| Type 2 diabetes mellitus (2-DM) has been a great public heath problem because of the harms of 2-DM and its various chronic complications, besides huge direct and indirect medical cost on them.2-DM is a disease of polygenic inherited heterogeneity with unknown cause and pathogenesy, insulin resistance (IR) was known to be one of the primitive pathogenesy presently. IR means the target organs or the organizations are insensitive to the insulin, thus it decreases the biological effect of the latter and leads to a series of changes in pathology and physical function. IR In recent years, the cases of diabetes, hypertension, obesity, disorder of blood fat, arteriosclerosis are increased every year since the living styleand dietary structure have been changed, and IR is assumed as the foundation and correlation of these diseases. Therefore, to cure IR has been the important component element in the process of preventing and curing 2-DM and its various chronic complications.There is no medicine which has been recognized as an ideal one to cure IR by most people, although researchers have studied it deeply. While Tradditonal Chinses Mmedicine (TCM) can cure diseases with its characteristic of various approach and target point, so they have the advantages in treating some metabolic diseases. And it has been proved from clinic by doctors of TCM with the method of treatment based on symptoms under the guidance of holism. But the metabolism of action is retarded, for most cases lack of experimental data for complementary proof. Early findings of our project showed that the water-soluble part of Radix Salviae Miltiorrhiae (WPR) has significantly Ameliorating IR in Rats with 2-DM, and HPLC analysis showed that the main component was salvianolic acid B (SB) which content was 53%, it suggested that SB may be the main active ingredient which ameliorated IR. There is no research report which showed that SB has the role of prevention or treatment to IR at home and abroad. Therefore, based on verifing the efficacy of WPR, our study intended to invest the effect of SB on glucose metabolism, lipid metabolism, oxidative stress, fat cell factor in type 2 diabetic rats, to make an evaluation of SB on ameliorating IR, and further to approach the mechanism of the action, so as to provide more exact experimental evidences on the treatment of IR and related diseases by SB.This research project covered two sides of content, one is on document research, and the other is on experimental research.1 Document Researchs:After comprehensive retrieval of document reports and systematic summary about IR at home and abroad, we comprehend the study on IR and SB commonly used on the whole. Document research includes the following four aspects:(1) Modern medicine studys on the relationship between 2-DM and IR, IR pathogenesis, treatment and evaluation of profile, etc.; (2) TCM studys on cognition of IR, treatment of simple recipe and compound recipe on IR, etc.; (3) Described the relationships between glucose transporter protein 4 (GLUT-4) and IR in 2-DM, GLUT-4 and free fatty acids (FFA), GLUT-4 and leptin, GLUT-4 and oxidative stress, etc.; (4) The sdudy progression about prevention other diseases related to IR with SB.2 Experimental Studies:2.1 The Study on the effect of WPR to Ameliorat IR in type 2 diabetic rats.Objective: to establish IR rat model of 2-DM, and to evaluat and verify the effect of WPR to ameliorat IR in type 2 diabetic rats.Methods:Reproduction IR rat models by the method of long-term high glucose and high fat diet plus a single dose of streptozotocin (STZ) intraperitoneal injection, to treat the IR rat models with WPR which the content of SB was 53% for 4 weeks and choose gliclazide as a positive control, in order to observe the effects of WPR on glucose metabolism, lipid metabolism, insulin sensitivity index, renal function, etc.Results:(1) Model Evaluation:Successful replication of IR rat model of 2-DM, the experimental animal models characterized by high blood sugar, high blood cholesterol, high insulin, low insulin sensitivity, which consistent with the clinical features of IR in 2-DM. The model had a high achievement ratio which was feasible, economic, and it can be an ideal vehicle to study IR in 2-DM. (2) Efficacy studies:Glucose metabolism results showed that WPR reduce the levels of FBG and Ins in IR rats, raised the level of ISI and ameliorated IR significantly; lipid metabolism results showed that WPR reduce serum content of TC and TG significantly. Renal function results showed that WPR reduce the serum BUN concentration significantly. All results shows WPR can ameliorate glucose and lipid metabolism, elevate insulin sensitivity and ameliorat IR in type 2 diabetic rats.2.2 The Study on the effect of SB to Ameliorat IR in type 2 diabetic IR rats.Objective:to evaluat the effect of SB to ameliorat IR in type 2 diabetic rats, and to explore its mechanism.Methods:To treat IR rat model of 2-DM with SB (93%) by the dose of 100,200 mg/Kg for 6 weeks, rosiglitazone as a positive control, to observe the effect of SB to ameliorat glucose metabolism, lipid Metabolism, ISI, renal function, oxidative stress, FFA levels, leptin, tumor necrosis factor-a (TNF-a) level, etc.Results:(1) Efficacy studies:Glucose metabolism results showed that the administration of SB for 6 weeks can significantly reduce the levels of FBG and Ins, raise the level of ISI and ameliorat IR in type 2 diabetic rats; Lipid metabolism results showed that SB can significantly reduce serum content of TC and TG. Renal function results showed that SB can decrease the serum BUN concentration. All results shows SB can ameliorate glucose and lipid metabolism, elevate insulin sensitivity and ameliorat IR in type 2 diabetic rats. (2) Mechanism studies:Fat cell factor test results showed that SB reduce the level of serum FFA, Laptin significantly, but TNF-a levels were not affected. Oxidative stress results showed that SB can decrease MDA content and increase SOD activity significantly. It promptes that SB could ameliorat IR and abnormal metabolism of glucose and lipid partly by the role of decreasing FFA damage to the body, ameliorating leptin resistance and antioxidation.2.3 The study on the protection of SB on IR rat model of 2-DM by long-term administration.Objective:To evaluat the protection of SB on IR rat model's pathological organ and tissue by long-term administration, and to invest its adjunctive therapy to gliclazide in type 2 diabetic rats.Methods:Based on IR rat model of 2-DM with prolonging the administration time, it resulted in some pathological changes on organ and tissue in type 2 diabetic rats. This experiment is to observe the effects of SB, SB combined with gliclazide to ameliorat IR rats'glucose metabolism, lipid Metabolism, ISI, oxidative stress, etc., and evaluat the protections on the main pathological organ and tissue in type 2 diabetic rats.Results: SB administrated for 10 weeks could reduce the levels of TC, TG and MDA, increase SOD activity significantly. The role of lowering blood sugar, lowering FFA level, and ameliorating IR were gradually weakened, but SB combined with gliclazide could enhance most of effects significantly. Meanwhile, SB could ameliorate the degree of pathological changes on pancreas, liver, kidney, and toes which was connected with its antioxidation. SB combined with gliclazide could enhance the effects of ameliorating glucose and lipid Metabolism, glycogen synthesis action, IR, and pathological changes on organ and tissue. It shows that SB can be used to assist the kind of diabetic drugs on insulin secretion.2.4 The inhibiting activity of SB to a-glucosidase in vitro.Objective:α-amylase, sucrase and maltase origined from mucous membrane of rat's small intestine were selected to test SB's inhibiting activity in vitro, in order to evalust the inhibiting effect of SB and to definite inhibition type.Methods:a-amylase, sucrase and maltase origined from mucous membrane of rat's small intestine were selected to build various a-glucosidase inhibitor screening models with different types of substrates, and choosed acarbose as a positive control, this research tried to measure the inhibitory activity on a-amylase, sucrase, maltase, and to definite their inhibition type by Lineweaver-Burk mapping. Results:There was no inhibitory activity of DB at high concentration (1.3088mg/ml) to a-amylase. DB had a little inhibitory activity to sucrase and maltase, and their IC50 were 0.6537,0.4921 mg/ml respectly, their inhibition types were both non-competitive inhibition.2.5 The influence of DB to the expressions of CLUT-4mRNA in skeletal muscle and adipose tissue in type 2 diabetic IR ratsObjective: To observe the expressions of CLUT-4mRNA in skeletal muscle and adipose tissue in type 2 diabetic IR rats, and to approach the molecular mechanism of IR.Methods:Based on previous pharmacodynamic study, to observe the expressions of CLUT-4mRNA of skeletal muscle and adipose tissue in type 2 diabetic IR rats by RT-PCR method.Results:SB could elevate the expressions of CLUT-4mRNA in skeletal muscle and adipose tissue, and eventually ameliorated IR in type 2 diabetic IR rats. Combined with document research and pharmacodynamics studies, we belives thar the effect of DB to elevate expressions of CLUT-4mRNA in skeletal muscle and adipose tissue may be mainly related to its actions of decreasing the levels of FFA and laptin and antioxidation.2.6 Conclusions(1) The method of long-term high glucose and high fat diet plus a single dose of STZ intraperitoneal injection can replicate IR rat models successfully. (2) WPR can ameliorate glucose and lipid metabolism, elevate insulin sensitivity and ameliorat IR in type 2 diabetic rats. (3) SB can ameliorat IR and abnormal metabolism of glucose and lipid, partly by the role of decreasing FFA damage to the body, ameliorating leptin resistance and antioxidation. (4) The role of lowering blood sugar, lowering FFA level, and ameliorating IR were gradually weakened, but SB combined with gliclazide could enhance most of effects significantly. (5) SB can ameliorate the degree of pathological changes on pancreas, liver, kidney, and toes, and it can be used to assist the kind of diabetic drugs on insulin secretion. (6) DB had a little inhibitory activity to sucrase and maltase, and their inhibition types were both non-competitive inhibition. (7) SB can ameliorate postreceptor defects of IR through elevate the expressions of CLUT-4mRNA in skeletal muscle and adipose tissue, and eventually ameliorates IR in type 2 diabetic IR rats. |
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