Study On Effect And Mechanism Of Xiao Yao Wan On Impaired Fasting Glucose Induced By A High-Fat Diet In Rats

BackgroundImpaired glucose regulation (IGR), also known as pre-diabetes, refers to elevated blood sugar which have not yet reached the diagnostic criteria for diabetes, including impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). In recent years, the incidence of diabetes and pre-diabetes is increasing year by year. The related epidemiological study showed that the age-standardized prevalences of total diabetes (which included both previously diagnosed diabetes and previously undiagnosed diabetes) and prediabetes were9.7%and15.5%, accounting for92.4million adults with diabetes and148.2million adults with prediabetes in china. The natural history of both IFG and IGT is variable, with25%progressing to diabetes,50%remaining in their abnormal glycemic state, and25%reverting to normal glucose tolerance (NGT)over an observational period of3-5years. It has been a consensus that diabetes is one of high-risk factors of cardiovascular and cerebrovascular events and the importance of IGR resulting in macrovascular disease is a growing concern. IFG and IGT, less prone to diabetic microangiopathy, can increase the risk of large vascular events. It is of great significance for treatment of IGR to prevent metabolic diseases such as diabetes, metabolic syndrome, cardiovascular disease, etc.It has been recognized that high fat diet is one of important environmental factors of obesity, type2diabetes and metabolic syndrome. Fat diet can lead to abnormal lipid metabolism, which have toxic effects on cells. The level of plasma free fatty acids (FFAs) or intracellular fat content increases more than the storage capacity and oxidation of FFAs of adipose tissue. Excessive FFAs deposite in the target organ of insulin action such as muscle and liver by the form of triglyceride (TG), which cause insulin resistance. While the deposition of excessive FFAs in the pancreatic islet can result in pancreatic βcell dysfunction. Insulin resistance and pancreatic β cell dysfunction ultimately can cause impaired fasting glucose, impaired glucose tolerance, diabetes and metabolic syndrome. In addition, adipocytokines secreted by fat cells, such as tumor necrosis factor a (TNF-a), adiponectin and leptin are often involved in insulin resistance, which regulate glucose metabolism and body energy balance by autocrine endocrine and neural pathways.The main drugs for IGR are biguanides, glucosidase inhibitors and insulin sensitizing agents. Because these drugs can cause edema, liver and kidney damage, gastrointestinal reactions and other adverse reactions, the application is subject to certain restrictions. Traditional Chinese medicine has certain advantages in improving impaired glucose regulation with characteristics of multi-target and relative safety. Traditional Chinese medicine believes that impaired glucose regulation is caused by improper diet, malaise and lack of exercise, which can lead to the disfunction of spleen and liver. The pathogenesis is characterized by spleen deficiency and Liver-Qi stagnation. Xiao Yao Wan comes from Xiao Yao San, which was published on " Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy", consisting of bupleurum, peony root, atractylodes, tuckahoe, angelica, licorice, ginger, mint Bawei drugs. Modern pharmacological studies indicate that Xiao Yao Wan has protective effect of liver and stomach and play a critical role in antidepressant and improvement of hyperlipidemia. Xiao Yao Wan is widely used in Hepatology, Gastroenterology, neurosis and other diseases, while it is rarely applied in impaired glucose regulation, diabetes and metabolic syndrome. We established a rat model of impaired glucose regulation induced by a high-fat diet. The present study was undertaken to investigate the preventive effects of Xiao Yao Wan on impaired fasting glucose, impaired glucose tolerance, providing laboratory evidence for the expansion of its clinical application.ObjectiveThe present study was undertaken to investigate the preventive effects and mechanism of Xiao Yao Wan on impaired fasting glucose, impaired glucose tolerance induced by a high-fat diet in rats, providing laboratory evidence for the expansion of its clinical application.Methods40male Wistar rats, weighting200～250g, were randomly divided into four groups (n=10):normal diet group, high fat diet group, high fat diet with Xue Zhi Kang (positive control) group, high fat diet with Xiao Yao Wan group. Normal diet group was fed with basal diet (energy ratio:10%fat,67%carbohydrates, protein23%), other groups were fed with high fat diet (energy:60%fat,20%carbohydrate,20%protein,5.24kal/g). Xue Zhi Kang was orally administrated at a dose of300mgKg-1day-1for8weeks. Xiao Yao Wan was orally administrated at a dose of SgKg-1day-1for8weeks. The rats in normal diet group and high fat diet group received equivalent volume of vehicle(saline). The body weight was measured every week. At4and8weeks, fasting blood glucose (FBG) was measured. At the end of the8-week treatment period, fasting serum insulin (FINS) were measured by enzyme-linked immunosorbent assay (ELISA), insulin resistance index (HOMA-IR) was calculated. Insulin tolerance test (ITT) was used to assess insulin sensitivity. Oral glucose tolerance (OGTT) test was performed. Serum total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) were measured. At the end of the experiment, a subset of rats from each treatment group was randomly distributed into2further groups. One group was injected with insulin(5IU/kg)15minutes before sacrifice, the other was not treated with insulin. Liver was quickly excised, immediately frozen in liquid nitrogen, and stored at-80℃for further analysis. The perirenal fat and epididymal fat pad were weighted and the relative perirenal fat weight (perirenal fat weight/body weight×100%) and relative epididymal fat weight (epididymal fat weight/body weight×100%) were calculated. Expression of p-Aktser473in liver of the group which had been injected with insulin was measured by Western blot.Statistical analysis:SPSS13.0software was used for Statistical analysis.Data were presented as means±SD. Statistical analysis was performed by one-way analysis of variance (ANOVA) or repeated measure ANOVA where appropriate.Differences between individual group means were analyzed by Fisher’s protected least squares difference test. A P value less than.05was considered to be statistically significant.Results1body weight and organ weightsBody weight of rats in high fat diet group increased more than normal group from2weeks to the end of experiment (P＜0.05). The relative perirenal fat weight and relative epididymal fat weight in high fat diet gruoup were significantly higher than normal group (P＜0.05). Xue Zhi Kang and Xiao Yao Wan had a tendency to slightly reduce body weight as compared with high fat diet group, but this effect failed to reach statistical significance(P=0.384;P:=0.067).The increased adiposity was prevented by Xiao Yao Wan but not by Xue Zhi Kang in HFHS-fed rats.2Fasting glucose, fasting insulin and HOMA-IRThe high fat diet significantly increased fasting glucose and fasting insulin (P <0.05). As a result of increased serum glucose and insulin levels, homeostatic model assessment values for insulin resistance (HOMA-IR), of high fat diet group was higher than that of normal diet group (P＜0.05). Xiao Yao Wan administered rats showed significant decrease in fasting glucose and fasting insulin relative to those in high fat diet group (P≮0.05). The insulin resistance indices of Xiao Yao Wan group was significantly reduced compared with the high fat diet group (P＜0.001). Xue Zhi Kang had a tendency to slightly reduce fasting glucose, fasting insulin and HOMA-IR as compared with high fat diet group, but this effect failed to reach statistical significance (P=0.901;P=0.106;P=0.209;P=0.080).3Insulin tolerance test(ITT)Insulin was less effective in lowering glucose level in the high fat diet rats compared with those in normal diet group (P＜0.05), while Xiao Yao Wan administration significantly enhanced insulin-mediated glucose-lowering, especially at90min and120min time points (P＜0.05). When the area under the curve (AUC) was compared between groups, Xiao Yao Wan administered groups showed significant reduction in the AUC compared with the high fat diet group (P＜0.05), while there was no significant difference for the AUC between Xue Zhi Kang-treated group and high fat diet group (P=0.310).4Oral glucose tolerance test (OGTT)Glucose challenge dramatically increased the blood glucose levels in high fat diet group compared with those in normal diet group, while Xiao Yao Wan and Xue Zhi Kang administration failed to prevent the blood glucose levels from rising. When the area under the curve (AUC) was compared between groups, Xiao Yao Wan and Xue Zhi Kang administered groups showed no effect on the AUC compared with the high fat diet group(P=0.538;P=0.565).5Serum lipid levelsIn high fat diet group, serum TC, TG, and LDL-C was significantly increased Compared with normal diet group(P<0.05), and serum HDL-C were significantly lower than normal diet group (P＜0.05). Xiao Yao Wan and Xue Zhi Kang administration rats showed significant decrease in serum TC and TG. Xue Zhi Kang can reduce serum LD-C, which Xiao Yao Wan failed to affect. Xiao Yao Wan and Xue Zhi Kang administered groups showed no effect on serum HDL-C (P=0.831;P=0.442). 6Effect of Xiao Yao Wan on Aktser473phosphorylationInsulin-induced Aktser473phosphorylation, which was decreased in high fat diet group, showed a significant improvement after treatment with Xiao Yao Wan (P＜0.05). However, Xue zhi kang administered groups showed no significant effect on Insulin-induced Aktser473phosphorylation in the liver (P=0.063)Conclusion1. A rat model of impaired glucose regulation induced by a high-fat diet for8weeks was successfully established, which is similar to human impaired glucose regulation caused by change of life style, indicating that life style is closely related to impaired glucose regulation.2. Xiao Yao Wan can improve obesity induced by a high-fat diet, regulate lipid metabolism, reduce the accumulation of abdominal fat and fasting insulin, enhance insulin-mediated glucose-lowering, showing significant improvement in impaired fasting glucose induced by a high-fat diet in rats. Xiao Yao Wan can enhance Insulin-induced Akt ser473phosphorylation in the liver, which may be one of the molecular mechanisms of Xiao Yao Wan improving impaired fasting glucose.3. Xiao Yao Wan and Xue Zhi Kang failed to improve impaired glucose tolerance induced by a high-fat diet in rats, which maybe due to the difference of pathogenesis between IFG and IGT, the effect of high-fat diet and the dose of Xiao Yao Wan.