山奈酚通过线粒体凋亡途径诱导肝星状细胞凋亡作用机制研究OA

Objective To investigate the molecular mechanism of kaempferol-induced apoptosis in hepatic stellate cells(HSC)via the mitochondrial pathway.Methods HSC were divided into five groups:blank control,trans-forming growth factor-β1(TGF-β1)(10 ng/mL)group,and groups treated with different concentrations of kaemp-ferol(8,16,32 µmol/L).Cell counting kit-8(CCK-8)assay was used to evaluate chrysin's effect on HSC prolif-eration.Transwell assay was used to assess chrysin's impact on HSC migration.Flow cytometry was used to detect apoptosis,reactive oxygen species(ROS)expression,and mitochondrial permeability transition pore(mPTP)opening across groups.5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylimidacarbocyanine iodide(JC-1)assay was used to test mitochondrial membrane potential.Western blot analysis was performed to determine the expression levels of mitochondrial apoptosis-associated proteins,namely B-cell lymphoma-2(Bcl-2),Bcl-2-associated X pro-tein(Bax),Cleaved-Caspase3,Cleaved-Caspase9,and sirtuin 3(SIRT3).Transmission electron microscopy was used to examine mitochondrial structural alterations.Molecular docking and kinetic simulations were used to ana-lyze the targeted interaction between kaempferol and NAD-dependent deacetylase sirtuin-3(SIRT3).Results Compared with the blank control group:in the TGF-β1 group,HSC proliferation and migration rates increased(P<0.01),ROS levels and mPTP opening both increased(P<0.05),the JC-1 red/green fluorescence ratio increased(P<0.01),and the expression of Cleaved-Caspase-3,Cleaved-Caspase-9 and Bcl-2 increased(P<0.05),whilst SIRT3 expression decreased(P<0.01);the number of mitochondria increased(P<0.05),and mitochondrial length decreased(P<0.01).Compared with the TGF-β1 group:treatment with all groups of kaempferol reduced HSC proliferation and migration rates(P<0.01),whilst simultaneously increasing the HSC apoptosis rate(P<0.01);HSC ROS levels and mPTP opening both increased(P<0.05);the JC-1 red/green fluorescence ratio de-creased(P<0.01),and the expression levels of Bax,Cleaved-Caspase-3 and Cleaved-Caspase-9 were upregulated(P<0.01),whilst the expression levels of Bcl-2 and SIRT3 were downregulated(P<0.01),and the number of mi-tochondria increased(P<0.05);mitochondrial length was reduced(P<0.01).Molecular docking and dynamics simulations revealed that the docking energy for kaempferol with SIRT3 was 108.89 kcal/mol;the RMSD curve sta-bilized after 20 ns;and RMSF analysis indicated that most residues exhibited minimal fluctuations.Conclusion Kaempferol induces HSC apoptosis through the mitochondrial pathway,potentially via targeting SIRT3 and disrupt-ing mitochondrial function.