Effects Of Xiongshao Capsule On Atherosclerosis Effects On The Distribution And Function Of HDL Subtypes

Atherosclerosis is the pathological basis of many cardiovascular and cerebr ovascular diseases. The pathogenesis of atherosclerosis mainly includes abnorma 1 lipid metabolic, inflammation, endothelial injury and oxidative stress, etc. Hig h density lipoprotein(HDL) can protect vessels and inhibit atherosclerosis throug h reverse cholesterol transport, anti-inflammatory, antioxidant, anti-thrombus, ant i-apoptosis and vascular expansion function. The latest study found that HDL 1 evel does not reflect its function of anti-atherosclerosis, and the function of H DL is closely associated with its subtypes, metabolism and composition. HDL s ubtype distribution and metabolism have become hot topics in the study of anti-atherosclerosis.ApoA-I is the main protein composition and structure basis of HDL, and i t is also the key point of HDL to complete its function such as the RCT, end othelial cell apoptosis resistance, anti-oxidation, and anti-inflammatory function, and ApoA-I plays significant role in resistance to AS. Fewer ApoA-I or structu re change of ApoA-I will make HDL weaker or unable, even promote AS. HD L could gradually mature through the process, prep 1-HDL→HDL3→>HDL2. As the large and mature subtype particle of HDL, HDL2 could promote transport of cholesterol to liver and tissues that produce steroid hormones. Low HDL21 evel is negatively related to the risk of coronary heart disease.ATP-binding cass ette transporters A1(ABCA1) can combine with HDL, and promote efflux of ce llular cholesterol, and affect the genetating of HDL particles in the RCT and li pid metabolism process. In addition to participating in lipid metabolism, ABCA 1 can inhibit expression of inflammation factors, and engage in oxidative stress to affect the AS process in a variety of ways. Scavenger receptor class B, ty pe I(SR-BI) could take in HDL cholesterol selectively, and give the HDL chole sterol to liver and steroid hormone generating tissues to complete the RCT; SR-BI could also mediate cholesterol efflux in peripheral cells, and participate in metabolism of many lipoproteins. Myeloperoxidase (MPO) and Paraoxonase 1(P ON1) are related proteins in inflammation, oxidative stress, and AS. MPO coul d oxidize LDL, and selectively oxidize and modificate ApoA-Ⅰ, which makes H DL weakened or lost its normal function, and accelerate the AS development. I nstead, PON1 could guarantee the antioxidant effect of system, and promote th e anti-atherosclerosis function of HDL. Lecithin cholesterol acyltransferase(LCA T) can esterify cholesterol, and transport esterified cholesterol to HDL, which makes HDL gradually mature and rich in cholesterol ester. LCAT is the key enzyme of human plasma protein metabolism, and it plays an important role in HDL metabolism.If the function of LCAT is damaged, synthesis of cholesterol ester will be inhibited, leading to high cholesterol; At the same time, the HD L mature process will be blocked, and the incidence of the AS will increase.In TCM, dyslipidemia is seen as the result of the interaction between intri nsic and extrinsic factors. The internal aspects mainly include deficiency of spl een, liver and kidney, and external causes include unhealthy diet, excessive dri nking, emotional disorders and lack of exercise. The mechanism of dyslipidemia i n chinese medicine mainly lies in two aspects of deficiency in origin and enrichment in symptom. The deficiency of spleen, liver and kidney are basic root, and d amp turbidity, phlegm, blood-stasis are superficial. The principles of treatment a re mainly soothing the liver to regulating qi, nourishing liver and kidney, invig orating spleen to promote digestion, activating blood and dispelling stasis, elimin ating phlegm and freeing channels. It has been confirmed that a variety of mo nomer composition, single Chinese medicine and compound could regulate bloo d fats. Many reports suggest Chinese medicine can improve the level of HDL, but the current research on fat-regulating Chinese medicine influence HDL fun ction via adjusting the its subtype distribution is insufficient, and there are few research reports that explore anti-atherosclerosis mechanism of TCM from the perspective of affecting the anti-inflammatory, antioxidant function of HDL.In t he early experiments we found that Xiongshao Capsule have an anti-atheroscler osis function, and in AS rabbit HDL increased as TC, LDL increased. We thou ght that HDL have heterogeneity, and increasing the HDL level could not nec essarily resist AS. On the basis of the previous studies, this experiment made a further study on HDL subtype, metabolism, component and function.Objective:This research establishde a rabbit model of atherosclerosis to investi .gate the possible mechanisms of Xiongshao Capsule(XSC) against AS from the perspective of regulating lipid metabolism and affecting HDL subtype distribut ion and function.Methods:1. Groups and administration60 male New Zealand rabbits were randomly divided into 5 groups, blank control group, model group, simvastatin group, XSC low-dose group, XSC hig h-dose group,12 each. Single high-fat fodder feeding method is used to establi sh the rabbit model of the AS. Drug administration methods:(1) the blank con trol group was feeded with ordinary fodder for 22 weeks; (2) the model group was feeded with high fat fodder for first 14 weeks, ordinary fodder for the n ext 8 weeks; (3)the simvastatin group was feeded with high fat fodder and sim vastatin in the first 14 weeks, ordinary fodder and simvastatin for the next 8 weeks,and dosage of simvastatin is 2 mg/Kg·d;(4)the XSC low-dose group wa s feeded with high fat fodder and XSC in the first 14 weeks, ordinary fodder and XSC for the next 8 weeks,and dosage of XSC is Ligusticum wallichii 1.5 g/Kg·d and Radix Paeoniae Rubra 0.75 g/Kg·d;(5)the XSC high-dose group was feeded with high fat fodder and XSC in the first 14 weeks, ordinary fod der and XSC for the next 8 weeks,and dosage of XSC is Ligusticum wallichii 3.0 g/Kg·d and Radix Paeoniae Rubra 1.5 g/Kg·d.2. Observation aorta plaque formation by pathological examinationThe thoracic aortas of the animals were taken after execution under anesth esia at the end of 22 weeks. Fatty streaks and grease spots on vessel wall wer e observed macroscopically, and the AS plaque formation was observed through HE staining.3. Detecting serum HDL and other blood fat indexes The blood samples were collected before and after drug administration for 14 weeks and 22 weeks. Serum high-density lipoprotein (HDL), its component apolipoprotein AI (ApoAI), and total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), apolipoprotei n B (ApoB) were tested with Automatic biochemical analyzer.4. Detecting HDL subtype particleWith serum samples, the serum HDL2 level was tested by Enzyme-linked immunosorbent assay (ELISA).5. Detecting reverse cholesterol transport function of HDLThe liver of the animals were taken after execution under anesthesia at th e end of 22 weeks, then were cryopreserved in liquid nitrogen. Trizol method was used to extract the liver total RNA, and liver ABCA1 mRNA, SR-BI m RNA expression were determinated by fluorescence real-time quantitative polym erase chain reaction (real-time PCR).6. Detecting antioxidant function of HDLSerum MPO activity was assayed by o-dianisidine method, and serum PO N1 activity was tested by Enzyme-linked immunosorbent assay (ELISA).7. Detecting metabolism of HDLReal-time PCR was used to determinate LCAT mRNA expression in liver.Results:1. In blank control group, the surface of aortic wall is smooth, and the e ndothelial cells are continuously and complete, without lipid deposition; In mod el group, aortic wall is covered by lipid plaque, in which plaques and lipidosis can be found in smooth muscle cells obviously, and a large number of foam cells accumulate under the intima.The fatty streak on the surface of aortic wa 11, formation of plaques under microscope, accumulating of foam cells under th e intima, and lipidosis in the cells of aortic wall are declining with the increas ing administration of drug in XSC low and high dose group.2. First we compared the values at different time points of the same grou p. After feeding with durgs and high-fat fodder for 14 weeks, in all groups ex cept the blank control group, serum TC, TG, HDL, LDL, VLDL, ApoA-I, Apo B have increased, the differences are statistically significant (P<0.05 or P<0. 01). After feeding with durgs for 22 weeks, in all groups except the blank con trol group, compared to the level before feeding with durgs, serum TC,VLDL, ApoB have decreased, while LDL in model group has increased, the difference s are statistically significant (P<0.05 or P<0.01).Then we compared the values between groups at the same time point. Aft er feeding with durgs and high-fat fodder for 14 weeks, compared with blank control group, serum TC, TG, HDL, LDL, VLDL, ApoA-I, ApoB of model gr oup have increased, the differences are statistically significant (P<0.05或P<0. 01), while differences of changes among treatment groups and model group ar e not statistically significant. After feeding with durgs for 22 weeks, compared with blank control group, serum TC, LDL, VLDL, ApoA-I of model group h ave increased, the differences are statistically significant (P<0.01); compared w ith model group, HDL of treatment groups are not statistically different; compa red with model group, serum TC, LDL, VLDL of treatment groups have decre ased, while ApoA-I have increased, the differences are statistically significant (P<0.05, or P<0.01); serum TC, VLDL, ApoA-I among XSC low-dose group, XSC high-dose group and simvastatin group are not statistically different.3. After feeding with durgs for 22 weeks, compared with blank control gr oup, serum HDL2 of model group has increased; compared with model group, the changes of serum HDL2 of simvastatin group and XSC high-dose group ar e greater, the differences are statistically significant (P<0.05).4. Compared with blank control group, the ABCA1 mRNA expression of model group is statistically significant (P<0.05). Compared with model group, the the ABCA1 mRNA expressions of treatment groups are statistically signific ant (P<0.05). Compared with blank control group, the SR-BI mRNA expressi on of model group is statistically significant (P<0.01). Compared with model group, the the SR-BI mRNA expressions of treatment groups are not statisticall y significant.5. After feeding with durgs and high-fat fodder for 14 weeks, compared w ith blank control group, the change of serum MPO activity of model group is statistically significant (P<0.01), and compared with model group, the changes of serum MPO activity of treatment groups are statistically significant (P<0.0 5). After 8 weeks’ fading period, compared with blank control group, the chan ge of serum MPO activity of model group is statistically significant (P<0.05), and compared with model group, the changes of serum MPO activity of treat ment groups are statistically significant (P<0.05). The differences of changes o f serum PON1 activity among all group are not statistically significant.6. Compared with blank control group, the LCAT mRNA expression of m odel group is statistically significant (P<0.01). Compared with model group, th e the LCAT mRNA expressions of treatment groups are statistically significant (P<0.01).Conclusions:1. XSC can inhibit the plaque formation in artery of rabbits with AS and reduce the deposit of lipids in the lining of blood vessels, and reduce the acc umulation of the foam cells under the intima.2. XSC can increase serum ApoA-I level of AS rabbit, and decrease seru m TC, LDL and VLDL level to regulate the lipids in blood.3. XSC can increase serum HDL2 level of AS rabbit. Since HDL2 is the mature subtype particle, XSC could promote maturation of HDL and affect HD L subtype distribution.4. XSC can increase ABCA1 mRNA expression in liver of AS rabbit, whi ch could promote the metabolism of cholesterol in liver, and promote RCT pro cess, while its adjustment of the SR-BI mRNA is not obvious.5. XSC could reduce serum MPO activity of AS rabbits to inhibit oxidati on of HDL by MPO, and protect the anti-inflammatory function of HDL to m aintain its normal lipid carrier role, but its effect on serum PON1 activity is n ot obvious.6. XSC can increase the LCAT mRNA expression in liver of AS rabbit, which could promote mature of HDL and affect the function of HDL,7. The anti-atherosclerosis mechanism of XSC may be related to promotin g HDL mature, increasing HDL mature particles and regulating blood lipids lev els.8. The anti-atherosclerosis mechanism of XSC may be associated with incr easing mRNA expression of RCT proteins, and protecting antioxidant function of HDL.