| Liver fibrosis is a pathological process of excessive deposition of extracellular matrix(ECM)after liver injury,which can progress to cirrhosis and hepatocellular carcinoma.Activated hepatic stellate cells(HSCs)are the main source of ECM and are the main effector cells in liver fibrosis.Studies have shown that senescent HSCs are found to exist during the repair phase of liver fibrosis,and further found that induction of senescence of activated HSCs can resist liver fibrosis.Therefore,induction of activated HSC senescence and reduction of ECM production is an important strategy for the treatment of liver fibrosis.As an important metabolic organ of the body,the liver has a specific role in regulating the metabolic processes of many substances and energy essential to the body,which also profoundly affects the pathological changes of the liver.Studying the mediation of HSC aging from the perspective of metabolic regulation is expected to obtain drug targets with high specificity,thus promoting the development of safe and efficient anti-liver fibrosis drugs.Recent studies have revealed that the manifestation of high glutamine catabolism occurs during HSC activation.In view of the key role of ASCT2 in glutamine transport,ASCT2 was first detected in the livers of clinical patients with different fibrosis grades(F0-1/no fibrosis,F2/mild fibrosis,F3-4/severe fibrosis)and was found to be highly expressed in the fibrous junctions of fibrotic livers,and ASCT2 expression had a significant positive correlation with fibrosis grade.Subsequently,two mouse models of liver fibrosis(CCl4 and BDL)also showed high ASCT2 expression in fibrotic livers.ASCT2 expression in activated HSC was determined by immunofluorescence costaining of ASCT2 with α-SMA,Alb,CK19,F4/80,and CD31.knockdown of ASCT2 in mice was constructed by AAV8 viral vector,and liver pathology assay,liver hydroxyproline assay,and serological indexes showed that knockdown of ASCT2 ameliorated CCl4-induced liver fibrosis in mice.ASCT2 expression in primary hepatic cells(HSCs,KCs and HEPs)was detected only in primary HSCs.Real-Time PCR,Western blot and immunofluorescence assays showed that inhibition of ASCT2 suppressed the expression of activation markers α-SMA,COL1α1,and COL1α1 in primary HSCs,HSC-LX2 and HSC-T6 cell lines.α1 expression,and induced resting indicator GFAP and LRAT expression in primary HSCs.So,could ASCT2 act as a regulator mediating activated HSC senescence?By ASCT2 siRNA or inhibitor Benser treatment of two HSC cell lines(HSC-LX2 and HSC-T6),EdU incorporation.SA-βgal staining,senescence markers p16 and p21 and cell cycle assays revealed that downregulation of ASCT2 induced activated HSC toward proliferative arrest and cell cycle arrest in the G1 phase of senescence and was associated with its translocation of glutamine was associated.Senescent cells can release many complex,multicomponent bioactive molecules collectively known as senescence-associated secretory phenotype(SASP).Here,RNA sequencing and subsequent Real-Time revealed that unlike high SASP under Etoposide treatment,ASCT2 inhibition-induced senescent HSC did not express pro-inflammatory SASP.Moreover,we found that SASP expression did not affect the senescence of HSC,but pro-inflammatory SASP produced by Etoposide had a paracrine effect that promoted migration of neighboring HSC.Further,it was observed by metabolic flow analysis that the pro-inflammatory SASP in senescent HSC was regulated by ASCT2mediated glutamine metabolism.Western blot revealed that ASCT2 inhibition regulated the formation of precursor IL-1α and influenced the secretion of IL-1α.Furthermore,results revealed that ASCT2 regulates the IL-1α/NF-κB feeding loop through binding to the K82 of precursor IL-1α,which in turn regulates the production of SASP in senescent HSC.Our group have been devoted to the discovery of the anti-liver fibrosis effects and mechanisms of natural products,and the discovery of natural inhibitors of ASCT2 from the treasure trove of natural drugs has become an important research direction.The direct binding of Atractylenolide Ⅲ to ASCT2 and the inhibition of ASCT2 expression were discovered by molecular docking and cellular heat transfer assays.Subsequent mode of action analysis and further validation revealed that the hydroxyl group(-OH)of atractylenolide Ⅲ was the key group for the inhibition of ASCT2 expression.Molecular dynamics and microscale thermophoresis experiments revealed that the Asn230 site of ASCT2 was the key site of ASCT2 inhibition by atractylenolide Ⅲ.Furthermore,by constructing a CCl4-induced liver fibrosis model in mice and giving Atractylenolide Ⅲ for 4 weeks after successful modeling,the results showed that Atractylenolide Ⅲ had good therapeutic effects on CCl4-induced liver fibrosis in mice,and Atractylenolide Ⅲ inhibited ASCT2 expression of HSC in liver of mice with liver fibrosis.The detection of SA-β-Gal staining of tissues and senescence indexes in primary HSCs indicated that Atractylenolide Ⅲ induced HSC senescence in the liver of liver fibrosis mice.Finally,we injected HSC-targeted ASCT2 shRNA or ASCT2 overexpression plasmid into hepatic fibrosis mice by tail vein injection,and then administered atractylenolide Ⅲ treatment.Real-Time PCR,Western blot and tissue immunofluorescence co-staining confirmed ASCT2 expression in HSCs of mouse liver.The detection of activation index,resting index and senescence index in primary HSCs,as well as SA-β-Gal staining and tissue immunofluorescence co-staining also demonstrated the importance of ASCT2 in HSC senescence induced by AtractylenolideⅢ,confirming the anti-liver fibrosis effect of Atractylenolide Ⅲ by inhibiting ASCT2induced HSC senescence.The results of this thesis reveal the importance of ASCT2 as a modifiable target in the treatment of liver fibrosis at the overall,cellular and molecular levels,and provide an experimental basis for the development of atractylenolide Ⅲ inhibition of ASCT2 as an effective drug against liver fibrosis. |
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