补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨

潘怡; 王振兴; 郭静; 张川峰; 王丽娟; 杨晗; 王飞

doi:10.13422/j.cnki.syfjx.20190602

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补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨

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- 补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨
- Regulatory Mechanism of Buyang Huanwu Tang on mTOR Protein that Mediates Autophagy in Mice with Pulmonary Fibrosis
- 中国实验方剂学杂志 2019年25卷第6期页码：23-31
- 作者机构：
  
  成都中医药大学　临床医学院，成都　 610075
- 作者简介：
  
  潘怡，在读博士，从事中医药防治呼吸系统疾病与老年疾病的实验研究，E-mail：904300087@qq.com
  杨晗，博士，副教授，从事中医药防治呼吸系统疾病与老年疾病的实验研究，E-mail：15882458490@163.com
  王飞，教授，博士生导师，从事中医药防治呼吸系统疾病与老年疾病的实验研究，E-mail：wangfei896@163.com
- 基金信息：
  
  国家自然科学基金青年基金项目(81503557)
- DOI：10.13422/j.cnki.syfjx.20190602
  中图分类号： R2-0;R22;R285.5;R289
- 收稿日期：2018-08-27，
  
  网络出版日期：2018-05-12，
  
  纸质出版日期：2019-03-20
- 稿件说明：
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潘怡, 王振兴, 郭静, 等. 补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨[J]. 中国实验方剂学杂志, 2019,25(6):23-31.

Yi PAN, Zhen-xing WANG, Jing GUO, et al. Regulatory Mechanism of Buyang Huanwu Tang on mTOR Protein that Mediates Autophagy in Mice with Pulmonary Fibrosis[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(6): 23-31.
潘怡, 王振兴, 郭静, 等. 补阳还五汤对肺纤维化小鼠中介导细胞自噬的mTOR蛋白的调控机制探讨[J]. 中国实验方剂学杂志, 2019,25(6):23-31. DOI： 10.13422/j.cnki.syfjx.20190602.

Yi PAN, Zhen-xing WANG, Jing GUO, et al. Regulatory Mechanism of Buyang Huanwu Tang on mTOR Protein that Mediates Autophagy in Mice with Pulmonary Fibrosis[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(6): 23-31. DOI： 10.13422/j.cnki.syfjx.20190602.

摘要

目的：

观察肺纤维化中介导自噬的哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin

mTOR)的表达、自噬在肺纤维化形成中的作用，探索补阳还五汤对肺纤维化的治疗机制。

方法：

144只C57BL/6小鼠随机分为6组，分别为假手术组，模型组，强的松组，补阳还五汤高、中、低剂量组，每组24只，假手术组小鼠给予气管注入等量的0.9%生理盐水，其余各组采用博莱霉素(bleomycin，BLM)气管注入法复制肺纤维化模型，造模后假手术组、模型组给予0.9%生理盐水(0.01 g·kg

－1

·d

－1

)，补阳还五汤高剂量组给予补阳还五汤(28.08 g·kg

－1

·d

－1

)，补阳还五汤中剂量组给予补阳还五汤(14.04 g·kg

－1

·d

－1

)，补阳还五汤低剂量组给予补阳还五汤(7.02 g·kg

－1

·d

－1

)，强的松组给予强的松(0.455 g·kg

－1

·d

－1

)灌胃，于造模后第7，14，28天分批提取样本。采用苏木素-伊红(HE)及马松(Masson)染色观察小鼠肺泡炎和纤维化程度；蛋白免疫印迹法(Western blot)检测小鼠肺组织mTOR，核糖体S6，微管相关蛋白1轻链3Ⅱ(microtubule-associate protein 1 light chain 3，MAP1LC3-Ⅱ)的表达；电镜检测观察小鼠肺组织自噬情况。

结果：

与假手术组比较，模型组小鼠在7，14，28 d肺泡炎及肺纤维化显著(

0.01)；与模型组比较，给药组各时间点肺泡炎和肺纤维化程度均有不同程度的改善(

0.05)，补阳还五汤高剂量组下降最明显(

0.05)。与假手术组比较，模型组小鼠肺组织mTOR，S6蛋白表达水平显著上调(

0.01)，与模型组比较，各给药组mTOR，S6蛋白表达下调(

0.01)；与假手术组比较，模型组小鼠肺组织LC3-Ⅱ表达水平下调(

0.01)，与模型组比较，各给药组LC3-Ⅱ上调(

0.01)。假手术组细胞形态良好，未见自噬。模型组细胞形态最差，存在个别自噬。各给药组细胞形态及自噬数量好于模型组，其中补阳还五汤高、中剂量组自噬数量最多。

结论：

在BLM所致小鼠肺纤维化形成的过程中，小鼠肺组织中mTOR蛋白被激活，自噬抑制，mTOR蛋白通过抑制自噬参与了肺纤维化的发病；补阳还五汤对BLM所致小鼠肺纤维化有一定的治疗作用，其机制可能与其下调介导细胞自噬的mTOR蛋白表达有关。

Abstract

Objective:

To observe the expression of mammalian target of rapamycin (mTOR) that mediates autophagy in pulmonary fibrosis and the effect of autophagy in the formation of pulmonary fibrosis

in order to explore the treatment mechanism of Buyang Huanwu Tang on pulmonary fibrosis.

Method:

Totally 144 C57BL/6 mice were randomly divided into 6 groups: sham operation group

model group

prednisone group

high-dose Buyang Huanwu Tang group

medium-dose Buyang Huanwu Tang group and low-dose Buyang Huanwu Tang group

with 24 mice in each group. The sham operation group was injected with the same amount of 0.9%saline. The remaining groups were treated with bleomycin tracheal injection to replicate the pulmonary fibrosis model. After modeling

sham operation group and model group were given 0.9%normal saline (0.01 g·kg

-1

·d

-1

)

group high-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang (28.08 g·kg

-1

·d

-1

)

medium-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang (14.04 g·kg

-1

·d

-1

)

low-dose Buyang Huanwu Tang group was given Buyang Huanwu Tang(7.02 g·kg

-1

·d

-1

)

and P group was given prednisone (0.455 g·kg

-1

·d

-1

) by gavage. The samples were taken in batches on the 7

and 28

days after modeling; degrees of alveolitis and fibrosis in mice were observed by hematoxylin-eosin (HE) staining and Masson staining. The mTOR protein

ribosomal S6 protein and microtubule associate protein 1 hight chain3-Ⅱ(MAP1LC3-Ⅱ) of mouse lung tissue were detected by Western blot; electron microscopy was used to observe the autophagy of lung tissue in mice.

Result:

Compared with the sham-operated group

the degrees of alveolitis and pulmonary fibrosis were significantly severer in the model group on 7

and 28

days (

0.01); compared with the model group

the degrees of alveolitis and pulmonary fibrosis were alleviated at each observation time in the drug-administered groups (

0.05). The decreased scores in high-dose Buyang Huanwu Tang group were the most obvious

with statistically significant differences (

0.05). compared with the sham-operated group

the expressions of mTOR and S6 protein in lung tissue of the model group were significantly up-regulated

and each drug-administered group showed down-regulations. Compared with the sham-operated group

the expression of LC3-Ⅱ protein in lung tissue of the model group was significantly down-regulated

and each drug-administered group showed anup-regulation(

0.01). The cells in the sham-operated group were well-formed

and no autophagy was observed. The model group had the worst cell morphology

with individual autophagy. The cell morphology and autophagy in each drug-administered group were better than those in the model group. The high-dose Buyang Huanwu Tang group and the medium-dose Buyang Huanwu Tang group had the highest number of autophagosome.

Conclusion:

The mTOR protein is activated in mice lung tissue

autophagy is inhibited

mTOR protein participates in the pathogenesis of pulmonary fibrosis by inhibiting autophagy; Buyang Huanwu Tang has a certain therapeutic effect on BLM-induced pulmonary fibrosis in mice

and its mechanism may be related to the down-regulation of mTOR protein expression that mediates autophagy.

关键词

Keywords

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