Studies Of The Effect And Mechanism Of Celastrol Against Atherosclerosis

A key determinant of atherosclerotic lesion occurrence is foam cell formation, which is associated with enhanced cholesterol in macrophages, and can be elicited by excess oxLDL uptake via scavenger receptors such LOX-1.Oxidative stress plays an important role in the pathogenesis of atherosclerosis. ROS have been implicated in the pathogenesis of virtually every stage of vascular lesion formation in atherosclerosis. Traditionally, macrophages have been assumed to be the source of the ROS in the vessel wall, and there is no doubt that these cells play an important role in vessel pathology. Previous studies showed that ROS can induce the expression of LOX-1. Other studies, stimulation of the endothelial mono layer by binding of oxLDL to LOX-1 produces additional ROS, suggesting a positive feedback loop between ROS and LOX-1. Celastrol is a triterpenoid compound extracted from the Chinese herb Tripterygium wilfordii Hook F. Previous research has revealed its anti-oxidant, anti-inflammatory, anti-cancer and immunosuppressive properties. In our study, we investigated the possible mechanism and effect of celastrol on oxLDL-induced oxidative stress, foam cell formation and atherosclerosis in apoE-/- mice fed with a high-fat/high-cholesterol diet (HFC) and whether the classical NF-?B signal pathway is involved in the antioxidative effect of celastrol.In vitro: RAW 264.7 cells were cultured overnight before the addition of celastrol (25-200nM) along with an appropriate volume of vehicle, as well as oxLDL (80?g/ml). After a further 24h incubation period, medium or cells or both were harvested and assayed. Detection of foam cell formation was practiced by oil red O staining and enzyme fluorescence method of lipid-laden macrophages. Western blot was used to detect the expression of LOX-1?iNOS?NF-?B and the phosphorylation of I?B. RT-PCR was performed to detect LOX-1 and inflammatory cytokine mRNA levels. NO production was determined by measuring the levels of a stable NO metabolite, nitrite, in the culture medium by Griess reaction. ROS generation was tested by DHE staining. Our present results showed that pretreatment with celastrol significantly attenuated oxLDL induced excessive expresstion of LOX-1 and generation of ROS. We also found that celastrol decreased I?B phosphorylation, attenuated excessive production of NO and proinflammatory cytokines such as TNF-a in ox-LDL stimulate RAW 264.7 cells. In vivo: Celastrol was administered (1 or 2 mg/kg body weight, i.p.) to twenty-six male 8-week-old apoE-/- mice fed a HFC. After 30 days, hearts of mice were embedded in tissue optimal cutting temperature (OCT) compound, serially sectioned, mounted on slides and then stained with oil red O solution for analysis of atherosclerotic lesions. Total plasma cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) concentration were determined using commercially available kits as per the manufacturer's instructions. Celastrol markedly attenuated atherosclerotic lesion size in aortic root from apoEv-/- mice. The lipid profile showed no significant different between apoE-/- mice fed a HFC-diet plus celastrol treatment for 4 weeks and apoE-/- mice without celastrol treatment. Frozen sections from the aorticroots of apoE-/- mice and control mice were stained with DHE with/without pretreatment with celastrol. Celastrol markedly suppressed superoxide levels in the aortic of apoE-/- mice. Confocal microscopy demonstrated that celastrol inhibited LOX-1 expression within the atherosclerotic lesions, while plasma oxLDL level in apoE-/- mice was decreased by celastrol. Atherosclerosis in these animals wascharacterized by a marked increase in plasma TNF-a and IL-6, which upregulation was successfully suppressed by celastrol treatment. Aortic NF-?B activity was also increased in apoE-/- mice and this effect was also reduced by celastrol treatment.The present results suggest that celastrol significantly attenuated oxLDL induced foam cell formation by inhibtting the expression of LOX-1 and oxidative tress. We also found that celastrol inhibited the NF-?B pathway, attenuated excessive production of NO and proinflammatory cytokines such as TNF-a in oxLDL stimulate RAW 264.7 cells. Moreover, celastrol reduced atherosclerotic plaque size in apoE-/-mice fed a HFC diet by inhibiting oxidative stress, independent of modulating plasma concentrations of cholesterol and triglyceride. Our findings pinpoint a novel mechanism for the anti-atherosclerotic effect of celastrol and suggest that celastrol may potentially be of therapeutic relevance in inhibiting human atherogenesis.