.5μmol/L,泼尼松龙25μmol/L,0.5%DMSO)、D组(淫羊藿苷15μmol/L,泼尼松龙25μmol/L,0.5%DMSO)、E组(淫羊藿苷150μmol/L,泼尼松龙25μmol/L,0.5%DMSO)、F组(朝藿定C 1.5μmol/L,泼尼松龙25μmol/L,0.5%DMSO)、G组(朝藿定C 15μmol/L,泼尼松龙25μmol/L,0.5%DMSO)、H组(朝藿定C 150μmol/L,泼尼松龙25μmol/L,0.5%DMSO)。所有培养液均含有0.5%DMSD。将各组幼鱼放置与24孔板中,每日更换培养液,在恒温环境28.5℃培养箱中,培养至第9天处死,以茜素红染色。用显微镜对斑马鱼颅骨腹侧进行观察,定量分析将成像染色区域。结果与S组比较,A组骨矿化累计光密度值降低(P<0.01);B组骨矿化累计光密度值升高(P<0.01)。而C、D、E组随浓度增加矿化面积呈微弱的递增趋势(P<0.05)。与A组比较,B组累计光密度值增高明显,差异有统计学意义(P<0.01),C、D组差异无统计学意义(P>0.05),而E组明显升高(P<0.05)。F、G组随浓度增加,矿化面积呈递增趋势,颅骨染色累计光密度值升高(P<0.05),H组内斑马鱼胚胎无存活。E、F、G组间比较,差异无统计学意义(P>0.05)。S组斑马鱼头颅骨染色清晰,脊椎骨及两旁鳃骨染色清晰。A组在相同染色区域的强度明显减少。B组在相同条件下呈现骨组织成骨加快,矿化面积明显增多,骨组织深染等特点。C、D、E、F、G组,在染色中颅骨矿化程度逐渐增加,椎骨及两侧腮骨矿化面积及染色强度递增,以椎骨改变最为显著,但均未达到B组的染色强度。结论斑马鱼骨质疏松模型是一种简单、高效的中药成分筛选模型,低浓度朝藿定C在该模型中的活性优于淫羊藿苷,高浓度朝藿定C的可能毒性作用还需要深入研究。

Objective To evaluate anti-osteoporotic activity of icariin and Epimedin C monomer under the same molarity in predinsolone-induced osteoporosis zebrafish. Methods Zebrafish larvae after 4-day fertilization were divided into group S E0.5% dimethyl sulfoxide （DMSO） ）, A （25 μmol/L predniso- lone, 0.5% DMSO）, B （2 lUlL salmon calcitonin, 25 μmol/L prednisolone,0.5% DMSO）, C （1.5 μmol/L icariin, 25 μmol/L prednisolone, 0.5% DMSO）, D （15 μmol/L icariin,25 μmol/L prednisolone, 0.5% DM- SO）, E （150 μmol/L icariin, 25 μmol/L prednisolone, 0.5% DMSO）, F （1.5 μmol/L Epimedin C, 25 μmol/L prednisolone, 0.5% DMSO）, G （15 μmol/L Epimedin C, 25 μmol/L prednisolone, 0.5% DM- SO）, H （150 μmol/L Epimedin C, 25 μmol/L prednisolone, 0.5% DMSO）. All culture solution contained 0.5% DMSO. All the young fishes were grown in a 24-well plate. The culture medium was changed every day. They were cultured in a incubator box at 28.5 ℃; and killed at day 9. Zebrafish skeleton was stained with alizarin red. The stained Zebrafish ventral skull was observed using microscope, and mineralized area was quantitatively analyzed. Results Compared with group S, accumulative integrated optical densi- ty（IOD）of the mineralized area significantly decreased in group A （P 〈0.01 ） ; accumulative IOD of the mineralized area significantly increased in group B （P 〈0.01 ）. The accumulative IOD of the mineralized area showed weakly increasing tendency in group C, D, and E along with increased concentration （P 〈 0.05）. Compared with group A, accumulative IOD obviously increased in group B with statistical difference （P 〈0.01）, but with no statistical difference as compared with group C or group D （P 〉0.05）. Statistical difference existed in accumulative IOD between group A and group E （P 〈0.05）. The mineralized area showed increasing tendency in group F and group G along with increased concentration （P 〈0.05）, and accumulative IOD obviously increased as well （P 〈0.05）. No Zebrafish embryo survived in group H. There was no statistical difference in Zebrafish embryo survival among group E, F, or G （P 〉0.05）. The staining of Zebrafish skull was clearly seen in group S, with vertebrae and bilateral branchial skeleton clearly seen. The intensity of staining in the same area was obviously attenuated in group A. The osteogenesis was speeded up under the same condition in group B, with obviously enlarged mineralized area and more darkly stained bone tissue. The mineralization of skull was gradually increasing during the stai- ning process in group C, D, E, F, and G. The mineralized area and the intensity of staining were gradually enhanced, and changes of vertebrae were most obviously seen in group C, D, E, F, and G, but they were not arrived at the stained intensity level in group B. Conclusions Osteoporosis Zebrafish model is a simple and efficient model for screening bioactive ingredients of Chinese herbs. The activity of Epimedin C at low concentration was better than icariin in this model. But possible toxicity of Epimedin C at high concentration needs to be further studied.