Study On Mechanism Of Felodipine And Irbesartan Suppressing Atherosclerosis

Part I Molecular Mechanisms of Felodipine Suppressing Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: Oxidative stress and inflammation processes are key components of atherosclerosis (As), from fatty streak formation to plaque rupture and thrombosis. Evidence has revealed that calcium channel blockers (CCBs) could retard atherogenesis, but the exact mechanisms have not been fully elucidated. The present study was undertaken to investigate the potential effects and molecular mechanisms of a calcium channel blocker felodipine on the process of As in high cholesterol-diet (HCD) apolipoprotein E knock-out (ApoE KO) mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus felodipine (HCD+Fel) group. They were given ND or HCD and randomized to no treatment or felodipine 5mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. Nuclear factor-κB (NF-κB) and inhibitors ofκB (IκB) were measured by western blot. Nicotinamide-adeninedinucleotide phosphate [NAD(P)H] oxidase subunits and inflammatory cytokines were measured by real-time reverse-transcription polymerase chain reaction (PCR) and western blot.Results: (1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and felodipine treatment didn't affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Fel group. (3) Felodipine treatment can decrease the activity of NF-κB in atherosclerotic lesion. (4) Felodipine treatment can decrease the expression of NADPH oxidase subunits (p47phox and Rac-1). (5) Felodipine treatment also decreased the expression of cytokines such as tumor necrosis-α(TNF-α), monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1).Conclusion: The results suggest that felodipine could inhibit As. This effect is partly related to the decrease of NF-κB activity, inhibition of oxidative stress and inflammatory signal transduction pathways, which leads to the decrease in the expression of inflammatory cytokines.Part II Molecular Mechanisms of Irbesartan Suppressing Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: As is a chronic inflammatory disease in which the renin-angiotensin system (RAS) plays an important role. Evidences indicate that the angiotensin type 1 receptor blockers (ARBs) can suppress atherogenesis, but the exact mechanisms have not been fully elucidated. The present study was undertaken to investigate the potential effects and molecular mechanisms of an ARB irbesartan on the process of As in HCD ApoE KO mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus irbesartan (HCD+Irb) group. Those mice were given ND or HCD and randomized to receive no treatment or irbesartan 10 mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. NF-κB, IκB, extracellular signal-regulated kinase 1/2 (ERK1/2), janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. NAD(P)H oxidase subunits and inflammatory cytokines were measured by real-time reverse-transcription PCR and western blot.Results:(1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and irbesartan treatment didn't affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Irb group. (3) Irbesartan treatment can decrease the activity of NF-κB as well as the activity of ERK1/2, JAK2, STAT3 in atherosclerotic lesion. (4) Irbesartan treatment attenuated the expression of NADPH oxidase subunits (p47phox and Rac-1) in the aortas. (5) Irbesartan treatment decreased the expression of cytokines such as TNF-α, interleukin 6 (IL-6), MCP-1 and VCAM-1 in the aortas.Conclusions: The results suggest that irbesartan can attenuate As. The effects maybe relate to the decrease of the activity of signal transcriptional factors such as NF-κB, ERK1/2, JAK2 and STAT3, thus leading to the inhibition of oxidative stress and inflammatory signal transduction pathways which eventually leads to the reduction of inflammatory cytokines expression.Part III Effect and Mechanism of Combination of Low Dose Felodipine and Irbesartan on Atherosclerosis in High Cholesterol-diet Apolipoprotein E Knock-out MiceObjective: To investigate the potential effect and mechanism of combination of low dose felodipine and irbesartan on the process of As in HCD ApoE KO mice.Methods: Adult male ApoE KO mice were divided into three groups randomly (n=15 each): normal diet (ND) group, HCD group and HCD plus felodipine and irbesartan (HCD+Fel+Irb) group. Those mice were given ND or HCD and randomized to receive no treatment or felodipine 2.5 mg/kg/d plus irbesartan 5mg/kg/d for 12 weeks. The plasma total cholesterol and triglyceride concentration were measured by autoanalyzer. Atherosclerotic lesion area in arotic root was evaluated by oil red O staining. NAD(P)H oxidase subunits and inflammatory cytokines were measured by real-time reverse- transcription PCR and western blot.Results:(1) The ApoE KO mice with HCD were associated with a marked increase in plasma lipid levels, and the drug treatment didn't affect the plasma lipid levels. (2) Atherosclerotic lesion size was larger in HCD group than those in ND group or HCD+Fel+Irb group. (3) HCD feeding increased the expression of NADPH oxidase subunits (p47phox and Rac-1) and cytokines such as TNF-α, IL-6, MCP-1 and VCAM-1 in the aortas. These changes were suppressed in mice that were treated with low dose of felodipine and irbesartan.Conclusions: The results suggest that combination of low dose of felodipine and irbesartan could also inhibit As, and this effect maybe partly relates to the inhibition of oxidative stress and inflammatory cytokines expression.