Protective Effect and Mechanism of Gandou Fumu Decoction on Oxidative Damage of HepG2 Cells Induced by CuCl2
10.13422/j.cnki.syfjx.20211108
- VernacularTitle: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
- Author:
Lu-lu TANG
1
;
Jing ZHANG
1
;
Tao-hua WEI
1
;
Hai-lin JIANG
1
;
Ting DONG
1
;
Yue YANG
2
;
Nan-nan QIAN
2
;
Wen-ming YANG
1
Author Information
1. The First Hospital Affiliated to Anhui University of Chinese Medicine，Hefei 230031，China
2. The Graduate School of Anhui University of Chinese Medicine，Hefei 230012，China
- Publication Type:Research Article
- Keywords:
Gandou Fumu decoction;
Wilson's disease;
HepG2 cells;
autophagy;
phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signal pathway
- From:
Chinese Journal of Experimental Traditional Medical Formulae
2021;27(12):48-56
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To explore the effect of Gandou Fumu decoction （GDFMD） on the oxidative damage of HepG2 cells induced by CuCl2 based on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） signaling pathway. Method:CuCl2 （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 + CuCl2 + normal rat serum）， a GDFMD group （HepG2 cells + CuCl2 + 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 （P<0.01）. Compared with the model group， the GDFMD group showed elevated activities of SOD and GSH-Px and reduced content of MDA （P<0.05， P<0.01）， while the inhibitor group exhibited weakened GSH-Px activity and up-regulated content of MDA （P<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 （P<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 （P<0.05， P<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 （P<0.05， P<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（P<0.05， P<0.01）. The expression of LC3Ⅱ protein in the model group was increased （P<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 （P<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 （P<0.01）， while the inhibitor group showed increased expression of Beclin-1 mRNA （P<0.05）. The expression of Beclin-1 and LC3Ⅱ mRNA in the GDFMD + NVP-BEZ235 group was elevated （P<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 CuCl2， with the underlying mechanism related to the activation of PI3K/Akt/mTOR signalling pathway.
