Study On The Effect And Mechanisms Of Huatan Jiangzhuo Decoction Regulating Lipid Metabolism In ApoE^-/- Mice With Phlegm Turbidity Syndrome Of Hyperlipidemia

ObjectiveDuplicating phlegm turbidity syndrome of hyperlipidemia animal model by high-fat diet,and evaluating the stability and repeatability of the animal model,in order to establish an evaluation standard of it and provide an animal model basis for further evaluation of the therapeutic effect researches of phlegm turbidity syndrome of hyperlipidemia.By the quantitative analysis of miR-33 and its target gene expression level of ATP binding cassette transporter A1 and G1,as well as 3-hydroxy-3-methylglutaryl-coenzyme A reductase and corresponding protein levels,revealing the effect of Huatan Jiangzhuo Decoction on regulating the lipid metabolism and its mechanisms on ApoE-/-mice model with phlegm turbidity syndrome of hyperlipidemia.Methods(一)Replication and evaluation of animal model of phlegm turbidity syndrome of hyperlipidemia and the preliminary experimentsEvaluating the stability and repeatability of the animal model of phlegm turbidity syndrome of hyperlipidemia and regarding as the preliminaryexperiments.1.Evaluating the stability of the animal model of phlegm turbidity syndrome of hyperlipidemia30 male SD rats of 8-week old were randomly divided into normal group and model group,normal group and model group were respectively fed with normal diet and high fat diet.At the end of the 4,6,8 weeks the blood were obtained through posterior orbital venous plexus respectively,and the levels of TC,TG,LDL-C and HDL-C were measured.The animals were given free access to food and water during the entire experimental period.Body weight was measures weekly,and food and water intake was recorded everyday during the course of the study.At the end of the experiment,all mice were sacrificed and tissue weights were measured and the liver and aortic were stained by HE.Finally,evaluating the model condition and its stability.2.Evaluating the repeatability of the animal model of phlegm turbidity syndrome of hyperlipidemia16 male SD rats of 8-week old were randomly divided into normal group and model group,respectively fed with normal diet and high fat diet for 8 weeks.The animals were given free access to food and water during the entire experimental period.Body weight was measures weekly,and food and water intake was recorded everyday during the course of the study.At the end of the experimental,all mice were sacrificed and the levels of TC,TG,LDL-C and HDL-C were measured.Evaluating the repeatability of the animal model and comparing the results with the first preliminary experiment.(二)Study on the effect and mechanisms of HuaTan JiangZhuo decoction(HTJZ)regulating lipid metabolism in ApoE-/-mice with phlegm turbidity syndrome of hyperlipidemia50 Apo-/-mice were randomly divided into normal control group of 8 rats and high-fat diet group of 42,fed with normal diet and high-fat diet for 4 weeks respectively,and at the end of 4th week,TC,TG,LDL-C,HDL-C were measured.Then the high-fat diet fed group were randomly divided into 7 groups by TC,model group,low dose of HTJZC,middle dose of HTJZC,high dose of HTJZC,and western medicine group.The animals were given free access to food and water during the entire experimental period.Body weight was measures weekly,and food and water intake was recorded everyday during the course of the study.After the 4 weeks’ therapy of dosing regimen respectively,all animals were sacrificed and blood was collected to measure the levels of TC,TG,LDL-C,HDL-C;liver tissue weights were measured and the liver and aortic were stained by HE;and the liver tissues were used for Q-PCR assay of miR-33,ABCA1,ABCCG1 and HMGCR gene and Western-blot assay of ABCA1,ABCCG1 and HMGCR proteins.Results(一)The results of preliminary experiments and replication of animal model of phlegm turbidity syndrome of hyperlipidemia1.The animal model of phlegm turbidity syndrome of hyperlipidemia is stableAt the end of 4th and 6th week of high-fat diet,the weight,average amount of diet and dringking showed no significant difference compared with normal group(P>0.05).At the end of the 8th week,the average amount of diet and dringking is higher than the normal group(P<0.05);the diet amount of model group showed no significant difference compared with normal group(P>0.05),however,the rats in the model group bit the feed from the 6th week and gradually aggravated,resulting a less amount of the actual intake compared with normal group.At the end of 4th week,the level of TC in model group increased significantly,compared with the normal group(P<0.05),while the level of TG,LDL-C,HDL-C increased without statistically significance(P>0.05).At the end of 6th week,the TC level still have statistically significance compared with the normal group(P<0.05),which decreased slightly compared with the level of 4 week’ s with no significant difference(P>0.05).At the the end of 8th week,the TC,TG,LDL-C,HDL-C showed no significant difference(P>0.05),compared with normal group.2.The animal model of phlegm turbidity syndrome of hyperlipidemia is repeatableHigh-fat diet can successfully establish phlegm turbidity of hyperlipidemia model for 8 weeks,the TC,TG,LDL-C level of model group of were higher than the normal group,and HDL-C level was lower than the normal group compared with the normal group(P<0.05).To copy the animal model of phlegm turbidity syndrome of hyperlipidemia with high-fat diet on SD rats,4 weeks is more appropriate with stability and repeatability.Methods and techniques are established in this preliminary experiment,and laying a solid foundation for further research on the regulation mechanism of lipid metabolism in ApoE-/-mice.(二)The result of the effect and mechanisms of HuaTan JiangZhuo decoction(HTJZ)regulating lipid metabolism in ApoE-/-mice with phlegm turbidity syndrome of hyperlipidemia1.The general situations of the miceAfter 4 week’ high-fat diet,the autonomic activity and the response to external stimulation showed no obvious change before modeling.Compared with the high-fat diet group,the weight and the average amount of drinking water for 4 weeks of normal control group had no significant difference(P>0.05);the average diet for 4 weeks was higher in normal control group,compared with the high fat diet group(P<0.05).At the end of 4th week,the high-fat diet groups had shown some part of the characteristics of phlegm turbidity syndrome of hyperlipidemia.From the beginning of administration,the frequency of autonomic activity reduced,and low-dose HTJZ group is more obvious.At the end of the administration,the average diet of normal control group,mediun-dose HTJZ group,western medicine group,miR-33 antagomir group,miR-33 inhibitor negative control group increased,compared with model group(P<0.05);the average drinking of normal control group,low-dose HTJZ group,medium-dose HTJZ group,high-dose HTJZ group and miR-33 inhibitor negative control group decreased,compared with model group(P<0.05).The final weight of mice compared with the model group,only high-dose HTJZ group decreased with significant difference(P<0.05),the others showed no significant difference(P>0.05).Most of the mice were in line with the characteristics of phlegm turbidity syndrome animal model.2.The blood lipid levels of miceAfter 4 weeks of high-fat diet for modeling,the lipid levels of high-fat diet groups were higher than the normal group(P<0.05),which explaining the model was established successfully.All the high-fat diet groups’baseline is consistent and with no significant diffenence of the lipid level(P>0.05).The TC,TG,LDL-C levels of high-fat diet group were higher than the normal control group before and after the administration,proving high-fat diet can significantly promote the blood lipid level of ApoE-/-mice and promote the rapid formation of hyperlipidemia model.After treatment for 4 weeks,the western medicine group showed good effect,whose level of TC,TG,LDL-C decreased and HDL-C level increased,compared with the model group and the level before administration.However,the effect of HTJZ decoction group was poor and most of the indexes of blood lipids were with no significant differences compared with model group(P>0.05),except the high-dose HTJZ decoction group,which could down-regulate the TG level.3.The pathological results of liver and aorticAfter 4 weeks of treatment,the mice liver weight and liver coefficient compared with the model group had no significant difference(P>0.05),but compared with the normal control group,high dose group,three chinese medicine groups,the liver coefficient increased with statistical significance(P<0.05).The hepatic steatosis of all high-fat diet groups were more serious than the normal control group,showing more fat vacuoles with different size,and some part of the nucleus move to one side,small part of them with punctiform necrosis and lymphocytes infiltration.The condition of hepatic steatosis got better while the dose of HTJZ increased.The structure of aortic intima,membrane and outer mold were slightly abnormal and the smooth muscle arranged irregularly slightly of all the groups.There were no obvious lipid deposition,fibrous cap formation and plaques in model group.4.The gene and corresponding proteins’ expressions of miR-33,ABCA1,ABCG1,and HMGCRCompared with the model group,the level of miR-33 gene decreased in the low-dose HTJZ group,high-dose HTJZ group and western medicine group;the level of ABCA1 and ABCG1 gene decreased in the high-dose HTJZ group with significant difference(P<0.05).The level of ABCA1 gene showed no signifant difference between all groups(P>0.05).The level of ABCA1 and HMGCR protein showed no significant difference compared with model group(P>0.05).Concusion(一)To dupicate the animal model of phlegm turbidity syndrome of hyperlipidemia with high-fat diet on SD rats,4 weeks is more appropriate with stability and repeatability.Methods and techniques are established in this preliminary experiment,and laying a solid foundation for further research on the regulation mechanism of lipid metabolism in ApoE-/-mice.(二)Making a preliminary evaluation of phlegm turbidity syndrome of hyperlipidemia animal model standard according to the clinical diagnostic criteria of results of phlegm turbidity syndrome and the two experiments:①abnormal lipid levels;② body fat;③ burnout;④ Decreased amount of dringking water and intake food.(三)High-fat diet could accelerate ApoE-/-mice to show dysl ipidemia,and the blood lipid levels were significantly increased.HTJZ can control the lipid levels of high-fat diet ApoE-/-mice,as well as decrease the weight of mice and improve the situation of anorexia.(四)The possible mechanism of HTJZ decoction may be inhibiting miR-33 and HMGCR gene expression,so that regulating the lipid metabolism.