二至丸治疗斑马鱼骨质疏松模型效益观察及破骨细胞自噬机制

Observation on the Effect of Erzhiwan on Zebrafish Osteoporosis Model and Study on Autophagy Mechanism of Osteoclast

    摘要
  • 摘要: 目的 观察二至丸对泼尼松龙诱导的斑马鱼骨质疏松模型的治疗疗效，并明确其对破骨细胞分化的自噬调控机制。方法 选取正常培养3 d后活力较好的斑马鱼幼鱼，用25 μmol/L泼尼松龙建立斑马鱼骨质疏松症模型，造模2 d后分组，分为空白组、模型组、依替磷酸二钠(ED)组(300 mg/L)、墨旱莲组(50 μg/mL)、女贞子组(50 μg/mL)和二至丸组(100 μg/mL)。4 d后，用0.2%钙黄绿素进行染色，拍照并统计斑马鱼脊椎骨荧光面积；实时荧光定量PCR检测斑马鱼成骨分化基因碱性磷酸酶（ALP）、骨形态发生蛋白2b（Bmp-2b）、Runt相关转录因子2（Runx2）和破骨分化基因组织蛋白酶K（CTSK）、活化T细胞核因子（NFATC-1）、抗酒石酸酸性磷酸酶（TRAP）的表达。培养RAW264.7细胞至80%~90%密度后分为空白组、LPS诱导组(10 μg/L)、二至丸诱导组（100 μg/mL）、墨旱莲诱导组(50 μg/mL)和女贞子诱导组(50 μg/mL)。经LPS诱导细胞破骨分化及药物干预4 d后，实时荧光定量PCR检测RAW264.7细胞TRAP、CTSK、NFATC-1和自噬相关基因5（ATG5），泛素结合蛋白62（p62），雷帕霉素靶蛋白（mTOR）和酵母ATG6同源物（Beclin）的mRNA表达。结果 与空白组比较，模型组椎骨荧光面积显著减少（P<0.01），成骨标志基因表达显著降低（P<0.05~0.01），破骨标志基因表达显著升高（P<0.05~0.01）；与模型组比较，ED组、女贞子组及二至丸组均显著改善了斑马鱼骨质疏松症（P<0.01），且二至丸组显著优于墨旱莲组及女贞子组（P<0.01）。qPCR显示相较于空白组，模型组成骨分化标志物表达显著降低（P<0.05~0.01），破骨分化标志物表达显著升高（P<0.05~0.01）。与模型组比较，ED组、墨旱莲组、女贞子组及二至丸组成骨标志基因表达显著升高（P<0.05~0.01），二至丸组破骨标志基因表达显著降低（P<0.01）。体外研究显示，LPS诱导的细胞破骨分化基因CTSK、TRAP和NFATC-1及自噬相关基因mTOR、ATG5、Beclin、p62表达均升高（P<0.01），二至丸组、墨旱莲组、女贞子组均可显著降低破骨分化基因和自噬相关基因的表达（P<0.05~0.01）。结论 二至丸可显著改善斑马鱼骨质疏松模型的临床症状，其机制可能与通过降低自噬相关基因表达从而抑制破骨细胞分化有关。

     

    Abstract: OBJECTIVE To observe the treatment efficacy of Erzhiwan on prednisolone-induced osteoporosis of zebrafish model and to clarify its regulation of autophagy mechanism on osteoclast differentiation. METHODS The young zebrafishes of good energy after 3 days， normal development were selected. The zebrafish osteoporosis model was established with 25 μmol/L prednisolone. The zebrafishes were divided into control group， model group， etidronate disodium group (300 mg/L)， Mohanlian group (50 μg/mL)， Nvzhenzi group (50 μg/mL) and Erzhiwan group (100 μg/mL). After 4 days， the calcein staining was adopted to measure the vertebral bone fluorescence area of zebrafish; qPCR was adopted to detect the osteogenic marker genes ALP， BMP-2b， Runx2 and osteoclast marker genes CTSK， TRAP， NFATC-1 expression. RAW264.7 cells were divided into control， LPS induction (10 μg/L)， EZW(100 μg /mL)， MHL(50 μg/mL)， and NZZ group (50 μg/mL) after they were cultured to 80%~90% density. The mRNA expression of TRAP， CTSK and autophagy-related genes 5 (ATG5)， ubiquitin-binding protein 62 (p62)， mammalian target of rapamycin(mTOR)，and Beclin were detected by qPCR. RESULTS Compared with the control group， the model group had significant declines in both vertebral bone fluorescence area and osteogenic marker gene expression and had significant rise in osteoclast marker genes（P<0.05，P<0.01）. Compared with the model group， the ED group， the Nvzhenzi group and the Erzhiwan group all remarkably improved zebrafish osteoporosis and acted better than both Mohanlian group and Nvzhenzi group（P<0.01）. qPCR showed that compared with the control group， the model group's osteogenic marker gene expression significantly declined and osteoclast marker genes rose conspicuously（P<0.05，P<0.01）. Compared with model group， the ED group， Mohanlian group and the Nvzhenzi group as well as Erzhiwan group had osteogenic marker gene expression rose significantly（P<0.05，P<0.01）， and Erzhiwan group has osteoclast marker genes reduced significantly（P<0.01）. The in-vitro study showed that the expression of osteoclast marker genes CTSK， TRAP， ATG5 and NFATC-1 induced by LPS as well as autophagy-related genes mTOR， ATG5， Beclin， p62 all rose（P<0.01）. The Erzhiwan group， Mohanlian group and Nvzhenzi group significantly decreased the expressions of osteoclast marker genes and autophagy-related genes（P<0.05， P<0.01）. CONCLUSION Erzhiwan can significantly improve the clinical symptoms of osteoporosis in zebrafish model， its mechanism may be related to the inhibition of the osteoclast-differentiation by reducing autophagy-related gene expressions.

     

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