基于斑马鱼模型的枸杞子改善骨质疏松活性部位筛选及其机制初探

Screening of active components and preliminary mechanism exploration of Lycii Fructus for improving osteoporosis based on the zebrafish model

利用斑马鱼幼鱼筛选枸杞子改善骨质疏松的活性部位,并通过斑马鱼成鱼骨质疏松模型验证筛选结果。以泼尼松龙为诱导剂构建斑马鱼幼鱼及成鱼骨质疏松模型,给予枸杞子不同提取物及化学部位共9组样品,通过茜素红染色后统计第一椎骨面积及光密度值,椎骨骨节数量以筛选枸杞子抗骨质疏松的活性部位。采用茜素红染色观察斑马鱼成鱼鳞片基质矿化及骨吸收情况;采用碱性磷酸酶(ALP)和抗酒石酸酸性磷酸酶(TRAP/TRACP)染色检测鳞片中成骨细胞和破骨细胞活性;采用qRT-PCR法测定鳞片中骨代谢相关基因alp、骨桥蛋白(opn)、成骨细胞特异性转录因子(sp7)、组织蛋白酶K (ctsk)、tracp、Runx家族转录因子2b (runx2b)的表达情况。结果显示,枸杞子各样品组均能够不同程度地改善斑马鱼幼鱼第一椎骨形成面积、染色光密度值和椎骨骨节数量,其中以枸杞子多糖部位效果最优。枸杞多糖能够显著抑制斑马鱼鳞片中骨吸收陷窝的形成,增强鳞片中ALP活性,降低TRAP活性,上调alp、sp7、opn基因,下调ctsk、tracp基因的表达。研究结果表明,枸杞多糖能够调节成骨细胞与破骨细胞活性,减少骨吸收,促进骨形成,增强骨密度,为枸杞子抗骨质疏松的主要活性部位。本研究为中药枸杞子“坚筋骨”传统功效提供了科学证据,为基于斑马鱼成鱼模型评价中药抗骨质疏松活性的研究提供了借鉴,也为老龄化社会人群骨健康需求提供了有益的启示。

Juvenile zebrafish were used to screen the active components of Lycii Fructus for improving osteoporosis.The screening results were further verified by zebrafish adult osteoporosis model and the action mechanism was explored.Prednisolone was used as the inducer to build osteoporosis models of juvenile and adult zebrafish,and 9 groups of samples of different extracts and chemical parts of Lycii Fructus were given.Alizarin red staining was applied for observing the scale matrix mineralization and bone resorption.The activities of osteoblasts and osteoclasts were detected using alkaline phosphatase(ALP) and tartrate resistant acid phosphatase(TRAP/TRACP) staining.The expressions of bone metabolism-related genes alp,osteoprotectin(opn),osteoblast specific transcription factor(sp7),cathepsin K(ctsk),tracp,and Runt family transcription factor 2b(runx2b) in each group were determined using quantitative polymerase chain reaction.The results showed that all components of Lycii Fructus improved the formation area of the first vertebrae,the staining light density value,and the number of vertebrae joints in juvenile zebrafish and the Lycium barbarum polysaccharide(LBP) treatment group exerted the best effect.In addition,LBP prevented the formation of bone resorption lacunae in zebrafish scales,increased ALP activity,decreased TRAP activity,up-regulated the alp,sp7,and opn genes,and lowered the expressions of ctsk and tracp genes.In conclusion,LBP regulated the activity of osteoblasts and osteoclasts,reduced bone resorption,promoted bone formation and enhanced bone density,which might be the main anti-osteoporosis active fraction of Lycii Fructus.This study provided modern scientific evidence for the scientific connotation of the traditional effect of "strengthening bones and muscles" of Lycii Fructus,provided the reference for the evaluation of the antiosteoporosis activity of traditional Chinese medicine based on zebrafish adult model,and provided beneficial enlightenment for the bone health needs of the aging society population.