The Anti-atherosclerotic Effect Of Pseudolaric Acid B In High-Fat Fed ApoE Knockout Mice:alterations In Circul- Ating And Atherosclerotic Plaque Monocytes/Macrophages And The Underlying Mechanisms

Objective: Atherosclerosis is the pathological basis of cardiovascular and cerebrovascular diseases. The ischemic and hemorrhagic events caused by atherosclerotic vascular remodeling are the major reasons accounting for disability and death all over the world. The chronic inflammation and lipid infiltration theory, the currently acknowledged doctrine, are thought to be the pathogenesis of atherosclerosis. Monocyte/macrophage subsets, an important defense of the innate immune system, were key players involved in the lipid metabolism of plaque and chronic inflammation. Their dynamic changes play an important role in initiation and development of the atherosclerosis. Pseudolaric acid B, an extract of traditional Chinese medicine, has been approved with pleotropic pharmacological effects, such as anti-tumor, antifertility, anti-angiogenic and anti-fungal effects. Recently, we show that PB could activate peroxisome proliferator activated receptor γ in mononuclear cells, while is an important molecular target involved in the process of inflammation reaction and lipid metabolism. Accordingly, in this study, we hypothesis that PB could regulate the process of chronic inflammation and lipometabolism by influencing the dynamic change of monocyte/macrophage system, and thus has therapeutic potential for atherosclerosis.Methods: In the first part, Apo E-/-mice(8 weeks) were used for in animal study. Mice, consuming high fat diet for 8 weeks, were randomly grouped into the baseline group(n=7), high fat diet group(n=7), PB treated with high fat diet intervention group(n=7), normal diet group(n=7) and PB treated with normal diet intervention group(n=7). The baseline group harvested quickly. Four weeks after intervention, we did additional experiments as followings,(1) the proportions of Peripheral blood monocyte subsets(Ly6G-CD11b+Ly6Chi, Ly6GCD11b+Ly6Clo) were analyzed by flow cytometry,(2) confocal microscopy was used to investigate the density of the proliferation macrophages in plaque of aortic root,(3) Oil Red O staining was used to explore the influence of PB and diet on the plaque size of aortic, common carotid artery and the aortic root,(4) plasma triglycerides and total cholesterol concentration were measured by enzyme linked immune sorbent assay(ELISA). In the second part, RAW264.7 cells(mouse macrophage cell line) were used for in vitro study. Flow cytometry was used to explore the effluence and Uptake of lipid in RAW264.7 cells by Dil-ox-LDL. RAW264.7 cells were intervened with oxidized low density lipoprotein for 24 hours, which would form foam cells, then divided into the model group, PB intervention group and PB with PPARγ antagonist(GW966.2) block group. The Foam cells were subject to the following experiments,(1) Oil Red O staining was used to investigate foam cell,(2) The gene expression of PPARγ, LXRα, ABC1 and CD36 was measured by real-time PCR.Results: The in vivo dietary intervention study showed that,(1) the proportion of Ly6 Chi monocyte subsets tended to be lower in the group of PB with high fat diet compared with high fat diet. Normal diet or PB with normal diet can decrease it,(2) there was a trend toward lower density of proliferative macrophage in the group of PB with high fat diet compared with high fat diet. Normal diet or PB with normal diet can decrease it.(3) The levels of plasma triglycerides and total cholesterol tended to be lower in the groups of PB with high fat diet compared with high fat diet. Normal diet or PB with normal diet can decrease it.(4) The load of atherosclerotic plaque in the root of aorta, common carotid arteries and aorta tended to be lower in the groups of PB with high fat diet or normal diet compared with high fat diet. PB with normal diet can decrease it. In second study found the following points.(1) PB can increase the lipid effluence level of macrophage.(2) PB can increase the gene expression of PPARγ, LXRα, ABC1 in foam cells.Conclusion: 1.The high fat diet could increase triglycerides and total cholesterol levels, increase the percentage of Ly6 Chi monocyte subtype, enhance the density of proliferative macrophage, and promote the formation of atherosclerosis in Apo E-/- mice. Diet intervention normalizes the lipid profile and the percentage of Ly6 Chi and the proliferation activity of macrophages in atherosclerotic lesions. PB with normal diet not only delay the progression of atherosclerosis but also reduce plaque burden. 2. PB could increase the gene expression of PPARγ, active the signal pathway of PPARγ-LXRα-ABC1 and PPARγ-CD36 to regulate the lipid droplet and efflux of foam cells, and inhibit the formation of foam cells. 3. We showed for the first time that PB combined with diet intervention could prevention and treatment of AS, and further explored the therapeutic potential for traditional Chinese medicine of Pseudolarixkaempferi.