狗肝菜乙酸乙酯提取物通过PI3K/AKT /mTOR /p70S6K信号通路诱导自噬减轻肝纤维化

Yuan LIU; Yan-meng BI; Ting PAN; Ting ZENG; Chan MO; Bing SUN; Lei GAO; Zhi-ping LYU

doi:10.1007/s11655-021-3298-5

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狗肝菜乙酸乙酯提取物通过PI3K/AKT /mTOR /p70S6K信号通路诱导自噬减轻肝纤维化

Original Article | Updated：2022-03-18

- 狗肝菜乙酸乙酯提取物通过PI3K/AKT /mTOR /p70S6K信号通路诱导自噬减轻肝纤维化
- Ethyl Acetate Fraction of Dicliptera chinensis (L.) Juss.Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/mTOR/p70S6K Signaling Pathways
- Chinese Journal of Integrative Medicine 2022年28卷第1期页码：60-68
- Affiliations：
  
  1.College of Integrated Traditional Chinese and Western Medicine, Jining Medical University, Jining ,Shandong Province(272000), China
  2.School of Traditional Chinese Medicine,Southern Medical University, Guangzhou (510515), China
  3.Department of Traditional Chinese Medicine, Affiliated Hospital of Jining Medical University, Jining ,Shandong Province (272000),China
- Author bio：
  
  Prof. LYU Zhi-ping, E-mail: lzp48241@126.com
- Funds：
  
  the National Natural Science Foundation of China(81673774);the Administration of Traditional Chinese Medicine of Shandong Province(2019-0447)
- DOI：10.1007/s11655-021-3298-5
  中图分类号：
- 纸质出版日期：2022-01-01，
  
  网络出版日期：2021-06-08，
  
  录用日期：2020-11-17
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Yuan LIU, Yan-meng BI, Ting PAN, 等. 狗肝菜乙酸乙酯提取物通过PI3K/AKT /mTOR /p70S6K信号通路诱导自噬减轻肝纤维化[J]. Chinese Journal of Integrative Medicine, 2022,28(1):60-68.

Yuan LIU, Yan-meng BI, Ting PAN, et al. Ethyl Acetate Fraction of Dicliptera chinensis (L.) Juss.Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/mTOR/p70S6K Signaling Pathways[J]. Chinese Journal of Integrative Medicine, 2022,28(1):60-68.
Yuan LIU, Yan-meng BI, Ting PAN, 等. 狗肝菜乙酸乙酯提取物通过PI3K/AKT /mTOR /p70S6K信号通路诱导自噬减轻肝纤维化[J]. Chinese Journal of Integrative Medicine, 2022,28(1):60-68. DOI： 10.1007/s11655-021-3298-5.

Yuan LIU, Yan-meng BI, Ting PAN, et al. Ethyl Acetate Fraction of Dicliptera chinensis (L.) Juss.Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/mTOR/p70S6K Signaling Pathways[J]. Chinese Journal of Integrative Medicine, 2022,28(1):60-68. DOI： 10.1007/s11655-021-3298-5.

摘要

目的:

基于肝星状细胞模型及肝纤维化小鼠模型探讨狗肝菜乙酸乙酯提取物抗肝纤维化的分子机制.

方法:

体外实验中

首先将肝星状细胞用PDGF预处理以确保激活

并用狗肝菜乙酸乙酯提取物分别处理24和48小时; 采用CCK8、EdU染色、电镜、免疫荧光染色和蛋白印迹等方法分别评估狗肝菜乙酸乙酯提取物对肝星状细胞的作用. 体内实验中

通过持续6周腹腔注射四氯化碳 (2μL/g

25%浓度

每周3次) 制备肝纤维化小鼠模型. 将40只8周雄性C57BL /6小鼠随机分为4组

包括对照组、模型组、阳性对照组 (秋水仙碱0.2 mg/kg) 和狗肝菜乙酸乙酯提取物 (0.5 g/kg) 组. 狗肝菜乙酸乙酯提取物和秋水仙碱组小鼠给予狗肝菜乙酸乙酯提取物 (0.5 g/kg) 或秋水仙碱 (0.2 mg/kg) 每日一次灌胃

持续6周; 对照组及模型组小鼠给予等量生理盐水灌胃. 通过生化检测和HE、Masson染色评估肝纤维化程度. 采用蛋白质印迹法测定α-平滑肌肌动蛋白 (α-SMA) 和微管相关蛋白轻链3B (LC3B) 的蛋白表达水平.

结果:

细胞模型和肝纤维化小鼠模型检测结果均显示

狗肝菜乙酸乙酯提取物可以抑制肝星状细胞活化

减轻α-SMA蛋白表达

促进 LC3-I到LC3Ⅱ的转换增加 (

0.05) . 病理组织学检查结果表明

模型组小鼠肝脏具有明显纤维化病理改变

狗肝菜乙酸乙酯提取物组可见肝脏胶原纤维的沉积减少. 巴弗洛霉素A1和雷帕霉素在肝星状细胞干预结果表明

抑制自噬会增强α-SMA蛋白表达水平 (

0.01) . 蛋白质印迹分析显示PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR及p-p70S6K水平均降低 (

0.05) .

结论:

狗肝菜乙酸乙酯提取物通过诱导自噬发挥抗肝纤维化活性

狗肝菜乙酸乙酯提取物可能是一种用于人类肝纤维化治疗的潜在药物.

Abstract

Objective:

To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction

Dicliptera chinensis

(L.) Juss. (EDC) in human hepatic stellate cells (HSCs)

in vitro

and in a carbon tetrachloride (CCl

)-induced hepatic fibrosis mouse model

in vivo

Methods:

For

in vitro

study

HSCs were pre-treated with platelet-derived growth factor (10 ng/mL) for 2 h to ensure activation and treated with EDC for 24 h and 48 h

respectively. The effect of EDC on HSCs was assessed using cell counting kit-8 assay

EdU staining

transmission electron microscopy

immunofluorescence staining

and Western blot

respectively. For

in vivo

experiments

mice were intraperitoneally injected with CCl

(2 μL/g

adjusted to a 25% concentration in olive oil)

3 times per week for 6 weeks

to develop a hepatic fibrosis model. Forty 8-week-old male C57BL/6 mice were divided into 4 groups using a random number table (

=10)

including control

model

positive control and EDC treatment groups. Mice in the EDC and colchicine groups were intragastrically administered EDC (0.5 g/kg) or colchicine (0.2 mg/kg) once per day for 6 weeks. Mice in the control and model groups received an equal volume of saline. Biochemical assays and histological examinations were used to assess liver damage. Protein expression levels of α-smooth muscle actin (α-SMA) and microtubule-associated protein light chain 3B (LC3B) were measured by Western blot.

Results:

EDC reduced pathological damage associated with liver fibrosis

downregulated the expression of α-SMA and upregulated the expression of LC3B (

0.05)

both in HSCs and the CCl

-induced liver fibrosis mouse model. The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhanced α-SMA protein expression levels (

0.01). The results also found that the levels of phosphoinositide (PI3K)

p-PI3K

AKT

p-AKT

mammalian target of rapamycin (mTOR)

p-mTOR

and p-p70S6K all decreased after EDC treatment (

0.05).

Conclusion:

EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.

关键词

Keywords

Dicliptera chinensis (L.) Juss.autophagyliver fibrosishepatic stellate cellsChinese medicine

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