| BackgroundDiabetes mellitus (DM) is a multi-factorial metabolic disorder characterized by chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion and/or insulin action. Far from now, there is not an effective method for diabetes control. Traditional Chinese medicine (TCM) has played a significant role in treatment of diabetes for centuries. It acts on reducing blood glucose, as well as adjusting the balance between Yin and Yang, so it can also prevent and control the chronic complications besides DM. Based on the ample clinical experience and comprehensive understanding of TCM and western medicine theories, we proposed the novel theory based on zang-fu differentiation for treatment of DM. The root of the theory is diabetes should be treated based on regulating the function of liver, spleen and kidneys. And we also constructed a series of formula according to the above theory, of which Jiang Tang Xiao Ke Granule (JTXKG) was one of them. Function of JTXKG is to deal with the qi and yin deficiency of liver, spleen and kidney, as well as excessive heat and blood stasis. JTXKG has been proved to be effect and safe in clinic, so that it has been chosen as the important drugs for diabetes treatment for study by New Drug Development Program. This researches in this dissertation is part of the above program.Objectives:In the present study, we intended to study the pharmacological action of JTXKG in experimental diabetic rats. The observed indictors includes blood glucose, serum lipid profiles, serum glycosylated hemoglobin, fasting serum insulin, glucose tolerance and insulin tolerance, oxidative stress, liver and pancreatic pathological changes, and AMPK signal pathway. So that to investigate the hypoglycemic effect of JTXKG and the related mechanisms, and to provide experimental proofs for treatment of diabetes with TCM methods. Methods:50male Sprague Dawley rats, weighing180-200g, were numbered according to the body weight.10of them were randomly selected as the normal control rats, which were fed with normal diet. The other40rats were fed with high fat diet for4weeks, and then a single intraperitoneal injection of a prepared solution of STZ (30mg/kg suspended in0.1mol/L citrate buffer at pH4.5) was applied to induce diabetic models. If volume of fasting blood glucose (FBG) was not less than16.7mmol/L after72hours of STZ injection, the diabetic models were successful. The32diabetic rats were randomly divided into three groups.(1) drug-untreated diabetic rats;(2) and (3) diabetic rats treated with pioglitazone1.5mg/kg and JTXKG9g/kg, respectively. Both pioglitazone and JTXKG were dissolved in distilled water and given to the rats via gastrogavage once a day. The normal and drug-untreated DM rats were administrated with the same volume of vehicle. Drug intervention lasted for4weeks.The body weight of the rats was record weekly. The fasting blood glucose (FBG) and the random blood glucose (RBG) before and after the treatment were detected. The oral glucose tolerance test (OGTT) and insulin tolerence test (ITT) were performed at the end of the study. We also measured the serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Cr), urea nitrogen (BUN), fasting insulin (FINS), superoxide dismutase (SOD), malondialdehyde (MDA) and nitric oxide (NO) and calculated the insulin sensitivity index (ISI). The pancreas and liver were removed and fixed for pathological observation.The mRNA levels of AMPKa, GLUT4and IRS-1and the protein expression levels of AMPKa, p-AMPKa, GLUT4and IRS-1in muscle tissues were detected. The mRNA levels of AMPKa and the protein expression levels of AMPKa, p-AMPKa, p-ACC in liver tissues were determined. The mRNA of leptin in adipose tissue was also measured.Results:Improvement of the symptoms:The typical symptoms of DM, such as polydipsia, polyphagia, polyuria, and emaciation of rats were relieved by JTXKG. Indicators of glucose metabolism:JTXKG reduced the FBG and RBG levels of the diabetic rats (P<0.05), and effect on reducing FBS was dose-dependent. The HbAlc and the serum insulin levels of the diabetic rats were not improved by JTXKG (P>0.05). JTXKG increased the ISI (P<0.05), and improved the glucose tolerance and the insulin tolerance of the diabetic rats.Indicators of lipid metabolism:Compared with the model group, serum TC, TG, LDL-C levels of the rats in JTXKG group decreased by33%,57%and44%, respectively, and the serum HDL level increased by69%. And the difference between groups was significant (P0.05).Liver and kidney function:JTXKG reduced the ALT level by50%(P<0.05), but it did not change the AST, BUN and Cr levels (P>0.05).Pathological changes:JTXKG protected the pathological changes of the pancreatic and liver tissue of the diabetic rats.Oxidative stress indicators:Compared with the model group, JTXKG increased the SOD activity by60%in diabetic rats, and it lowered serum MDA and NO levels by34%and52%, respectively. The difference between the groups was statistical significance (P<0.05).Influence on AMPK signal pathway:The AMPK-a mRNA and protein expression in liver and skeletal muscle of the diabetic rats were lower than the normal rats. However, JTXKG increased the AMPK-a mRNA level, as well as the AMPKa and p-AMPKa protein expression in liver and muscle tissues. JTXKG up-regulated the mRNA level of GLUT4and IRS-1in the muscular tissues of diabetic rats. It enhanced the protein expression of GLUT4and IRS-1in the muscular tissues of diabetic rats. JTXKG significantly increased p-ACC protein expression in the liver tissue of diabetic rats. The mRNA expression of leptin in adipose tissue of diabetic rats was also increased by JTXKG.Conclusion:1JTXKG can reduce the blood glucose levels, regulate glucose metabolism, improve the insulin sensitivity, and maintain glucose homeostasis of the diabetic rats.2JTXKG can decrease serum lipid profiles and regulate lipid metabolism of the diabetic 3JTXKG can improve the ability of anti-oxidative stress, adjust the oxidative stress state of the diabetic rats.4JTXKG can regulate the AMPK-GLUT4-ISR-1signal pathway, so that it relieved insulin resistance in the muscle tissue of the diabetic rats, which may be one of the mechanism of JTXKG treating diabetes.5JTXKG can regulate the AMPK-ACC signal pathway in the liver, through which it can promote fatty acid oxidation and regulate lipid metabolism. This may be another mechanism of JTXKG on diabetes.6Theory of controlling DM by regulating the function of liver, spleen and kidney together is not only theoretically scientific but also supported by experimental data.The innovation of the study is to research on the pharmacological and anti-oxidative effect of JTXKG on diabetic rats, which was induced by high fat diet combined with streptozotozin. The constructed principal of JTXKG, which is controlling DM by regulating the function of liver, spleen and kidney together, was innovated. And we further studied the influence of JTXKG on AMPK signal pathway, which is one of the most important pathway in glucose and lipid metabolism, so as to reveal the possible working mechanisms of JTXKG and provide the experimental data proof for the novel theory in DM treatment. |
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