Study On ApoE Knockout Mice Of Atherosclerosis And Visfatin Expression By Dingxin Recipe

Background:Since the1980s, along with the improvement of people’s li ving standard, the change of lifestyle, and China’s population is aging, corona ryheart disease has become China’s most common cardiovascular diseases, bec ome a danger to our country people’s healthy serious public health problem. Atherosclerosis (Atherosclerosis. AS) is the main basis of Coronary heart dise ase. The pathogenesis of AS is not yet clear. People have put forward lipid i nfiltration theory, arterial smooth muscle cell proliferation, blood clots source said thetheory, damage responses in the theory hypothesis. AS atherosclerosis research unceasingly thorough, increasingly, people are realizing that inflammat ion is important factors in the process of AS.AS early as the end of last cent ury Ross is put forward AS is a kind of inflammatory disease, more and mor e research confirmed that inflammation in AS happened in development plays an importantrole, and throughout AS patch formation, and growth to all the process rupture. Vulnerable plaques that inflammation in the reaction prompted unstable patch formation and the rupture, and based on this, the activation c ell components happened and mediated thrombosis, is most acute coronary sy ndrome happened main reasons. Therefore, intervention inflammation and inter vention lipid metabolism are the main therapy for the prevention and control of be the atherosclerotic disease.Visfatin is a newly found associated with inflammation of the fat cells factor, Visfatin and before in lymphocytes found in known as a" precursor B cell colony enhancement factor (" pre-B cell colony-enhancing factor, PBEF) immune system proteins for the same material.Visfatin has many biological activities, such as an alog insulin like effect, increased insulin sensitivity, involved in the inflammatory response, delayed neutrophil apoptosis, regulation of lipid metabolism.Although the Visfatin in the body the specific mechanism of action has not been studied in depth, but its findings related to the pathogenesis of the disease and provide a new idea for the treatment.Study finds type2diabetes patients with plasma Visfatin levels and vascular endothelial cell function in significantly negative correlation.Vascular endothelial injury is one of the initial factors of AS. Patients with the metabolic syndrome Visfatin plasma significantly increased, and accompanied by carotid atherosclerosis of patients rise is more obvious. Visfatin in with obesity, diabetes, metabolic diseases such as coronary heart disease patients, the level also rises. Patients with coronary heart disease of the plasma Visfatin significantly increased, especially in patients with acute coronary syndrome rise is more apparent, Visfatin is independent of the known coronary heart disease risk factors and a new risk factors. By means of a case-control study got the same conclusion, the plasma Visfatin level was significantly higher than that in the control group, and the Visfatin and hs-CRP were significantly positive correlation, multiple regression analysis confirmed the plasma levels of Visfatin are independent risk factors of myocardial ischemic episodes.Clinical studies have shown, the Visfatin is elevated in plasma of patients with coronary heart disease associated with monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6), high sensitivity C-reactive protein (,hs-CRP) and other inflammatory mediators in the elevated, in vitro cell level experimental get similar results.In addition, Visfatin can enhance monocyte strains of matrix metalloproteinase-9(MMP-9) and peripheral blood mononuclear cells in tumor necrosis factor-alpha (TNF-a), interleukin-8,(IL-8) expression.The above studies show that, Visfatin at the AS inflammatory reaction plays an important role in the process.C-reactive protein (CRP) is on acute injury, infection or other inflammatory stimuli in the acute phase reaction products, plasma CRP concentration can be predicted in patients with unstable angina pectoris after healing.In unstable angina patients, CRP>3u g/ml patients after1years survival rate were significantly reduced than CRP<3u g/ml patients.For stable angina patients, coronary artery stent implantation for72hours after the measurement of plasma CRP levels, and for1consecutive years of follow-up, the CRP>5u g/ml survival of patients with CRP <5u g/ml were also significantly lower.In healthy individuals, the concentration of CRP monitoring can predict future cardiovascular disease risk prospective study is a milepost type results.Started in1982," the physicians’ health study" was measured in1086healthy men CRP level, in which543men later development for myocardial infarction, stroke or venous thrombosis, another543men in eight years of follow-up no reports of cardiovascular disease. Thereafter, menopause women for prospective studies, compared the other inflammatory markers, such as SAP, IL26, sICAM21etc, is the most powerful CRP found future cardiovascular disease risk predictors of content.TNF-a in flammatory factor family is active and easily measured in the peripheral circulation, which has many biological effects, mainly involved in the inflammatory response cascade process. TNF-α can direct injury of endothelial cells, and promote the proliferation of vascular smooth muscle cells, so as to promote AS plaque formation.TNF-α can also induce platelet adhesion, enhanced leukocyte chemotaxis, promote coagulation, inhibit fibrinolysis, promotes monocyte adhesion to endothelium and reduced activity of vascular smooth muscle cells and participate in atherosclerosis development, almost completely inhibit vascular smooth muscle cell expression of interstitial gene so that plaque instability direction.In acute coronary syndrome patients with anti-inflammatory treatment were found anti-inflammatory drugs can reduce the levels of TNF-α. TNF-α is clearly related to plaque inflammation.People use TNF-α and other inflammatory cytokines as a future cardiovascular event risk factors and predictive factors.Medicine for preventing coronary heart disease curative effect, traditional Chinese medicine against atherosclerosis mechanism having a multi-level, multi-angle, multiple target feature, in which inflammation is one of the important mechanisms.Chinese medicine used to prevented coronary heart disease. Chinese medicine against atherosclerosis mechanism having a multi-level, multi-angle, perspectives, and the characteristics of targets,in which inflammation is one of the important mechanisms.Many Chinese scholars think, from the pathogenesis of Chinese medicine theory perspective, AS has" Qi, blood stasis, phlegm "characteristic, Qi and Phlegm Blood stasis is one of main methods of TCM Prevention and treatment of AS.DingXin party is my school affiliated hospital of traditional Chinese medicine recipe, a beneficial qi and activate blood circulation dry wet expectoranting function. Clinical studies have shown its good curative effect on coronary heart disease. Preliminary experiments show that, centering on ischemia and reperfusion injury has a good protective effect.Objective:This experiment used ApoE gene knock out mice with high fat feed copy AS animal model, with DingXin party to intervene, monitoring in AS development process in serum and organization of Visfatin protein expression, in order to understand the Visfatin in AS in the role. Observation of Dingxin Recipe on AS plaques and the influence on Visfatin and inflammatory factor CRP, TNF-a expression of intervention effect, for the prevention and treatment of AS provides a new train of thought.Methods:Thirty-five male8-week-old ApoE-/-mice were randomly divided into five equal groups:model group,low-dose Dingxin Recipe [9.30g/(kg·d)] group,medium-dose Dingxin Recipe [18.59g/((kg-d)] group,high-dose Dingxin Recipe [37.18g/(kg·d)] group, Simvastatin[5mg/(kg·d)] group,Wildtype mice were used as normal control group. Each group of mice free drinking water, according to the mouse body mass (weekly measurements of1), with a correction dose,12weeks after cessation of the drug intervention, to end the experiment.Abdominal anesthesia mice, eye removal method get blood specimen, done heart infusion, independent artery from the root the abdominal aorta at the end of the aorta separation article, partly in4%paraformaldehyde fixed, partly in preserved in liquid nitrogen.The tissues was paraffin imbedded and made into slices.Used HE dyeing to observe in aortic pathology and calculate for each group of patch area percentage. Separation of serum, detection of total cholesterol (TC), triglycerides (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) content.We detected Visfatin、CRP、TNF-αin serum by ELISA,and observed the effect of DingXin Recipe on expression of serum Visfatin、CRP、TNF-α. Total protein was extracted from aortic tissue for Western blot to observe the changes of Visfatin expression the effect of DingXin Recipe. The experimental data are completely random design of measurement data, so the use one-way ANOVA for analysis,homogeneity of variance with LSD method and missing variance with Dunnett’s T3method.The experiment data were expressed mean±standard deviation(x±s),and the correlation between variables was analyzed by multiple linear stepwise regression analysis,P<0.05indicated statistical significance.SPSS13.0software was used for statistical analysis.Results:1.The effect of DingXin Recipe on ApoE-/-mice body weight and growth After the end of the experiment, mice in each group increased body weight. Normal control group, model group, Simvastatin group, low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group add weight values were (3.83±0.93)g、(7.70±1.01)g、(6.87±0.82)g、(4.97±0.58)g、(4.87±0.70)g、(4.61±0.49)g.Treatment groups of the add weight value were lower than model group,which low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group weight gain compared with the model group had statistical significance (P<0.01); Simvastatin group weight gain compared with the model group had no statistical significance(P>0.05).We observed feeding and drinking were normal in mice. Mice in model group hair color is generally lack of gloss, activity is poorer, excretion is normal. Simvastatin group, low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group the growth of mice improved,comparing with model group.2. The change of plasma biochemical parameters in mice Model group lipid levels significantly higher than normal control group (P<0.01). Treatment groups serum TC, TG, HDL, LDL levels were lower than that of the model group. Compared with model group, the serum TC, TG, Hdl, LDL of high-dose Dingxin Recipe group and simvastatin group ignificantly reduced (P<0.05).There was no significant difference between the two groups (P>0.05).High-dose Dingxin Recipe group lipid levels was lower than low-dose Dingxin Recipe group and medium-dose Dingxin Recipe group, among which TG were significantly reduced (P<0.05).3. The effect of DingXin Recipe on ApoE-/-mice aortic atherosclerotic plaque lesions histopathology The observation of normal control group lining is smooth, aorta wall thickness uniformity, endometrium, membrane and membrane structure is clear with no AS lesions.Model group of arterial wall is uneven, there are a lot of foam cell and cholesterol crystallization in the atherosclerotic plaque lesions, large lipid core, foam cell and cholesterol crystallization, and the pipe wall adhesion is not close. Plaque volume is larger, fused into pieces, endometrial extensive and obvious thickening, fibrosis, vascular wall to lumen with prominent, smooth muscle shrinking significantly, epicardial visible inflammatory cell infiltration.Vascular internal elastic membrane surrounding the area was significantly increased than that in normal control group.Compared with the model group, each group of mice artery root AS lesions have different levels of control.Compared with the model group, simvastatin group, medium-dose Dingxin Recipe group, High-dose Dingxin Recipe group root artery atherosclerotic plaque area of the differences were statistically significant (P<0.01). Low-dose Dingxin Recipe group with model group aortic root atheromatous plaque area were no significant difference (P>0.05). High-dose Dingxin Recipe group with medium-dose Dingxin Recipe group aortic root atheromatous plaque area were statistically significant difference(P<0.01).High-dose Dingxin Recipe group with Simvastatin group aortic root atheromatous plaque area were no significant difference (P>0.05).4.The effect of DingXin Recipe on ApoE-/-mice serum CRP levels Compared with normal control group, model group serum CRP were significantly increased (P<0.01). Simvastatin group, low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group serum CRP levels were significantly lower than those in model group (P<0.01). The high dose Dingxin Recipe group serum CRP levels are below the simvastatin group, there was no statistically significant difference between the two groups(P>0.05). The high dose DXR group CRP levels were lower than low-dose Dingxin Recipe group and medium-dose Dingxin Recipe group,he difference was statistically significant (P<0.05).5. The effect of DingXin Recipe on ApoE-/-mice serum TNF-a levels Compared with normal control group, model group serum TNF-awere significantly increased (P<0.01). Simvastatin group, low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group serum TNF-alevels were significantly lower than those in model group (P<0.01). The the simvastatin group serum TNF-a levels are below high dose Dingxin Recipe group, there was no statistically significant difference between the two groups(P>0.05). The high dose Dingxin Recipe group TNF-a levels were lower than low-dose Dingxin Recipe group and medium-dose Dingxin Recipe group,he difference was statistically significant (P<0.05).6. Western blot were detected in ApoE-/-mice aortic tissue of Visfatin protein expression Through the analysis of the bands of purpose that image, groups of aortic tissue of Visfatin protein expression significant difference (P<0.01). Compared with the normal control group, model group aortic tissue of Visfatin expression was significantly higher, the difference was statistically significant (P<0.01).Simvastatin group, medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group of aortic tissue Visfatin levels were lower than model group, the difference were significant (P<0.05);Simvastatin group and high-dose Dingxin Recipe group aortic tissue Visfatin expression difference does not have statistical significance (P>0.05).High-dose Dingxin Recipe group of aortic tissue Visfatin levels were lower than low-dose Dingxin Recipe group,medium-dose Dingxin Recipe group(P<0.05).7. The effect of DingXin Recipe on ApoE-/-mice serum Visfatin levels Mice in model group serum Visfatin was significantly elevated than normal control group,the difference was statistically significant (P<0.01)Simvastatin group, medium-dose Dingxin Recipe group,high-dose Dingxin Recipe group serum Visfatin levels were lower than model group(P<0.01)and higher than normal control group(P<0.01), the differences were statistically significant. The high dose DXR group with the simvastatin group serum Visfatin level was no statistically significant difference(P>0.05).The high dose DXR group Visfatin levels were lower than low-dose Dingxin Recipe group and medium-dose Dingxin Recipe group,he difference was statistically significant (P<0.01).8. Correlation analysis on ApoE-/-mice serum and tissue Visfatin expression with serum CRP, TNF-α level ApoE-/-mice serum Visfatin level with serum CRP level were positively related to relationship, relationship with statistical significance(P<0.01); serum Visfatin level with serum TNF-α level were no statistically significant difference(P>0.05). ApoE-/-mice tissue Visfatin expression with serum TNF-α level were statistically significant (P<0.01); ApoE-/-mice tissue Visfatin expression with serum CRP level were no statistically significant difference(P>0.05).Conclusions:1. Different dose of Dingxin recipe can reduce ApoE-/-mice aortic pathological lesions, the high-dose Dingxin Recipe group significantly reduce the degree of luminal stenosis, plaque is smaller, better than simvastatin group.2. Different dose of Dingxin Recipe can significantly decrease the course of AS the ApoE-/-mice weight gain, DingXin Recipe treatment groups ApoE-/-mice lipid levels decreased significantly, illustrate centering on ApoE gene deficient mice have lipid-lowering and effects of weight loss, delay the progress of AS.3. Mice in model group serum CRP, TNF-a level, blood lipid levels were significantly increased compared with the normal control group.After treatment, high-dose Dingxin Recipe group serum CRP TNF-a level and lipid levels are down significantly, and shows than control drug simvastatin more outstanding results, confirmed by centering on the inhibitory effect of AS.The Dingxin Recipe is possibly by decreasing ApoE-/-mice inflammatory response to anti AS and plaque stabilization effect.4. Visfatin in model mice serum and tissue is highly expressd, further confirme by Visfatin and AS exist definite correlation, which may be involved in the occurrence and development of AS. ApoE-/-mice serum and tissues Visfatin with serum CRP, TNF-a were positively related, suggesting Visfatin involved in the inflammatory response.5. Medium and high dose DingXin Recipe therapy can reduce ApoE-/-mice serum and tissue Visfatin expression, prompting DingXin Recipe on ApoE-/-mice AS inhibition is associated with reduced levels of Visfatin expression correlation.