| Part One Literature ResearchNow a days, diabetes mellitus (DM) incidence increasing year by year, has brought the world a siren, a serious threat to human health. As health promotion of human longevity and affluent lifestyle becomes popular, the prevalence of diabetes in the past 10 years in the form of double increase, the next 20 years is estimated that nearly 600 million worldwide with diabetes. And now, for type 2 diabetes mellitus (T2DM) pathogenesis has not been fully analyzed, its main focus on the pathogenesis is around insulin resistance and generating a plurality of signal transduction abnormalities when insulin binding after with the receptor. Thus, focused on solving the abnormality of type 2 diabetes of insulin signal transduction pathways, improving insulin resistance, is the key to find a new way out in type 2 diabetes treatment of the development history. More and more studies show that Chinese medicine in the prevention and treatment of type 2 diabetes and its complications is on a clear distinct advantage. TCM has documented a long history of Diabetes named Xiaoke disease, and also Xiaodan, Zhongxiao, Gexiao and etc, past famous physicians and later scholars has accumulated a wealth of clinical experience on the Diabetes research, lots of experimental studies, and full access to research results. Jiangtangsanhuang tablet is invented by famous professor Xiongmanqi, according to the summary of experience from the Shanghanlan, the formula has been used and studied over twenty years, is widely used in clinical of our hospital and has definite effect to qi and yin deficiency, blood stasis and heat accumulation in Diabetes. In this literature and experimental study are meant to observe the holistic theory from macro to micro in diabetes influenced by Jiangtangsanhuang tablet, to further proof its mechanism and therapeutic effect.Part Two Experimental researchObjective:Using STZ diabetic rats to observed modeling process and analyzed syndromes of Qi, Xue, Yin and Yang, and the impact of serum GLP-1 and FFA influence by Jiangtangsanhuang tablet in diabetic rats, and the protein expression effect of GLP-1 and PDX-1 in large intestine and pancreas, as well as the ultrastructural morphological changes in the pancreas of diabetic rats, and to describes the mechanism of Qi-Yin Deficiency therapy of Jiangtangsanhuang tablet in diabetes, providing new evidence for enhancing islet function.Methods:The 82 male SPF SD rats were fed a normal diet for 5 days feeding adaptation, and then randomly divided into two groups, normal group(n=10) and continuing normal diet feeding; model group(n=72) with a high sucrose high fat diet(HSFD) feeding a month later, according to 35mg/kg given by intraperitoneal injection of streptozocin(STZ), normal group were injected with the same amount of citric acid buffer. After 72 hours, rats with fasting blood glucose≥16.7mmol/1, randomly divided into five groups: model group(n=13, dose:equal amount of drinking water), Jiangtangsanhuang tablet high-dose group (n=12, dose:1575mg/kg/d), Jiangtangsanhuang tablet middle-dose group (n=12, dose:1181.25mg/kg/d), Jiangtangsanhuang tablet low-dose group (n=13, dose:787.5mg/kg/d) and Jienuo Wei group (n=13, dose:10.5mg/kg/d), the observation time is six weeks. Weekly observations of body weight and fasting blood glucose; measuring water and food intake, excretion; quantitative analysis RGB values of tongue, claws and tail; measuring grasping-force and open-field test twice a week; after six weeks, weighing organs of heart, liver, spleen, kidney and pancreas to calculating the organ index. The common syndromes standard value of DM rats as follows:qi deficiency degree= actual value of level grille numbers per rat/average level grille numbers of normal group X 0.3+value of vertical stand numbers per rat/average vertical stand numbers of normal group X 0.2+value of grasping-force per rat/ average grasping-force of normal group X 0.5; Diagnostic syndromes standard:Compared with the normal group,> 1.25 for qi exuberant,<0.75 for qi deficiency,<0.5 for severe qi deficiency,<0.25 for qi exhaustion. Evaluation quantitative index of the heat excess or deficiency usually use tongue R value (reflecting the degree of red), claw R value, tail R value as a regular quantitative index, each constituent accounted for 30%, 50%,20%, respectively. Yang-qi degree:value of tongue R index per rat/ average tongue R index of normal group X 0.3+value of claw R index per rat/average claw R index of normal group X 0.5+value of tail R index per rat/average tail R index of normal group X 0.2; Diagnostic syndromes standard:Compared with the normal group,) 1.02 for excessive heat,<0.95 for yang deficiency,<0.90 for yang exhaustion,<0.80 for yang collapse. Yin deficiency degree= value of body weight per rat/ average body weight of normal group; Diagnostic syndromes standard: Compared with normal group,>1.2 for plump body,<0.9 for yin deficiency, <0.8 for yin exhaustion,<0.7 for yin collapse. Stomach heat degree= value of water intake per rat/average water intake of normal group X 0.5+value of food intake per rat/average food intake of normal group X 0.5; Diagnostic syndromes standard:Compared with normal group,> 2 for stomach heat,> 3 for stomach fire,> 4 for excessive stomach fire. Using an enzyme-linked immunosorbent assay (ELISA) to detect serum GLP-1 and FFA content; Using Western-blot test to detect the protein expression of GLP-1 and PDX-1 in large intestine and pancreatic tissue; Using transmission electron microscopy (TEM) to observe the ultrastructural morphological changes of pancreas tissue in diabetic rats.Results:First, based on the above safety evaluation, the food intake, water intake, body weight, defecation, urine, fasting blood glucose and etc, it can be concluded:1. Using the STZ diabetic rat model is more suitable for clinical type 2 diabetes research. In food intake, water intake, defecation, fasting blood glucose and body weight, compared with the normal, the model group experienced six weeks are not significantly improved, the difference was significant (P<0.01). Animal model appeared "three" typical symptoms, FPG also maintained a high level, but not obvious reduction in weight.2. Chinese medicine of high, medium and low dose group and western medicine group of food intake, water intake, defecation and FPG value both slowed down compared with the previous, but have not returned to normal levels, which the Chinese low-dose group and western medicine group having the most obvious change(P<0.01). The low-dose group was clinically normal dose, medium-dose group was 1.5 times the clinical dose, high-dose was 2 times the clinical dose group, all observed indicators show that the best effect of low-dose group, indicating the presence of significant dose-effect relationship between traditional Chinese medicine, and the current clinical usage should be the best dose points.3. The side effects of traditional Chinese medicine group is relatively small, western group of hypoglycemic effect is good, but in the third week treatment there will be ups and downs, and in terms of weight gain, the effect of the chinese medicine group are better than the Western medicine group, chinese medicine high, medium and low dose group grew more obviously, but the weight gain of Western medicine group is not obvious (P<0.05).Second, the model rats from just modeling to the 2nd weeks of modeling, mainly shows the qi, yang, yin deficiency, from 2nd weeks to 6th weeks, syndromes basically stable, show that qi and yin deficiency with excessive stomach heat; treatment of the qi, yin deficiency and stomach heat, the effect of Chinese Medicine low-dose group is better (P <0.01), the Western Medicine group efficacy was not obvious; in terms of heat excess or deficiency, each group did not change significantly.Third, the model group, serum FFA and GLP-1 content compared to the normal group, there are significant differences (P<0.01), after treatment, the difference in the low-dose group and the normal group were very close(P<0.05), low-dose group could significantly increase the content of GLP-1, reducing FFA content. The study also found that the large intestine PDX-1 group and GLP-1 group, pancreatic PDX-1 group and GLP-1 group, Jiangtangsanhuang tablet low-dose group in GLP-1 and PDX-1 have the best protein expression, and compared with the normal group, no significant difference, while high-dose group was the weakest performance.Fourth, the pancreatic islet cells in normal rats can seen many endocrine granules in the pancreatic endocrine cell cytoplasm, in cell cytoplasm can seen mitochondrial, mitochondrial vacuolar degeneration has not occurred, rough endoplasmic reticulum also pathological changes did not occur, the oval shape of the nucleus, the nucleus Visible euchromatin and heterochromatin, often more chromatin. But the model group can see the islet endocrine cells, significantly reducing slurry particles in the cytoplasm of mitochondrial swelling significantly, disappear within mitochondria, rough endoplasmic reticulum also undergone significant swelling, clearly visible nucleus, the nucleus of irregular shape, nuclear more often stained cells can be seen around a small number of red blood cells, vascular congestion significantly. Through therapy, you can find three groups of traditional Chinese medicine and western medicine group can make a lot of secretory granules in the cytoplasm of pancreatic endocrine cells, so that the edge of the nucleus is clearly visible, nearly circular shape, the nucleus was visible lump of heterochromatin and light euchromatin dyed, cytoplasmic organelles within the number of rough endoplasmic reticulum and free ribosomes also increased. TCM three groups of low-dose group the best, but compared with the western group, in part to improve the cytoplasm of mitochondrial swelling, the effect of traditional Chinese medicine group better.Conclusion:1.A comprehensive food intake in rats, water intake, body weight, defecation, urine output, and so on all aspects of fasting glucose safety evaluation, using STZ diabetic rat model more suitable for simulation of clinical type 2 diabetes.2. The hypoglycemic effect despite western group of the best, but there are some side effects of treatment, the efficacy of the best low-dose group, and the relatively small group of the side effects of traditional Chinese medicine as a whole group.3. Jiangtangsanhuang tablet on STZ diabetic rats after 2 weeks of modeling, the effect is more obvious, the whole side Qi and activating blood circulation and use, increase fluid and diarrhea hereinafter Shi Yin of the model are in line with the two diabetic rats virtual, basic pathogenesis stomach.4. Jiangtangsanhuang tablet can increase the content of GLP-1 in serum, decreased serum FFA content, pancreas and intestine increase the expression of PDX-1 and GLP-1, and increased insulin secretion, restoring or improving 0-cell function and accelerate glucose oxidation, accelerate glucose into the cell, promote muscle glycogen synthesis, inhibition of gluconeogenesis, so as to correct the lipid disorders.5. Traditional Chinese medicine has a good low-dose STZ injection against cell damage caused by 3, reducing its role in apoptosis or mortality, resulting in insulin/glucagon secretion is suppressed or serious imbalances reversed, thereby improving glucose and lipid metabolism serious disorders, slowed the development and progression of type 2 diabetes. |
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