Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl<sub>2</sub>

Lu-lu TANG; Jing ZHANG; Tao-hua WEI; Hai-lin JIANG; Ting DONG; Yue YANG; Nan-nan QIAN; Wen-ming YANG

doi:10.13422/j.cnki.syfjx.20211108

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Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl₂

Pharmacology | 更新时间：2021-06-25

- Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl₂
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 27, Issue 12, Pages: 48-56(2021)
- 作者机构：
  
  1.安徽中医药大学第一附属医院，合肥 230031
  2.安徽中医药大学研究生院，合肥 230012
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20211108
  CLC： R2-0;R22;R285.5;R289;R33
- Received：05 February 2021，
  
  Published Online：15 April 2021，
  
  Published：20 June 2021
- 稿件说明：
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唐露露,张静,魏涛华等.肝豆扶木汤对CuCl₂诱导的HepG2细胞氧化损伤的保护作用和机制[J].中国实验方剂学杂志,2021,27(12):48-56.

TANG Lu-lu,ZHANG Jing,WEI Tao-hua,et al.Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl₂[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(12):48-56.
唐露露,张静,魏涛华等.肝豆扶木汤对CuCl₂诱导的HepG2细胞氧化损伤的保护作用和机制[J].中国实验方剂学杂志,2021,27(12):48-56. DOI： 10.13422/j.cnki.syfjx.20211108.

TANG Lu-lu,ZHANG Jing,WEI Tao-hua,et al.Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl₂[J].Chinese Journal of Experimental Traditional Medical Formulae,2021,27(12):48-56. DOI： 10.13422/j.cnki.syfjx.20211108.

摘要

目的

基于磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路探讨肝豆扶木汤（GDFMD）对氯化铜（CuCl

）诱导的人肝癌细胞（HepG2）氧化损伤的保护作用。

方法

HepG2细胞分为空白组，模型组，GDFMD组，PI3K/mTOR抑制剂NVP-BEZ235（10 nmol·L

-1

）组，GDFMD+NVP-BEZ235（10 nmol·L

-1

）组。采用200 μmol·L

-1

CuCl

构建模型组铜负荷HepG2细胞，酶联免疫吸附测定法（ELISA）观察细胞超氧化物歧化酶（SOD），谷胱甘肽过氧化物酶（GSH-Px）活性和丙二醛（MDA）含量；免疫荧光观察细胞微管相关蛋白1轻链3（LC3）蛋白表达水平；蛋白免疫印迹法（Western blot）检测细胞磷酸化PI3K（p-PI3K）/PI3K，磷酸化Akt（p-Akt）/Akt，磷酸化mTOR（p-mTOR）/mTOR，Beclin-1，LC3Ⅱ/LC3Ⅰ，p62表达；实时荧光定量聚合酶链式反应（Real-time PCR）检测细胞PI3K，Akt，mTOR，Beclin-1，LC3Ⅰ，LC3Ⅱ，p62 mRNA表达。

结果

与空白组比较，模型组细胞SOD，GSH-Px活性下降，MDA含量显著升高（

0.01）；与模型组比较，10% GDFMD含药血清组SOD，GSH-Px活性升高，MDA含量下降（

0.05，

0.01）；NVP-BEZ235组GSH-Px活性下降，MDA含量升高（

0.05）。与空白组比较，模型组p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，p62表达水平显著降低，Beclin-1，LC3Ⅱ/LC3Ⅰ表达水平显著升高（

0.01）；与模型组比较，10% GDFMD组p-PI3K/PI3K，p-Akt/Akt，p-mTOR/mTOR，p62表达水平升高，Beclin-1，LC3Ⅱ/LC3Ⅰ表达水平下降（

0.05，

0.01）；NVP-BEZ235组p-PI3K/PI3K，p-mTOR/mTOR表达水平下调，Beclin-1，LC3Ⅱ/LC3Ⅰ表达水平升高（

0.05，

0.01）。与10% GDFMD组比较，10% GDFMD+NVP-BEZ235组p-Akt/Akt，p-mTOR/mTOR表达水平下降，Beclin-1，LC3Ⅱ/LC3Ⅰ表达水平升高（

0.05，

0.01）。与空白组比较，模型组LC3Ⅱ蛋白表达显著升高（

0.01）；与模型组比较，10% GDFMD组LC3Ⅱ蛋白表达降低，与10% GDFMD组比较，10% GDFMD+NVP-BEZ235组LC3Ⅱ蛋白表达升高。各组PI3K，Akt，mTOR mRNA相对表达量差异无统计学意义，与空白组比较，模型组p62 mRNA相对表达量显著降低，Beclin-1，LC3Ⅰ，LC3Ⅱ mRNA相对表达量显著升高（

0.01）；与模型组比较，10% GDFMD组p62 mRNA相对表达量升高，Beclin-1，LC3Ⅰ，LC3Ⅱ mRNA表达水平下调（

0.01）；NVP-BEZ235组Beclin-1 mRNA相对表达量升高（

0.05）。与10% GDFMD组比较，10%GDFMD+NVP-BEZ235组Beclin-1，LC3Ⅱ mRNA相对表达量升高（

0.01）。

结论

GDFMD能抑制CuCl

诱导的HepG2细胞过度自噬，减轻细胞氧化损伤，其机制可能与激活PI3K/Akt/mTOR信号通路有关。

Abstract

Objective

To explore the effect of Gandou Fumu decoction （GDFMD） on the oxidative damage of HepG2 cells induced by CuCl

based on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway.

Method

CuCl

（200 μmol·L

-1

） was used to induce a copper-loaded HepG2 cell model. HepG2 cells were divided into a blank group （HepG2 cells + blank rat serum）， a model group （HepG2 cells + CuCl

+ normal rat serum）， a GDFMD group （HepG2 cells + CuCl

+ GDFMD-medicated rat serum）， an inhibitor group （HepG2 cells + NVP-BEZ235 + normal rat serum）， and a GDFMD + NVP-BEZ235 group （HepG2 cells + NVP-BEZ235 + GDFMD-medicated rat serum）. ELISA method was used to determine superoxide dismutase （SOD）， glutathione peroxidase （GSH-Px） activity， and malondialdehyde （MDA） content. The expression of microtubule-associated protein 1 light chain 3 （LC3） was detected by immunofluorescence. Phospho-PI3K/PI3K （p-PI3K/PI3K）， p-Akt/Akt， p-mTOR/mTOR， Beclin-1， LC3Ⅱ/LC3Ⅰ， and p62/Actin were determined by Western blot. PI3K， Akt， mTOR， Beclin-1， LC3Ⅰ， LC3Ⅱ， p62 mRNA expression was measured by real-time polymerase chain reaction （PCR）.

Result

Compared with the blank group， the model group displayed decreased activities of SOD and GSH-Px and increased content of MDA （

0.01）. Compared with the model group， the GDFMD group showed elevated activities of SOD and GSH-Px and reduced content of MDA （

0.05，

0.01）， while the inhibitor group exhibited weakened GSH-Px activity and up-regulated content of MDA （

0.05）. Compared with the blank group， the model group showed diminished expression of p-PI3K/PI3K， p-Akt/Akt， p-mTOR/mTOR， and p62， and increased expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ （

0.01）. The expression of p-PI3K/PI3K， p-Akt/Akt， p-mTOR/mTOR， and p62 was elevated， and the expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ declined in the GDFMD group （

0.05，

0.01）， while the p-PI3K/PI3K and p-mTOR/mTOR expression was down-regulated and the Beclin-1 and LC3Ⅱ/LC3 I expression was increased in the inhibitor group （

0.05，

0.01） as compared with those in the model group. Compared with the GDFMD group， the GDFMD + NVP-BEZ235 group showed down-regulated expression of p-Akt/Akt and p-mTOR/mTOR and up-regulated expression of Beclin-1 and LC3Ⅱ/LC3Ⅰ（

0.05，

0.01）. The expression of LC3Ⅱ protein in the model group was increased （

0.01） as compared with that in the blank group. The expression of LC3Ⅱ protein was lower in the GDFMD group than in the model group， and higher in the GDFMD + NVP-BEZ235 group than in the GDFMD group. No significant difference in the expression of PI3K， Akt， and mTOR mRNA was observed among the groups. Compared with the blank group， the model group displayed lowered expression of p62 mRNA， and elevated expression of Beclin-1， LC3Ⅰ， and LC3Ⅱ mRNA （

0.01）. Compared with the model group， the GDFMD group exhibited increased expression of p62 mRNA， and declining expression of Beclin-1， LC3Ⅰ， and LC3Ⅱ mRNA （

0.01）， while the inhibitor group showed increased expression of Beclin-1 mRNA （

0.05）. The expression of Beclin-1 and LC3Ⅱ mRNA in the GDFMD + NVP-BEZ235 group was elevated （

0.01） as compared with that in the GDFMD group.

Conclusion

GDFMD may inhibit the excessive autophagy and alleviate the oxidative damage of HepG2 cells induced by CuCl

， with the underlying mechanism related to the activation of PI3K/Akt/mTOR signalling pathway.

关键词

Keywords

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⁰

Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl2

DOI：10.13422/j.cnki.syfjx.20211108

摘要

Abstract

关键词

Keywords

references

Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl₂