| Objective:To study the effects of berberine on type 2 diabetic rats and expound the treatment of berberine on type 2 diabetic rats; observed the results of berberine on the heart and kidneys of type 2 diabetic rats, to understand the effects of berberine on complications of type 2 diabetic rats; Analysis the expression of PI3-K and GLUT4 in skeletal muscle and liver, to discussion the possible mechanism of berberine on the type 2 diabetes rats.Materials and Methods:Part I:Effect of berberine on glucose and lipid metabolism in type 2 diabetic rats. After 3 days adaptation fed,90 clean male (SD) rats were randomly divided into normal diet group (CON),15 rats; high fat/high carbohydrate diet group (HFS),75 rats. The rats were given the corre-sponding diet and weigh the body weight of rats weekly to observe the effects of HFS on the increase of body weight. After 8 weeks, the group fed by high fat/high sugar diet was further divided into 2 groups randomly: one group, of 15 rats, continue to fed by high fat/high carbohydrate diet (HFS); another group, of 60 rats, in fasting conditions, intravenou injection of freshly prepared and pre-cooling of the STZ 25mg·kg-1(pH =4.5). after injection, rats were free to eat and drink water. After 72h, the non-fasting blood glucose concentration was higher than 11. lmmol·L-1 were seen as successful induction of type 2 diabetes, then randomly divided into 4 groups and were given different treatment factors:diabetes mellitus model group (T2DM group),2ml 0.9% NaCl·d-1 gavage; berberine group (BBR group), 100mg kg-1·d-1 BBR gavage; Metformin hydrochloride group (MET group),50 mg·kg-1·d-1MET gavage; The Integrative Medicine group (B+M group), 100mg·kg-1·d-1BBR+50 mg·kg-1·d-1MET gavage (30'interval);The CON group and the HFS group, in fasting conditions,give same dose of intravenous injection of 0.1 mmol·L-1 citrate buffer solution (pH=4.5), and then give 2ml 0.9% NaCl·d-1 gavage respectively.after modeling, on 3d, 4w,8w and 12w, examine venous blood glucose, blood lipids (TC, TG) and insulin concentrations; observe the general status of the rats; record the changes of body weight and water intake. PartⅡ:BBR on type 2 diabetic renal pathological changes. In addition to no HFS group, the other condi-tions, including animal raise, modeling, group and treatment are the same as the first part, respectively. On 4w, 8w and 12w, after modeling, draw the materials from after general anesthesia. Morphological changes in rat kidney were observed in optical microscope by HE staining; GLUT4 and PI3-Kp110 were examed by immunohistochemical staining; 2 alco-hol-based substance were detect by periodic acid-Schiff reaction (PAS); The ultra-structural changes of rat kidney were observed by electron microscope; tubular density, tubular and glomerular capsule basement membrane thick-ness were surveyed by stereological analysis. PartⅢ:Effects of berbe-rine on myocardial of type 2 diabetic rats. Animal husbandry, modeling, group, processing method and sampling time with the second part. Using HE staining to observed cardiac morphology in optical microscope; Using immu-nohistochemical staining to exame the expression of GLUT4 and PI3-Kp110 subunit; Using periodic acid-Schiff reaction to detect 2 alcohol-based substances in myocardium of each group. PartⅣ:The study of the mechanism of BBR treatment on type 2 diabetic rats. Animal husbandry, modeling, group, processing method and sampling time with the second part. Using immunohi-stochemical staining to survey PI3-Kp110 subunit and GLUT4 in skeletal muscle, heart and liver expression; Using western blotting method to detect PI3-K and GLUT4 in skeletal muscle and liver.Results:1. The effect of berberine on glucose and lipid metabolism in type 2 diabetic rats. 1.1 The chenge of blood glucose:Compared to the same period of T2DM rats, the blood glucose levels of BBR rats were significantly reduced (P<0.01). In 4w, blood glucose decrease 25%,8w decrease 43% and 12w decrease 36%. The most obvious effect occured in 4w and between 8w-12w, blood glucose fluctuations in the interval of 1Ommol·L-1～12 mmol·L-1 level. BBR is of a significant hypoglycemic effect. In 4w, the blood glucose values of the rats intervened by MET and B+M are less than that of BBR group (P<0.05) and after this period, the blood glucose values of the rats intervened by MET and B+M decreased slowly. In 12w, the blood glucose values of the rats intervened by BBR less than that of the rats intervened by MET and B+M, but there is no significant difference between them (P>0.05).1.2 Changes of body weight:After high fat-high sugar feeding, the body weight increased significantly faster than that of the normal diet group (P<0.01). After modeling, the weight of T2DM group began to loss weight and in 4w, the body weight was significantly lower than that of the other 3 group (P<0.01). The body weight of BBR group, MET group and B+M group are decreased to some extent varying degrees but no significant difference between them (P> 0.05).1.3 Changes of blood lipids:After modeling, blood fat are increased gradually. In 12w, the value of glycerin trilaurate is nearly 2.5 times as the initial value. The value of glycerin trilaurate of 3 treatment group is increased slowlly and in 12w, the value of glycerin trilaurate is nearly 1.7 times as the initial value. There is no significant difference between them (P> 0.05). The total cholesterol concentration of T2DM group was rising rapidly. In 12w, the total cholesterol concentration was 3 times as the initial value. Serum total cholesterol concentration of 3 treatment group is increased significantly slow and in 12w, the total cholesterol concentration was 1.3 times as the initial value and there is no stati-stically significant (P>0.05).1.4 changes of insulin:serum insulin of HFS group increased slowly and in 12 w, it increased by 30% compared to the initial value and that of CON group stable. After modeling, the changes of serum insulin was signifi-cantly (P<0.01), fell by an average 60%. After treatment, despite the rise in insulin levels, but not statistically significant(P>0.05).1.5 Insulin sensitivity index:insulin sensitivity index of BBR, MET, and B+M group are increased gradually and in 8w, there is statistically signi-ficant (P>0.05) between 3treatment group and T2DM group respectively, but there is no statistically significant among 3 treatment group (P>0.05). Insulin sensitivity index of HFS group decreased slowly, while, CON group 's maintain stable and in 12w, there is statistically significant between the two group's (P<0.05).2. Effects of berberine on kidney of type 2 diabetic rats.2.1 Results of HE staining:Glomerular basement membrane, tubular basement membrane of T2DM group thickening in varying degrees, distaltubular epi-thelial cell swelling significantly, local cytoplasm showed vacuolar degeneration, nuclear dissolution disappeared (oncosis); Tubular base-ment membrane of BBR group thicker then that of CON group and there are no obvious changement in proximal tubule and distal tubule cells.2.2 PAS staining:Compared with CON, BBR, MET, B+M, and T2DM group were positive, including glomerular basement membrane and tubular basement membrane stained to rose pink. In proximal tubule epithelial cells of T2DM group can be seen abundant huge rose pink colour glycogen granules red roses, distal convoluted tubular epithelial cell swelling significantly and local cytoplasm showed vacuolar degeneration;there are no significant change in proximal tubule and distal tubule epithelial cells of BBR group. Stereological measurements show that the density of tubular of T2DM group is significantly higher than that of other 4 groups, renal tubular and glomerular capsule basement membrane thickening to certain extent. The results show that BBR has a protective effect on kidney.3. Effects of berberine on myocardial of type 2 diabetic rats3.1 Results of HE staining:In CON group, myocardial fibers arranged or-derly, myocardial horizontal grain be seen clearly and nucleus with normal morpho-logy; In T2DM group, myocardial fibers arranged disorderly, with varying degrees of hypertrophy, myocardial horizontal grain be seen punch-drunk, nuclear lightly stained and the gap was widened; BBR group's has no significant change.3.2 PAS staining:Myocardial fibers of CON group arranged orderly and myocardial cells be seen a small amount of red particles (+);Myocardial fibers of T2DM group displaied hypertrophy, arranged disorderly, the gap was widened, the cytoplasm dyed purple and red particles very dense (++++);Myocardial fibers of BBR group arranged orderly, no hypertrophy, cytoplasmic dyed dark red and red particles denser (+++). It hints that BBR with the competence of lowering Carbohydrate material accumulation and improve pathological changes of myocardial.4. The mechanism probe of BBR on the type 2 diabetes of rats4.1 Immunohistochemistry showed that GLUT4 was expressed, to some extent, in the rat renal proximal tubule, distal tubule and mesangial; in glo-meruli, glomerular capsule was negative expression and weak expression in the collection tube. GLUT4 expression of BBR group higher than that of T2DM group and lower than that of CON group and is of statistically significant (P<0.05).4.2 The expression of PI3-K protein gradually increased with the treatment time in skeletal muscle of BBR group, there is significantly different (P<0.01) compared with the T2DM group's; The expression of PI3-K protein of MET group did not change significantly. 4.3 Western blot detection of GLUT4 protein expression:With the extension of course, GLUT4 expression in skeletal muscle of T2DM group gradually decreased and is of a significant difference between different time points 's (P<0.01); There were significant differences (P<0.05)between GLUT4 protein expression of BBR group, T2DM group's and CON group's respe-ctivly, GLUT4 group's higher than that of T2DM group but lower than that of CON group in the same period. It tip that BBR hypoglycemic effect may be due to enhanced GLUT4 protein expression. Conclusion:1. BBR can significantly reduce blood glucose and blood lipids in diabetic rats and can enhance sensitivity of insulin sensitivity in the target tissue.2. BBR can slow down the process of pathological changes in kidney of type 2 diabetes and can reduce the deposition of carbohydrate in glomerular, tubular basement membrane and swelling tubular epithelial cells.3. BBR has a protective effect on myocardium and can reduce the carbohydrate deposition and fibrous connective tissue proliferation.4. The effect of BBR lower blood glucose and lipids is quite the same as MET and the role of treatement in pathological changes of the kidney and heart of diabetic changes is superior to MET.5. The effects of BBR on Type2 Diabetes Mellitus rats may be due to it 's enhance the expression level of PI3-K and GLUT4 in the target tissue.
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