Study On The Efficacy And Mechanism Of Pu'er Tea Ⅱ In Improving Glucose And Lipid Metabolism Disorder And Atherosclerosis In ApoE^-/- Mice

Dysfunction of glycolipid metabolism and atherosclerosis(AS)are the main pathological basis of cerebrovascular diseases,coronary heart disease,thromboembolism and other cardiovascular diseases.These deseases have the long course and complex pathogenesis,which affect human’s health seriously.The main characteristics of dysfunction of glycolipid metabolism are hyperlipidemia and diabetes.A large number of basic and clinical studies had shown that hyperlipidemia can cause in rising of total cholesterol(TC),triglycerides(TG)and low density lipoprotein cholesterol(LDL-C),and result in lipid accumulation,which lead to the formation of atherosclerosis.High blood sugar condition of diabetes can significantly improve the vascular endothelial cell function,and accelerate the occurrence of atherosclerosis.Traditional Chinese medicine has a long history in China and plays an important role to treat hyperlipidemia and diabetes deseases.The study of small molecule active substances in traditional Chinese medicine may provide a promising prospect to improve dysfunction of glycolipid metabolism and atherosclerosis.Pu-erh tea is produced using leaves of Camellia assamica(Mast.)Chang,and it is a traditional beverage in the southwestern regions of China.It is manufactured by piling crude pu-erh tea under high temperature and humidity,this process is called post-fermentation.During this process,many regionally dominant fungi are involved in the enzymatic reactions of major compounds of tea such as catechins,gallic acid and caffeine.It is reported that Pu-erh tea have a variety of pharmacological activities,such as anti-hyperlipidemia,anti-diabetics,anti-oxidation,anti-tumor,anti-bacterial,anti-inflammatory,and anti-virus effects.The chemical compositions of the Pu-erh tea were systematically studied in our early research.We separated and identified 35 compounds including many new compounds.Then we confirmed that the new compounds puerinⅠ(P1),puerinⅡ(P2),puerin Ⅲ(P3)and puerinⅣ(P4)are microbial metabolic products in the process of fermentation in Pu-erh tea.Since Pu-erh tea can significantly improve the glucolipid metabolic disorder,compound P2 had been chemically synthesized for the molecular mechanism on glucolipid metabolic disorder and atherosclerosis.We wish the study can provide the theoretical support for the development and clinical application of Pu-erh tea.This experiment wasdivided into two parts:(Ⅰ)Pharmacodynamic studies of P2 on ApoE-/-mice induced by high-sugar and high-fat diet.The ApoE-/-mice were divided into five groups:the chow diet-treated group(CG),the high-fat and high-sucrose diet(HFHS)-treated group(HG),the 10 mg/kg/d ezetimibe-treated group(EZG),the low and high-dose P2-treated groups(P2-50 and P2-100,respectively),those mice in P2L and P2H groups received 50 and 100 mg/kg/d of P2 for 6 successive weeks,respectively.The fasting blood glucose(FBG),intraperitoneal insulin tolerance test(ITT),and intraperitoneal glucose tolerance test(IPGTT)were conductedto evaluate the drug efficacy onimproving blood glucose.The total cholesterol(TC),triglyceride(TG),high-density lipoprotein(HDL-C),Non-high-density lipoprotein(Non-HDL-C)and other related indicators were studied to estimated it’s lipids-lowering effect.In addition,the anti-atherosclerotic effects of P2 were evaluated by the plaque area of aortic outflow tract after oil red O staining and HE staining.The results showed that P2 could dose-dependently reduced FBG level and improved ITT and IPGTT levels compared with the mice in high-fat and high-sucrose diet-treated group.At the same time,the TC,TG and Non-HDL-C levels had been significantly decreased after P2 treatments.Furthermore,the epididymal fat weights and atherosclerotic plaques of mice treated by P2 at 100 mg/kg dose were also reduced remarkably.(Ⅱ)The mechanism of P2 on improving glycolipid metabolism disorder and atherosclerosis.Firstly,we study the lipids-lowering mechanism of P2 onHepG2 cells.The results showed that P2 could promote the clearance of LDL on HepG2 cells in dose-dependent manner.Then we revealed that P2 could obviously increase the protein expression of LDLR and decreased the levels of PCSK9,thus reducing the LDL levels in blood circulation.Secondly,we found that P2 at 50 μM and 100 μM could significantly inhibit the adhesion between HUVEC and THP-1 cells induced by ox-LDL in concentration dependence,and down-regulated the expression of VCAM-1 and MCP-1 in HUVEC cells stimulated by ox-LDL.And we found that P2 at 100 μM could significantly inhibit the formation of foam cell,and down-regulated the expression of SR-A1 in THP-1 cells stimulated by ox-LDL,to inhibiting the development of atherosclerosis.For the hypoglycemic activity of P2,we found P2 could significantly inhibit the activity of α-glucosidase enzyme in vitro,which remarkably improved the glucose levels of hyperglycemic mice.In conclusion,P2 had decreased level of LDL by accelerating the clearance of cholesterol,reduced the atherosclerotic plaque areas by inhibiting the adhesion between monocyte and endothelial cells,and improved hyperglycemia by inhibiting the activity ofα-glucosidase enzyme.P2 could improve glucolipid metabolism disorder and atherosclerosis in multi-path,and display well applied prospects.