Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast

Deng TAN; Yu ZHANG; Nong-shan ZHANG; Jun LIU; Shi-wei WAN; Long HAN; Peng-hua FANG; Wen MIN

doi:10.13422/j.cnki.syfjx.20200641

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Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast

Pharmacology | 更新时间：2021-02-09

- Effect of Bushen Tongluo Formula on Osteoporosis of Zebrafish and Study on Autophagy Mechanism of Osteoclast
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 7, Pages: 79-85(2020)
- 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：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20200641
  CLC： R2-0; R289; R285.5
- Received：21 September 2019，
  
  Published Online：07 December 2019，
  
  Published：05 April 2020
- 稿件说明：
移动端阅览
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：

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

references

J E COMPSTON , M R MCCLUNG , W D LESLIE ． Osteoporosis [J]． Lancet ， 2019 ， 26 ( 6 ): 364 - 376 .

李冲，李伟华，王亭亭，等 . 1088李医务人员骨质疏松流行病学研究 [J]．中国骨质疏松杂志， 2015 ， 21 ( 10 ): 1217 - 1220 .

J FARR , J ROWSEY , B ECKHARDT ． Independent roles of estrogen deficiency and cellular senescence in the pathogenesis of osteoporosis: evidence in young adult mice and older humans [J]． J Bone Miner Res ， 2019 ， 34 ( 8 ): 1407 - 1418 .

D LAHA , M DEB , H DAS ， et al . KLF2(kruppel like factor 2 [lung])regulates osteoclastogenesis by modulating autophagy [J]． Autophagy ， 2019 ， 1596491 : 1 - 13 .

H KISHIMOTO , K NOGUCHI , K TAKAOKA ． Novel insight into the management of bisphosphonate-related osteonecrosis of the jaw (BRONJ) [J]． Swed Dent J ， 2019 ， 55 ( 1 ): 95 - 102 .

B H J COELINGH , C VERHOEVEN , A E DUTMAN ， et al . The use of high-dose estrogens for the treatment of breast cancer [J]． Maturitas ， 2017 ， 95 : 11 - 23 .

闵文，黄桂成，马勇，等．补肾通络对原发性骨质疏松症中医证候影响的临床研究 [J]．南京中医药大学学报， 2010 ， 26 ( 5 ): 344 - 346 .

韩龙，闵文，吴铭杰，等．补肾通络方促进骨质疏松症大鼠骨形成机制 [J]．中国中西医结合杂志， 2019 ， 39 ( 1 ): 97 - 102 .

闵文，韩龙，韩诗雨，等．补肾通络方对骨质疏松大鼠骨吸收的影响 [J]．中国老年学杂志， 2018 ， 38 ( 19 ): 4743 - 4746 .

杨国柱，段涛，卢丽，等．桃红四物汤对泼尼松诱导斑马鱼骨质疏松的治疗作用 [J]．中国骨质疏松杂志， 2017 ， 23 ( 8 ): 1007-1010，1021 .

林紫微，杨菲，黄天一，等．芍药苷干预成骨细胞凋亡抗骨质疏松作用研究 [J]．南京中医药大学报， 2019 ， 35 ( 4 ): 426 - 431 .

吴佼，李勇，南刚，等．荧光显微成像在医学细胞生物学实验教学中的应用探索 [J]．现代生物医学展， 2017 ， 17 ( 12 ): 2364 - 2367 .

葛静，凌洁，宁青，等．基于斑马鱼M-Act/Tox联合评价箭叶淫羊藿的代谢-效/毒作用 [J]．中草药， 2019 ， 50 ( 7 ): 1614 - 1620 .

I KIM , J H KIM , K KIM ， et al . The IRF2BP2-KLF2 axis regulates osteoclast and osteoblast differentiation [J]． BMB Rep ， 2019 ， 52 ( 7 ): 469 - 474 .

H C OWEN , I VANHEES , J GUNST ， et al . Critical illness-induced bone loss is related to deficient autophagy and histone hypomethylation [J]． Intensive Care Med Exp , 2015 , 3 ( 1 ): 52 .

G JIA , J R SOWERS ． Autophagy: a housekeeper in cardiorenal metabolic health and disease [J]． Biochim Biophys Acta ， 2015 ， 1852 ( 2 ): 219 - 224 .

C J DESELM , B C MILLER , W ZOU ， et al . Autophagy proteins regulate the secretory component of osteoclastic bone resorption [J]． Dev Cell ， 2011 ， 21 ( 5 ): 966 - 974 .

K H KIM , M S LEE ． Autophagy-a key player in cellular and body metabolism [J]． Nat Rev Endocrinol ， 2014 ， 10 ( 6 ): 322 - 337 .

Y WANG , N ZHANG , L ZHANG ， et al . Autophagy regulates chromatin ubiquitination in DNA damage response through elimination of SQSTM1/p62 [J]． Mol Cell ， 2016 ， 63 ( 1 ): 34 - 48 .

刘飞祥，林子璇，徐道明，等．左归丸防治骨质疏松症的动物模型和评价方法 [J]．中国实验方剂学杂志， 2019 ， 25 ( 18 ): 16 - 23 .

谭峰，樊巧玲，卞玉群，等．左、右归丸对去卵巢骨质疏松症大鼠模型骨密度及骨代谢的影响 [J]．中国实验方剂学杂志， 2015 ， 21 ( 9 ): 137 - 140 .

袁翰．补肾通络方激活HIF/VEGF信号通路促进骨质疏松大鼠模型的血管生成与抑制骨量流失 [D]．南京：南京中医药大学， 2017 .

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