Effects And Mechanisms Of Liuwei Dihuang Pills On Prevention Of Diabetes Mellitus In OLETF Rats

BACKGROUND:The incidence rate of diabetes mellitus roars up year by year and diabetes has become the third disease following the cardiovascular diseases and tumor which damage people's health seriously. With the knowledge of insulin, the development and application of drugs stimulating secretion of insulin and insulin-sensitizing drugs etc. , more methods are available in the treatment of diabetes. However, although progress has been made in the treatment of diabetes, we still can't control the development of diabetes and its complications. So preventing diabetes becomes one of the most important issues in order to solve the problem radically. Whether diabetes can be prevented and what is the most economical, safe, effective way to prevent diabetes becomes a must to tackle this problem. The evidence-based research of DPP and stop-NIDDM showed type 2 diabetes mellitus which accounts for 90% of all patients with diabetes can be prevented and the risk of development of diabetes can be decreased by 31%-58%.However, researches above uncover some problems to some extent, such as the compliance of patients, the side-effects and high price of thedrugs, the high cost of diabetes prevention and so on. Finding more economical, little side-effect and more effective drugs to prevent diabetes from occurring in susceptible populations or individuals become indispensable in pharmacological interventions.The mechanism of type 2 diabetes mellitus has not been acknowledged yet. B-cell dysfunction and insulin resistance is the most two important reasons for the onset of type 2 diabetes mellitus. B -cell dysfunction, as one of the most important pathogenesis of type 2 diabetes, is coming into spotlight gradually. Some even argued 3 -cell dysfunction is more important than insulin resistance in the development of diabetes. In the renowned research UKPDS, whether the patients were under intensive treatment, their blood glucose became higher due to the 3 -cell dysfunction with the progress of diabetes. Some researches showed cell apoptosis is the main reason which contributes to the decrease of the number of islet 3 -cell, which leads to hyperglycemia. Since the 1990's of last century till now, a lot of researches in animal and human beings told the apoptosis of 3 -cell in pancreatic islet happens in the period of pre-diabetes and becomes higher after the onset of diabetes. The apoptosis of 3 -cell leads to the imbalance of the mass of 3 -cell and the development of diabetes. In the research of Liuwei Dihuang preventing diabetes, it's important to assess the effects of the drug on the apoptosis of 3 -cell and the probable mechanism.Insulin resistance is another factor of type 2 diabetes mellitus. It is now generally recognized diabetes will get through the stage of IGT(impaired glucose tolerance), in which stage insulin resistance is the main character. Insulin resistance is a pathophysiologic condition that the reactivity of organ to insulin is decreased. It is the congenerous risk of many clinical diseases (such as hypertension and atherosclerosis), especially in endocrine and metabolism diseases. Researches show that manyhormone and cytokines secreted by adipose tissue play a very important role in insulin resistance. As one of cytokines secreted by adipocytes, adiponectin was found in 1994, and many experiments show that it plays an important role in energy metabolism and insulin resistance. Many research show that there is a negative correlation between plasma adiponectin concentration and constituent ratio of body fat, waist-to-hip ratio, fasting blood glucose, postprandial 2h blood glucose, and there is a positive correlation between plasma adiponectin concentration and insulin sensitivity. Plasma adiponectin level had decreased in earlier period of obesity and decreased continuously after type 2 DM happened. The decrease of plasma adiponectin has a close affinity to insulin resistance. Adiponectin may become a valid medicine for curing insulin resistance and DM. So it is necessary to study the effects of prophylactic agent on insulin resistance and the expression of adiponectin mRNA to elucidate the pathogenesis of insulin resistance and type 2 DM and the mechanism of prophylactic agent.Now the researches about prevention of type 2 diabetes include DPP and stop-NIDDM principally. With the development of combination of TCM with Western medicine about treatment of diabetes, Chinese herbs and their active component have come into spotlight. It is feasible to find effective drug to prevent diabetes. In recent years, many scholars have carried out researches in prevention of diabetes with Chinese herbs and found Liuwei Dihuang Pills can regulate glucose metabolism and immune function, anti-damage, stabilize membrane, etc. as well as increase glycogen, decrease the activity of G-6-PDH, decrease the blood glucose, TG, FFA and anti-oxidation. These researches provide the theoretical proof for the prevention of diabetes with Liuwei Dihuang Pills. We think it is very probable for Liuwei Dihuang Pills to prevent type 2 diabetes, which is achieved by intervening the P-cell dysfunction and insulin resistance.OLETF (Otsuka Long- Evans Tokushima Fatty) rats were used in this study to observe the effects of Liuwei Dihuang Pills on incidence rate of diabetes, and explore the probable mechanism ,such as P-cell dysfunction and insulin resistance in order to provide theoretical and experimental proofs.Part 1 Effects of Liuwei Dihuang Pills on prevention of diabetes in OLETF rats OBJECTIVE:To study the effects of Liuwei Dihuang Pills on preventing diabetes in OLETF (Otsuka Long-Evans Tokushima Fatty) rats explores the probability of Liuwei Dihuang Pills in preventing diabetes in laboratory animals.METHODS:1 OLETF rats, spontaneous NIDDM model rats, with their lean nondiabetic counterparts, Long-Evans Tokushima Otsuka (LETO) rats, were supplied at 4 weeks of age. Forty male OLETF rats were randomly divided into two groups, Liuwei Dihuang Pills treated group (Group Liuwei) and the control group (Group Control). Ten male LETO rats were in normal control group (Group LETO). Rats of Group Liuwei were given Liuwei Dihuang pills (2.4g ? kg~'d'1) by intragastric administration from 8 weeks of age on. Except for Group Liuwei rats, all rats were given distilled water by intragastric administration.2 The blood glucose was determined by OGTT (oral glucose tolerance test) to diagnose diabetes. 15 hours before OGTT (5:00 PM, the day before), food was removed from the cage. 2g/kg glucose was administered intragastricly to conscious rats. Blood was obtained from the tail before and 30, 60, 90 and 120 min after the administration of glucose and was analyzed for glucose with life scan ultra glucose photometer. Diabetes was diagnosed when both peak blood glucose > 16.7 mmol/Land 120 min blood glucose >ll.lmmol/L. IGT (impaired glucose tolerance) was diagnosed when either was determined.3 Body weight and food intake were monitored every week. Rats were sacrificed at 8, 32 and 40 weeks. Blood glucose, serum triglyceride, serum cholesterol, plasma FFA (free fatty acid), plasma insulin were determined.RESULTS:1 The food intake in both OLETF rats groups are significantly higher than group LETO rats (p<0.01). The food intake of Liuwei Dihuang pills treated rats was not different with that of group control. Body weight was significantly greater in OLETF rats compared with LETO rats since 6 weeks of age (p<0.05) till 40 weeks of age when the research came to an end. There was no difference of body weight between Group Liuwei and Group Control (p>0.05).2 The 2hPG of rats in Group Control rise at 23 weeks of age (10. 1 ± 1. 8mmol/L), whereas it rise at 30 weeks of age in Group Liuwei (9. 4 + 2. 3). 2hPG of Group Liuwei was significantly lower than that of Group Control (p<0.01) and same with the glucose area under curve (AUC) (p<0. 05). AUC in both Group OLETF rats are higher than that of Group LETO.3 OLETF rats in Group Control developed diabetes since 23 weeks of age and the incidence rate of diabetes became higher with the time goes by until got to 92.9% at 40 weeks of age when the research came to the end. Rats in Group Liuwei developed diabetes since 30 weeks of age and incidence rate was 28.6% at 40 weeks, significantly lower than Group Control (p<0.01). No rats have developed diabetes or IGT in Group LETO.4 The serum triglyceride, serum cholesterol, plasma FFA was significantly lower inGroup Liuwei than that of Group Control (p<0.05). CONCLUSIONS:1 Liuwei Dihuang Pills can decrease the extent of and delay the occurrence of hyperglycemia in OLETF rats.2 Liuwei Dihuang Pills can decrease the incidence rate of type 2 diabetes significantly which doesn't depend on the loss of body weight.3 Liuwei Dihuang Pills can decrease the serum triglyceride, serum cholesterol, plasma FFA and plasma insulin.Part 2 Effects of Liuwei Dihuang Pills on apoptosis of p-celi in OLETF rats OBJECTIVE:To observe the apoptosis of 0 -cell and the apoptosis related gene and protein expression in OLETF rats and study the effects of Liuwei Dihuang Pills on the apoptosis of P -cell and its probable mechanism.METHODS:1 Rats were sacrificed at 8, 32 and 40 weeks old of age. Pancreas were removed and weighed with water absorbed away by filter paper after rats were sacrificed. The parts were stored in liquid nitrogen, 4% formaldehydum polymerisatum and glutaral.2 The pancreatic tissues were stained with hematoxylin and eosin to observe the morphological change. The paraffin sections were stained with the method ofimmunohistochemistry and TUNEL to examine apoptotic P-cells and examine P-cell mass with anti-insulin antibody and calculate the apoptosis rate of P-cell.3 Determine the expression of apoptosis related protein Bcl-2 and Bax with immunohistochemistry.4 The RNA was abstracted and the RNA expression of apoptosis related gene Bcl-2 and Bax were determined by RT-PCR.5 To observe the change of P-cell with electron microscope. RESTULTS:1 No significant difference was found among the weight of pancreas in different group as well as the ratio of pancreas/body weight among different groups (p>0.05). The ratio of pancreas/body weight was significantly higher than that of the other two groups (p<0.01).2 Hematoxylin and eosin staining disclosed severe fibrosis in pancreatic islet of Group Control rats in 32 weeks of age and it is severer in 40 weeks of age which featured abnormal round appearance and disorganized islet with atrophy islet observed. There were relatively normal round islets in rats of Group Liuwei.3 P-cell apoptosis in Group Control were significantly higher than that of Group LETO in different ages (p<0.01). The percentage of islet P-cell apoptosis at 32 or 40 weeks in Group Control was significantly higher than that of 8 weeks of age. The percentage of apoptosis was lower in Group Liuwei than Group Control (p<0.01), but higher than Group LETO (p<0.01).4 The expression of protein Bcl-2 are higher in Group Liuwei as well as the expression of Bcl-2 mRNA (p<0.05). The expression level of protein and mRNA of Bax are significantly lower than Group Liuwei than Group Control.5 The apoptotic cell was observed with electron microscope. Organelle became swelling in Group Liuwei.CONCLUSIONS:1 Liuwei Dihuang Pills can help keep the normal structure of pancreatic islet in OLETF rats.2 Liuwei Dihuang Pills decrease apoptosis level of P-cell in OLETF rats.3 Liuwei Dihuang Pills can change the expression level of apoptosis related gene in protein and mRNA, up-regulate the expression of anti-apoptosis gene and down-regulate the pro-apoptosis gene.Part 3 Effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats OBJECTIVE:To study the effects of Liuwei Dihuang Pills on insulin resistance in OLETF rats, and observe the influence on plasma adiponectin level and the expression level of adiponectin mRNA in adipose tissue of OLETF rats. Other indexes related to insulin sensitivity were measured to identify the effects of Liuwei Dihuang Pills and to provide beneficial evidence of Liuwei Dihuang Pills on prevention of diabetes mellitus.METHODS:1 Rats were sacrificed at 8^ 32 and 40 weeks old of age. Fasting blood glucose, serum triglyceride, serum cholesterol, serum FFA, plasma insulin and plasma adiponectin concentration were determined.2 Visceral fat weight was calculated by the weight of greater omentunu epididymidis fatand perirenal fat.3 The adiponectin mRNA expression level in adipose tissue was tested by RT-PCR.4 Insulin sensitivity index (ISI) was calculated according to the fasting blood glucose and fasting plasma insulin concentration.5 The correlation between plasma adiponectin level and other related indexes were analyzed. RESULTS:1 Compared with Group LETO, rats of Group Control had significantly higher body weight, visceral fat weight, fasting plasma insulin concentration, serum lipid and lower ISI, but the FBG had not obvious change.2 Rats of Group Control had significantly lower serum adiponectin and the expression level of adiponectin mRNA in adipose tissue decreased compared with rats of Group LETO.3 Compared with Group LETO, Group Liuwei had lower plasma insulin concentration, serum lipid and higher ISI, but the FBG had not obvious change.4 The plasma adiponectin concentration and the expression level of adiponectin mRNA in adipose tissue in Group Liuwei was obvious higher than those of Group Control.5 There was a positive correlation between plasma adiponectin concentration and ISI, while there was a negative correlation between plasma adiponectin level and body weight, visceral fat weight, fasting plasma insulin, serum TG, FFA, CHOL.CONCLUSIONS:1 Plasma adiponectin level is positively correlated to insulin sensitivity in OLETF rats.2 The level of adiponectin is negatively correlated to visceral fat weight, fasting plasma insulin, serum FFA and triglyceride.3 Liuwei Dihuang Pills can increase the plasma level of adiponectin and the expression level of adiponectin mRNA in adipose tissue.4 Liuwei Dihuang Pills can decrease hyperlipemia and improve insulin resistance.