Study on the Anti-Liver Fibrosis Mechanism of Atractylenolide Ⅲ Regulating ASCT2-Mediated Mitochondria-Lysosome In-teraction to Induce Hepatic Stellate Cell Senescence
10.14148/j.issn.1672-0482.2024.0341
- VernacularTitle:白术内酯Ⅲ调控ASCT2介导的线粒体-溶酶体互作诱导肝星状细胞衰老的抗肝纤维化机制研究
- Author:
Qiuyu FU
1
;
Feixia WANG
;
Feng ZHANG
;
Shizhong ZHENG
;
Jinbai FU
Author Information
1. 南京中医药大学药学院,江苏 南京 210023
- Keywords:
atractylenolide Ⅲ;
ASCT2;
hepatic stellate cell;
senescence;
liver fibrosis;
glutamine metabolism
- From:
Journal of Nanjing University of Traditional Chinese Medicine
2024;40(4):341-349
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To explore the anti-liver fibrosis effect and mechanism of Atractylenolide Ⅲ-induced hepatic stellate cell(HSC)senescence.METHODS ASCT2 siRNA and Atractylenolide Ⅲ(40 μmol·L-1)acted on human hepatic stellate cells LX2 respectively to inhibit ASCT2,MTT was used to evaluate cell viability,EdU method was used to detect cell proliferation,and se-nescence associated-β-galactosidase(SA-β-Gal)staining was used to detect cell senescence;Western blot was used to detect chan-ges in the LC3-Ⅱ/Ⅰ ratio in LX2 cells,laser confocal detection was used to detect changes in LC3 autophagy flow and error protein accumulation,and the fluorescence of the lysosomal marker LAMP1 was also observed to detect lysosomal function and quantity;kits were applied to detect ROS and MDA levels as well as SOD activity in LX2 cells,and flow cytometry was used to analyze mitochondrial ROS levels and membrane potential.A CCl4-induced mouse liver fibrosis model was constructed.Atractylenolide Ⅲ was administered at 20,30,or 40 mg·kg-1.HE,Masson,and Sirius Red staining were used to observe liver tissue damage and collagen deposition.Western blot was used to detect the expression levels of P21 and P16 in mice in each group,and SA-β-Gal staining and immunohistochemistry were used to analyze the situation and origin of senescent cells.RESULTS After inhibiting ASCT2,the viabil-ity of LX2 cells decreased and senescence increased(P<0.01).Meanwhile,the autophagy function was enhanced and the number of lysosomes was increased but the function was weakened.After adding chloroquine(CQ)to clear lysosomes,the cell viability and auto-phagy function increased(P<0.01).After inhibiting ASCT2,the levels of MDA and ROS in LX2 cells increased,and the activity of SOD decreased(P<0.01).Among them,the level of mitochondrial ROS increased and the membrane potential decreased(P<0.01).After adding rotenone,the cellular redox homeostasis was improved,and the number of lysosomes was restored(P<0.01).In vivo experimental results showed that compared with the model group,Atractylenolide Ⅲ improved liver tissue structural damage and collagen deposition,induced HSC senescence in liver tissue of mice with liver fibrosis,and inhibited HSC activation marker α-smooth muscle actin(α-SMA),promoted the expression of senescence indicators P16 and P21(P<0.01).CONCLUSION Atractylenol-ide Ⅲ induces an increase in mitochondrial ROS and a decrease in membrane potential by inhibiting ASCT2,which further promotes the enhancement of HSC autophagy function,increases the number of lysosomes and weakens their function,thereby inducing the se-nescence of activated HSCs.
