The Study On The Protective Effect Of Danggui Buxue Tang In ApoE Knockout Mice Atherosclerosis And Its Mechanism

Part Ⅰ Contents of chemieal ingredients in Danggui Buxue TangObjective:To establish a high performance liquid chromatography-tandem mass spectrometry method and determine the contents of chemical ingredients such as astragaloside Ⅳ,ferulic acid,calycosin and formononetin in Danggui Buxue Tang.Method:Astragali mongholici Radix and Angelica sinensis Radix were mixed in a ratio of 5:1 and placed in the extracting tank with the extracting and concentrating unit for extracting.The extract was concentrated in vacuum at 80℃.The concentrated liquid was put into the vacuum dryer box and dried at 60℃.Chromatographic separations were performed with Waters C18 column(2.1 *50mm,1.7um).The mobile phases consisted of acetonitrile(A)and water(B)which contain 0.1%formic acid(v/v).The flow rate:0.4 ml/min;column temperature.(?)5℃.Electrospray ion source(ESI)was used to scan in positive ion mode.The flow rate of NZ is 789 L/h,the temperature of solvent removal is 261℃,the temperature of ion source is 100℃,and the capillary voltage is 3.0 kV.Cone hole voltage 40V,compensation voltage 80V.Result:Based on the developed method,a good linearity of each marker ingredient was observed in a relatively wide concentration with the correlation coefficient larger than 0.995.Both reproducibility and stability were also satisfactory.In the recovery assay,four compounds showed mean recovery rates in the range of between 95%and 100%,indicating an acceptable accuracy of the method.The contents of astragaloside IV,ferulic acid,calycosin and formononetin in Danggui Buxue Tang water extract concentrate were 30.39、21.65、558.34 and 224.11 p g/g,RSD<1.8%.Conclusion:In general,the proposed method exhibited a satisfactory linearity,reliable accuracy and good precision and could be used for further analysis of the DBT samples.The content of 4 compounds in Danggui Buxue Tang was stable.The stability of the quality of Danggui Buxue Tang is of great significance for experiments and the further study of the target drug ingredients.Part Ⅱ The effect of Danggui Buxue Tang on Atheroselerotic Lesion of ApoE Knockout MieeObjective:To observe the effect of Danggui Buxue Tang on the formation of atherosclerotic plaque in ApoE knockout mice.Method:Forty-eight male ApoE knockout mice aged 6 weeks fed with high-fat diet after 2 weeks of adaptive feeding.ApoE-/-mice aged 8 weeks were randomly assigned to one of the following four subgroups(n=12 each):(1)Model group(mod),(2)low-dose DBT group(DBT-L),(3)middle-dose DBT group(DBT-M),(4)high-dose DBT group(DBT-H).Animals in DBT groups were treated with DBT(7.4 mg/g/d,14.8 mg/g/d or25.6 mg/g/d);p.o.while model group were treated with corresponding volumes of normal saline after 8 weeks of high-fat diet.The period of drug administration continued for 8 weeks.After treatment,the mice were euthanized under excessive anesthesia,their heart and sort were separated.The atherosclerotic lession were accessed by oil red 0 staining.Result:Compared with the model group,the atherosclerotic lession area of aorta and aortic sinus in the Danggui Buxue Tang group were significantly reduced.Conclusion:Danggui Buxue Tang can significantly inhibit the formation of atherosclerotic plaque in aorta of ApoE knockout mice fed with high fat diet.Partlll The mechanism of Danggui Buxue Tang on lipid metabo l i sm in ApoE knockout miceObjective:Using ApoE gene knockout mice as hyperlipidemia mice model,to study the mechanism of Danggui Buxue Tang on lipid metabolism in ApoE knockout mice.Method:Forty-eight male ApoE knockout mice aged 6 weeks fed with high-fat diet after 2 weeks of adaptive feeding.ApoE-mice aged 8 weeks were randomly assigned to one of the following four subgroups(n=12 each):Cl)Model group(mod),(2)low-dose DBT group(DBT-L),(3)middle-dose DBT group(DBT-M),(4)high-dose DBT group(DBT-H).Animals in DBT groups were treated with DBT(7.4 mg/g/d,14.8 mg/g/d or25.6 mg/g/d);p.o.while model group were treated with corresponding volumes of normal sal ine after 8 weeks of high-fat diet.The period of drug administration continued for 8 weeks.Blood lipid levels of total cholesterol(TC),triglyceride(TG)、low density lipoprotein cholesterol(LDL-C)、high density lipoprotein cholesterol(HDL-C)、Free cholesterol(FC)and free fatty acids(FFA)in each experinental group were examined at different time.Liver lipid deposition was assessed by oil red 0 staining and determing the levels TC,FC and TG.Quantitative Real-time PCR was used to detect the expression of mRNA of genes related to lipid metabolism in liver.The expression of lipid metabolism-related protein in liver was detected by Westen blot.Result:Danggui Buxue Tang significantly reduced the levels of TC,TG,LDL-C and FC in ApoE-/-mice serum,and increased the level of HDL-C in serum.The effect of DBT on FFA was not obvious.Compared with the m.odel group,the lipid deposition in liver of DBT groups decreased significantly.RT-PCR showed that Danggui Buxue Tang could significantly increase the mRNA expression of apoAI,ABCA1,SR一BI,LCAT,ABCG5,ABCG8,CYP7al,SREBP-2 and LXR a.Western blot results showed that apoAI,ABCA1,SR-B1,SREBP-2 and LXR α protein expressions in liver of DBT groups were significantly higher than those in model group.Conclusion:Danggui Buxue Tang can inhibit the formation of atherosclerotic plaque by regulating blood lipid metabolism.The mechanism of lipid regulation may associated with DBT can significantly increase the expression of genes related to liver cholesterol uptake and reverse cholesterol transport,such as apoAI,ABCA1,SR-B1,LCAT,ACAT,and can also promote the expression of cholesterol excretion related genes ABCG5,ABCG8 and CYP7a1 through upregulating the expression of LXR α and SREBP-2.