Figure 1.
Celastrol reduces oxLDL induced lipid accumulation in RAW264.7 cells.
RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Representative photographs showing RAW 264.7 cells stained with oil red O (A). Measurement of CE in RAW 264.7 cells (B). ##p<0.01, c.f. no treatment,*p<0.05, c.f. treatment with oxLDL (n = 5).
Figure 2.
Celastrol inhibits oxidative stress in RAW 264.7 cells induced by oxLDL.
RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Quantification of LOX-1 mRNA was carried out by real-time PCR (A). Western blot analyses and quantification of LOX-1 protein expression (B). ROS generation monitored by DHE staining (C) and quantification of superoxide production (D). ROS scavenger tempol (10, 100, 1000 µmol/L) and oxLDL (80 µg/mL) were incubated with RAW 264.7 cells for 24 hours. LOX-1 protein expression was analyses and quantification (E). Western blot analyses and quantification of NADPH oxidase p47-phox protein expression (F). Quantification of MPO activity (carried out by a colorimetric activity assay kit) (G). ##p<0.01, ###p<0.001, c.f. no treatment, *p<0.05, **p<0.01, ***p<0.001 c.f. treatment with oxLDL (n = 4–5).
Table 1.
GSH/GSSG.
Figure 3.
Celastrol suppresses oxidative stress in RAW 264.7 cells by inhibiting NF-κB signaling pathway.
RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Representative examples of Western blots and quantification of IκBα phosphorylation (A) and IκBα degradation (B). Western blots and quantification of nuclear NF-κB p65 protein expression (C). #p<0.05, ###p<0.001 c.f. no treatment, *p<0.05, **p<0.01, ***p<0.001 c.f. treatment with oxLDL (n = 4).
Figure 4.
Celastrol inhibits oxLDL induced NO production and inflammatory gene expression in RAW 264.7 cells.
RAW 264.7 cells were exposed to oxLDL (80 µg/mL) in the presence or absence of celastrol (50–200 nM) for 24 h. Western blot analysis and quantification of iNOS protein expression (A). The iNOS inhibitor 1400w (200 µmol/L) and oxLDL (80 µg/mL) were incubated with RAW 264.7 cells for 24 hours. Representative photographs showing RAW 264.7 cells stained with oil red O (B). Quantification of NO, IL-6, TNF-α (carried out by real-time PCR )(C–E). ##p<0.01, ###p<0.001, c.f. no treatment, *p<0.05, **p<0.01, ***p<0.001, c.f. treatment with oxLDL (n = 4–5).
Figure 5.
Celastrol attenuates atherosclerotic lesion size and aortic superoxide formation in apoE−/− mice.
Celastrol (1 or 2 mg/kg/d, i.p) attenuated atherosclerotic lesion formation. The lesion size was determined by oil red O staining of the aortic sinus (40×magnification) (A). Quantitation of lesion area in oil red O stained aortic sections by Image-Pro Plus software. CeT: celastrol (B). Aortic superoxides were measured by staining aortic root sections with DHE with or without pretreatment with celastrol (C). *p<0.05, c.f. apoE−/− mice (n = 5–8).
Table 2.
Lipid profile.
Figure 6.
Celastrol decreases LOX-1 expression within the atherosclerotic lesions, plasma oxLDL and cytokines levels, aortic NF-κB activity in apoE−/− mice.
Representative cross-sections of aortic root stained with specific antibodies directed against LOX-1 (green), or DAPI (blue). Dotted lines indicate the boundary of lesion and Aortic tunica intima (A). Plasma levels of oxLDL, IL-6, TNF-α and aortic NF-κB activity were determined by ELISA (B–E). CeT: celastrol. ###p<0.001, c.f. WT, *p<0.05, c.f. apoE−/− mice (n = 5–8).