Mechanism of chrysophanol in inhibiting ox-LDL-induced macrophage foaminess through NF-κB/HMGB1-PI3K/Akt/mTOR pathway
The aim of this study was to investigate the underlying mechanism of chrysophanol (Chr) in reducing inflammation and foam cell formation induced by oxidized low-density lipoprotein (ox-LDL) and to investigate the targets and pathways related to effects of Chr on coronary atherosclerosis,providing a theoretical basis for the development of new clinical drugs.RAW264.7 macrophages were cultured in vitro,and after determining the appropriate concentrations of Chr and ox-LDL for treating RAW264.7 macrophages using a cell counting kit-8 (CCK-8),the macrophages were treated with different concentrations of Chr (10,15 μmol·L-1) and ox-LDL (with or without 80 mg·mL-1) for 24 h.RAW264.7 macrophages were divided into four groups:control group,model group (80 mg·mL-1 ox-LDL),treatment group (80 mg·mL-1 ox-LDL+10 μmol·L-1 Chr),and treatment group (80 mg·mL-1 ox-LDL+15 μmol·L-1 Chr).Lipid accumulation in each group was detected by oil red O staining.CD36 expression was analyzed by flow cytometry.Western blot was used to detect the expression of scavenger receptor class A1 (SR-A1),scavenger receptor class B type Ⅰ (SR-B1),autophagy-related protein 5 (Atg5),Beclin-1,autophagy adaptor protein p62 (P62),the ratio of microtubule-associated protein light chain 3(LC3)Ⅱ to LC3Ⅰ (LC3Ⅱ/LC3Ⅰ),nuclear factor kappa B P65 (NF-κB P65),inhibitor of κB kinase β (IKKβ),nuclear factor of κB inhibitor (IκB),high mobility group box protein 1 (HMGB1),phosphatidylinositol 3-kinase (PI3K),protein kinase B (Akt),and phosphorylated mammalian target of rapamycin (mTOR).Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA expression levels of ATP-binding cassette transporter A1 (ABCA1),ATP-binding cassette transporter G1 (ABCG1),interleukin-1β (IL-1β),tumor necrosis factor-α (TNF-α),HMGB1,inducible nitric oxide synthase (iNOS),arginase 1 (Arg1),macrophage galactose-type lectin-1 (Mgl-1),and NF-κB P65.Immunofluorescence analysis was performed to determine the localization of HMGB1 in RAW264.7 cells in each group.The autophagy inhibitor 3-methyladenine (3-MA) was added as a control for reverse validation,and the RAW264.7 macrophages were divided into four groups again:control group,model group (80 mg·mL-1 ox-LDL),treatment group (80 mg·mL-1 ox-LDL+15 μmol·L-1 Chr),and inhibitor group (80 mg·mL-1 ox-LDL+15 μmol·L-1 Chr+3-MA).The results showed that Chr effectively reduced foam cell formation by regulating the expression levels of SR-A1,ABCA1,ABCG1,the LC3Ⅱ/LC3Ⅰ ratio,Atg5,Beclin-1,and p62,and inhibited the NF-κB/HMGB1-PI3K/Akt/mTOR signaling pathway.Moreover,the inhibitory effects of Chr on autophagy and the NF-κB/HMGB1-PI3K/Akt/mTOR pathway were reversed by the autophagy inhibitor 3-MA.In conclusion,Chr exhibits therapeutic potential for the treatment of atherosclerosis by inducing autophagy and modulating the NF-κB/HMGB1 and PI3K/Akt/mTOR pathways to inhibit the formation of macrophage inflammatory foam cells.
万方数据
维普
