| Insulin resistance, as a common dangerous factor causing many metabolic diseases,refers to the situation in which is lower than biologic effect was produced by normal dose of insulin, and usually exists in the early stage of disease. Thus, to interfere and treat insulin resistance means significantly for the prevention of various kinds of metabolic disease, especially diabetes mellitus.Obesity (mainly abdominal obesity) is critical for the occurrence and development of insulin resistance. Obesity can lead to IR passing endocrine, adipocytokines, inflammatory reaction and intra-cellular signal pathway. Peroxisome proliferators activatedre-ceptor (PPAR) is a superfamily member of intranuclear acceptor transcription factor, regulating the expression of target gene. With diverse biological effect, PPAR can advance lipocyte differentiation and lipogenesis, reinforce sensitivity of insulin in body, regulation glycometabolism equilibrium in corpore, restrain inflammatory factor production and inflammation formation, influencing tumorous growth, cadiovascular protective effect. Free fatty acids (FFA) from the decomposition of fat and adipocytokines often relate to obesity mediated IR.Obesity, PPARs and IR forms a complex relationship of mutual constraint via various kinds active factors,among which bioactivity difference of different acceptor type of PPARs (as PPARαand PPARγ) to break or maintain this kind of balance to great degree. Just as PPARγsimple agonist brings the problem of fat increase while ameliorating IR and T2DM, PPARαsimple agonist can hardly lower the Blood Glucose while improving blood fat。Hence,the key to the problem is to find out a kind of medicine that can better maintain the agitation effective of PPAR.In a long period of clinical practice, a great quantity herbal medicine and herbal medicine were find in Chinese traditional medicine that can cure and improve IR, among which the traditional Chinese medicine compound recipe of suanwei based on TCM theory of"sour in taste acting on the liver","sour taste prevailing over sweet taste"functions well in improving blood Glucose and fat without increasing body weight. Our previous studies show that Ursolic acid (UA), as a kind of active substance contained in the traditional Chinese medicine compound recipe of suanwei, plays a significant role in regulating PPARαand PPARγimportant protein in downstream passageway.Therefore,we propose that UA possible can achieve above-mentioned efficacy through appropriate activation PPARs different acceptors in appropriate proportion. Our research, mainly focusing on fat,liver and muscle that are important in IR,will explore the feasible mechanism in vitro and in vivo via cell and zoopery using molecular biology method. By investigating the mechanism of acidity active component UA in the improvement of IR, this research aims at proving the instruction of traditional Chinese medicine theory of"sour taste prevailing over sweet taste"in preventing IR and DM, and providing experiment evidence for prevention and curing DM as well as for the development of relevant medicine.PurposeTo determine if UA can combine with PPARαand PPARγ, and the difference between fenofibrate and Rosiglitazone in combination rate. To examine UA's influence on the expression variation of aP2, CAP, Perilipin, MMP-1 in 3T3-L1 cell IR model,and to discuss the role played by UA in adipocyte differentiation at IR. To observe the influence of UA on the glucose and Lipid Metabolism of Spontaneous T2MD KKAy Mice. To observe the influence of UA on FFA, TNF-α, Adiponectin in serum and related gene and protein in liver and muscular tissue of Spontaneous T2MD KKAy Mice.MethodTo construct PPARα, PPARγexpression plasmid and PPRE luciferase reporter genes plasmid to instantaneously transfection positive control PPARαand PPARγas well as PPRE regulatory luciferase expression plasmid with reference plasmid pRL-TK respectively to HepG2 cell with liposome, adding into UA,Rosiglitazone,fenofibrate of different density,detecting double- luciferase in order to acquire relative luciferase activity, the activity of which indicates the activation intensity of the interference medicine PPARαand PPARγ.To induce and establish 3T3-L1 cell model with high glucose/hyperinsulinism, we apply 10-5mol/L UA interfere in 3T3-L1 cell IR model, adopting RT-PCR method to detect mRAN expression of aP2, CAP, Perilipin, MMP-1 genes as well as the Westen-blot method to detect the expression and Perilipin phosphorylation of aP2, CAP, Perilipin, MMP-1 protein.35 KKAy mice were separate 5 groups according to randomized block design, which are control group, Rosiglitazone group, fenofibrate group, UA high dose group and UA low-dose group respectively, with C57mice as normal control group, 7 mice per group. After being interfere in for 4 weeks, detect changes of body weight, liver weight, abdominal fat weight, fasting blood glucose, blood fat tetrachoric, glucose tolerance, liver glycogen content and gluconeogenesis in each group; computing LI, AI, HOMA-IR; observing pathological change of hepatic tissue (HE staining) in each group.Using the same zoopery method as in part 4,adopting ELESA method to detect FFA, TNF-α, Adiponectin content in serum;adopting RT-PCR method to detect the mRAN expression of PEPCK,IRS-2 and GLUT2 genes; adopting Westen-blot method to detect expression of PEPCK,IRS-2 and GLUT2 and the transport of GLUT4 in muscular tissue of mice and to observe the expression of PPARα, PPARγin liver and muscular tissue via immunohistochemisty.The Perilipin phosphorylation of 3T3-L1 cell IR model interfered by UA decreased obviously,presenting significant difference (P<0.01) from IR group. CAPmRNA and protein expression increases obviously (P<0.01).Results2×10-(5)mol/L2×10-7mol/L UA, Rosiglitazone and fenofibrate can induce PGL3-PPRE-Luc Transcriptional Activation,Rosiglitazone and fenofibrate Transcriptional Activation PPARγand PPARαexpression plasmid respectively, with statistically significant difference(P<0.01)。The fact that UA of high concentration can transcriptionally activate PPARγand PPARαexpression plasmid while UA of low concentration presents no obvious effect (P<0.01) on HepG2 cell expressing PPARγindicates that UA of different concentration may differs in their affinity with different PPARs. The topmost agitate effective is 38.6% and 51.6% Rosiglitazone and fenofibrate respectively.UA and ROS can ameliorate IR of spontaneous T2MD KKAy mice to varying degrees, cutting down HOMA-IR; improving oral glucose tolerance. UA and FEN can both decrease body weight, LI, AI, grow downwards abdominal fat of spontaneous T2MD KKAy mice; improving TC,TG,LDL-C and HDL-C of serum,increasing liver glycogen, presenting statisticly significant difference (P<0.05 or P<0.01) comaring to model control group obviously difference exisit between UA high dose group and low-dose group (P<0.05).FFA, TNF-αand Adiponectin content in serum in the UA group have changed, showing statistically significant difference comapring to control group (P<0.01), playing obvious role PEPCK expression and phosphorylation of IRS-2 (P<0.01) in livers, and improves GLUT4 transport in muscular tissue as well as improve the expression of PPARαin livers (P<0.01).ConclustionUA of concentration 2×10-5mol/L2×10-6mol/L can acticate PPARγand PPARαin vitro to certain degree,which may be a kind of PPARα/γweak double excitomotor. The mechanism of UA in improving adipocyte differentiation in 3T3-L1 cell IR model might be relevant with inhibition Perilipin phosphorylation and raising the expression of CAP. UA can decrease body weight of spontaneous T2MD KKAy mice,regulate glucose and Lipid Metabolism and ameliorate IR. The effects of UA in improving spontaneous T2MD KKAy mice IR and in regulating blood sugar and fat might be closely related with cytokine,Expression of protein and phosphorylation that regulate correlated passageway, the role of which is accomplished via the double activation of PPARγand PPARα.
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