补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制

谭登; 张玉; 张农山; 刘军; 万仕炜; 韩龙; 方彭华; 闵文

doi:10.13422/j.cnki.syfjx.20200641

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补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制

药理 | 更新时间：2021-02-09

- 补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制
- Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast
- 中国实验方剂学杂志 2020年26卷第7期页码：79-85
- 作者机构：
  
  1.南京中医药大学　附属医院，南京　 210029
  2.南京中医药大学　翰林学院，江苏泰州　 225300
- 作者简介：
  
  [第一作者]　谭登，在读硕士，从事中医药防治骨质疏松症研究，E-mail: tandeng1993@126.com
  *闵文，博士，教授，从事中医药防治骨质疏松症研究，E-mail: wenge1977@126.com
- 基金信息：
  
  国家自然科学基金面上项目(81774335;81473390);江苏省研究生科研与实践创新计划项目(KYCX19_1212)
- DOI：10.13422/j.cnki.syfjx.20200641
  中图分类号： R2-0; R289; R285.5
- 收稿日期：2019-09-21，
  
  网络出版日期：2019-12-07，
  
  纸质出版日期：2020-04-05
- 稿件说明：
移动端阅览
谭登, 张玉, 张农山, 等. 补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制[J]. 中国实验方剂学杂志, 2020,26(7):79-85.

Deng TAN, Yu ZHANG, Nong-shan ZHANG, et al. Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(7): 79-85.
谭登, 张玉, 张农山, 等. 补肾通络方对骨质疏松斑马鱼效应评价及破骨细胞自噬机制[J]. 中国实验方剂学杂志, 2020,26(7):79-85. DOI： 10.13422/j.cnki.syfjx.20200641.

Deng TAN, Yu ZHANG, Nong-shan ZHANG, et al. Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(7): 79-85. DOI： 10.13422/j.cnki.syfjx.20200641.

摘要

目的：

评价补肾通络方对斑马鱼骨质疏松模型的干预效应，并明确其对破骨细胞分化的自噬调控机制。

方法：

选取新生3 d健康斑马鱼幼鱼260尾，25 mmol·L

-1

泼尼松龙(Pred)干预3 d，通过钙黄绿素染色确认建模成功，分为空白组，Pred组(25 mmol·L

-1

)，依替磷酸二钠组(300 mg·L

-1

)，补肾组(180 mg·L

-1

)，通络组(30 mg·L

-1

)和补肾通络组(210 mg·L

-1

)各40尾。药物干预4 d后采用钙黄绿素染色后拍照，统计斑马鱼脊椎骨荧光面积；实时荧光定量PCR(Real-time PCR)检测斑马鱼碱性磷酸酶(ALP)，骨形态发生蛋白-2b(BMP-2b)，Runt相关转录因子2(Runx2)，组织蛋白酶K(CTSK)，抗酒石酸酸性磷酸酶(TRAP)及活化T细胞核因子(NFATC-1)mRNA表达。培养RAW264.7细胞至80%～90%密度后分为空白组，Rankl诱导组(10 μg·L

-1

)，补肾组(180 mg·L

-1

)，通络组(30 mg·L

-1

)和补肾通络组(210 mg·L

-1

)。经Rankl诱导细胞破骨分化及药物干预4 d后，采用FITC标记鬼笔环肽染色检测肌动蛋白环表达，Real-time PCR检测RAW264.7细胞TRAP，CTSK和自噬相关基因5(ATG5)，自噬相关基因7(ATG7)，泛素结合蛋白62(p62)mRNA表达。

结果：

在体内研究中，与空白组比较，Pred组斑马鱼椎骨面积及ALP，BMP-2b，Runx2表达显著下降(

0.01)，CTSK，TRAP，NFATC-1表达显著升高(

0.01)。与Pred组比较，通络组椎骨面积明显增加(

0.05)，而补肾通络组椎骨面积显著增加(

0.01)，补肾组，通络组及补肾通络组ALP，BMP-2b，Runx2表达均显著升高(

0.01)，补肾通络组CTSK，TRAP，NFATC-1表达明显降低(

0.05，

0.01)。体外研究显示，与空白组比较，Rankl诱导组肌动蛋白环数目显著增多(

0.01)，CTSK，TRAP和ATG5表达显著升高(

0.01)，ATG7，p62表达明显升高(

0.05)。与Rankl诱导组比较，补肾组、通络组及补肾通络组均明显减少了肌动蛋白环数目及CTSK，TRAP表达(

0.05，

0.01)，而补肾通络组同时降低了ATG5，ATG7，p62表达(

0.05，

0.01)。

结论：

补肾通络方能够显著改善泼尼松龙诱导的斑马鱼骨质疏松症，并通过降低自噬相关基因表达抑制破骨细胞分化。

Abstract

Objective:

To evaluate the intervention effect of Bushen Tongluo formula on zebrafish osteoporosis model and to clarify its regulation of autophagy mechanism on osteoclast differentiation.

Method:

The 260 young zebrafishes (3 dpf) were selected

the zebrafish osteoporosis model was established with 25 mmol·L

-1

prednisolone (Pred) for 3 days

confirming the successful model by calcein staining. The zebrafishes were divided into control group

Pred group (25 mmol·L

-1

)

etidronate disodium (ED) group (300 mg·L

-1

)

Bushen (BS) group (180 mg·L

-1

)

Tongluo (TL) group (30 mg·L

-1

)

and Bushen Tongluo (BSTL) group (210 mg·L

-1

)

40 tails per group. After intervened with medicine for 4 days

the calcein staining was adopted to count the vertebral bone fluorescence area of zebrafish

Real-time PCR was adopted to detect the mRNA expression of akaline phosphatase (ALP)

bone morphogenetic protein 2b (BMP-2b)

runt-related protein 2 (Runx2) and cathepsin K (CTSK)

phosphorus and tartrate-resistant acid phosphatase (TRAP)

nuclear factor of activated T-cells

cytoplasmic-1 (NFATC-1). Divided RAW264.7 cells into control

Rankl induction (10 μg·L

-1

)

BS (180 mg·L

-1

)

TL (30 mg·L

-1

)

and BSTL group (210 mg·L

-1

) after they were cultured to 80%-90% density. The expression of actin ring was detected by phalloidin cytoskeleton staining. The mRNA expression of TRAP

CTSK and autophagy-related genes 5 (ATG5)

autophagy-related genes 7 (ATG7)

and ubiquitin-binding protein p62 (p62) were detected by Real-time PCR.

Result:

Compared with the control group

in vivo

the vertebral area and ALP

BMP-2b

and Runx2 expression of zebrafish in the Pred group were significantly decreased (

0.01)

and CTSK

TRAP

and NFATC-1 expression of zebrafish were significantly increased (

0.01). Compared with the Pred group

the vertebral area of the TL (

0.05) and BSTL group (

0.01) increased significantly. The expressions of ALP

BMP-2b

and Runx2 in the BS

and BSTL group were significant increased (

0.01). The expression of CTSK

TRAP

and NFATC-1 in BSTL group were significant decreased (

0.05

0.01). Compared with the control group

in vitro

the number of actin rings (

0.01) and CTSK

TRAP

ATG5 (

0.01) expression and ATG7

p62 (

0.01) expression were significantly increased in the Rankl-induced group. Compared with the Rankl induction group

the number of actin rings and CTSK and TRAP expression were significantly decreased in the BS

and BSTL group (

0.05

0.01)

while the expression of ATG5

ATG7

and p62 were significant decreased in the BSTL group (

0.05).

Conclusion:

BSTL can significantly improve prednisolone-induced zebrafish osteoporosis and inhibit osteoclast differentiation by reducing autophagy-related gene expression.

关键词

Keywords

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