葛根芩连汤对2型糖尿病大鼠的表征作用及其与GLP-1相关的生物学基础

发布时间：2018-03-12 15:43

本文选题：表征　切入点：葛根芩连汤　出处：《北京中医药大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的观察葛根芩连汤对2型糖尿病大鼠宏观表征、肛温、抓力及相关指标是否具有改善作用,探索葛根芩连汤对模型动物中医相关指标的作用及其与中医临床既往报道的一致性,为丰富中药药效评价体系、体现中医药治疗特色奠定基础；分析多项胰高血糖素样肽(GLP-1)相关指标,从GLP-1相关机制的角度探索葛根芩连汤对2型糖尿病的可能作用机制,为利用GLP-1相关机制降糖中药增添新的中药复方方法 1.高脂喂养8周后,腹腔注射30mg/kg链脲佐菌素(STZ)诱导构建2型糖尿病大鼠模型,造模成功后,按大鼠按体重和血糖进行随机分组,分为4组：2型糖尿病模型组、葛根芩连汤剂量1组(9.10g/kg, GGQL1组)、葛根芩连汤剂量2组(18.20g/kg, GGQL2组)、西格列汀组(0.01g/kg),另设正常对照组。 2.分组后对各组大鼠肛温、抓力、饮食量、胰岛素耐量、葡萄糖耐量进行检测,其后进行上述药物的干预,灌胃6周,期间,模型组及给药各组仍以高脂喂养,正常组普通饲料喂养。每天给药一次,进行一般情况观察。 3.对大鼠体重、饮食量、空腹血糖值、抓力、肛温进行动态检测；给药3周、6周,对各组大鼠进行胰岛素耐量、葡萄糖耐量检测；给药6周,对各组大鼠进行表征量表采集。 4.给药6周后,腹腔麻醉大鼠,腹主动脉取血并分离血清,应用终点法测定血清中TG和TC值；取大鼠胰腺、回肠,甲醛固定,备用。 5.取甲醛固定组织胰腺、回肠,采用石蜡包埋技术,制成石蜡切片备用；采用石蜡切片HE染色技术观察胰腺、回肠组织病理结构改变。 6.取大鼠胰腺石蜡包埋切片,应用免疫组织化学检测方法,检测各组大鼠胰岛内胰岛素的表达；采用TUNEL法检测胰岛细胞,观察胰岛B细胞凋亡情况。 7.取大鼠回肠石蜡包埋切片,应用免疫组织化学方法检测各组大鼠GLP-1、GIP的表达。结果 1.体重：模型组体重显著降低,GGQL1组体重值略有降低,GGQL2组基本保持稳定； 2.空腹血糖：GGQL1组、GGQL2组于给药4周、6周显著降低,血糖值低于模型组(P0.05)； 3.饮食量：GGQL1组给药后1-3周内的饮食量低于模型组(P0.05)；GGQL2组于给药1周后的饮食量均低于模型组(P0.05)； 4.表征观察：与模型组相比,GGQL1组大鼠耳血管可见度适中、耳浅粉色、尾淡白、大便黑黄(P0.01/6),GGQL2组大鼠舌体适中、耳血管可见度适中、大便黑黄(P0.01/6),活动度增加、舌润滑、耳浅粉色、鼻淡红、爪粉红不润、尾黄中有白(P0.05/6)； 5.抓力：GGQL1组抓力值升高,给药1周、4周抓力值高于模型组(P0.05),GGQL2组抓力值升高,给药后4周、5周抓力值高于模型组(P0.05),且于给药后5周高于GGQL1组； 6.肛温：GGQL1组、GGQL2组肛温值呈降低趋势,于给药5周时,GGQL1组肛温值低于模型组(P0.05)； 7.胰岛素耐量：给药各组其血糖下降速率较高,血糖变化AUC值明显低于模型组(P0.05),而与正常组无显著性差异； 8.葡萄糖耐量：给药各组血糖变化速率高于模型组,血糖变化AUC值高于模型组,其中GGQL2组明显高于模型组(P0.05)； 9. TG、TC:GGQL1组TG、TC值均低于模型组但差异无统计学意义GGQL2组TG值(P0.05)低于模型组,TC值差异无统计学差异； 10.HE染色：与模型组相比,GGQL1组、GGQL2组胰岛细胞均有所改善胰岛较有规则,皱缩胰岛少见,胰岛细胞中空泡变性细胞有所减少,胰岛细胞有所增加；回肠染色略有增加,由环状黏膜肌层、黏膜下层、肌层组成的环层有所增厚,环状较规则,皱襞分布紧密,内部空腔相对减小 11.免疫组化、凋亡结果：GGQL1组、GGQL2组胰腺内细胞凋亡率明显低于模型组(P0.05),胰岛素IOD/Area则高于模型组(P0.05),肠内GLP-1平均光密度值高于模型组(P0.05),GIP平均光密度值低于模型组(P0.05)。结论 1葛根芩连汤对2型糖尿病大鼠表征作用葛根芩连汤可维持2型糖尿病大鼠的体重,减轻体重减轻症状；降低2型糖尿病大鼠的空腹血糖值,减轻高血糖症；减少2型糖尿病大鼠的饮食量,减轻多食症状；增加2型糖尿病大鼠轻响抬头率,改善眼睛黯淡无神、唇少光泽不润、毛干燥、爪干涩不润,改善大鼠活动减少、舌少津液、抓取反抗、鼻少光泽不润,改善倦怠乏力、津液亏虚症状；增加2型糖尿病大鼠抓力,改善乏力症状；改善2型糖尿病病程中肛温升温阶段,改善烦热症状。由此可知,葛根芩连汤可以从不同程度改善2型糖尿病的症状。 2葛根芩连汤与GLP-1相关的生物学基础葛根芩连汤可改善胰岛素抵抗,增加2型糖尿病大鼠注射胰岛素30min后的血糖下降速率,增加2型糖尿病大鼠口服葡萄糖后血糖变化趋势；调节血脂,有降低TG、TC值的趋势作用；改善胰岛、回肠形态；增加胰岛内分泌胰岛素的胰岛B细胞面积和胰岛素分泌量；增加回肠内GLP-1水平、减少GIP水平；减少胰岛细胞内的细胞凋亡率。葛根芩连汤除增加GLP-1水平、减少GIP水平、调节血脂的作用外,其他作用均与GLP-1的生物学机制相一致,故而认为,葛根芩连汤可能通过GLP-1的相关机制起到治疗2型糖尿病的作用。
[Abstract]:objective
Observation of Gegenqinlian Decoction on rats with type 2 diabetes mellitus macro characterization, rectal temperature, grip and related indicators are improved, the role of Gegenqinlian Decoction on the related indexes of TCM animal model to explore the consistency and clinical reports, to enrich the efficacy of traditional Chinese medicine evaluation system, lay the foundation of TCM treatment characteristics a number of analysis; glucagon like peptide (GLP-1) related indicators, from the GLP-1 point of view to explore the related mechanism of Gegenqinlian Decoction on the possible mechanism of type 2 diabetes, add in medicine is a new mechanism of traditional Chinese medicine by GLP-1
Method
1. high fat diet for 8 weeks after intraperitoneal injection of 30mg/kg streptozotocin (STZ) induced rat model of type 2 diabetes, after the success of the model, according to the rats with the body weight and blood glucose were randomly divided into 4 groups: type 2 diabetes model group, Gegen Qinlian Decoction group (1 9.10g/kg GGQL1, group), Gegenqinlian Decoction group (2 18.20g/kg, GGQL2 group), Sig Leo Dean group (0.01g/kg), and normal control group.
2. groups of rats after rectal temperature, grip strength, diet, insulin tolerance, glucose tolerance test, followed by the drug intervention period by gavage for 6 weeks, the model group and the treatment groups with high fat diet, normal group fed with normal diet. Administered once a day, the general situation of the observation.
3., weight, diet, fasting blood glucose, grasping force and Anal temperature were dynamically detected in rats. After 3 weeks, 6 weeks, the rats were tested for insulin tolerance and glucose tolerance. After 6 weeks, the rats were collected for each group.
4. after 6 weeks of administration, the abdominal aorta blood was taken out and the serum was separated from the abdominal anesthetized rats. The TG and TC values in serum were determined by endpoint method, and the pancreas, ileum and formaldehyde were fixed in rats.
5. formaldehyde was used to immobilate tissue pancreas and ileum. Paraffin sections were made by paraffin embedding technology. Paraffin sections and HE staining were used to observe the pathological changes of pancreas and ileum.
6. the paraffin embedded sections of rat pancreas were taken. Immunohistochemistry was used to detect the expression of insulin in the islets of rats. The islet cells were detected by TUNEL and the apoptosis of islet B cells was observed.
7. the paraffin paraffin embedded section of rat ileum was sectioned and the expression of GLP-1 and GIP was detected by immunohistochemistry.
Result
1. weight: the weight of the model group decreased significantly, the weight value of the GGQL1 group decreased slightly, and the GGQL2 group remained stable.
2. fasting blood glucose: group GGQL1, group GGQL2 was given 4 weeks, 6 weeks decreased significantly, and blood sugar was lower than that in model group (P0.05).
3. diet: the diet in group GGQL1 was lower than that in model group (P0.05) in 1-3 weeks after administration (group GGQL2), and in group GGQL2 after administration, the diet was lower than that of model group (P0.05).
4. characterization observation: compared with the model group, GGQL1 group of rats with moderate ear vascular visibility, ear pale pink, pale yellow tail, black stool (P0.01/6), GGQL2 group rats tongue moderate, ear vascular visibility is moderate, black and yellow stool (P0.01/6), increased mobility, tongue lubrication, ear pale pink, the nose is reddish, pink claw does not run, yellow tail in white (P0.05/6);
5., grasping power: the grasping force value of group GGQL1 increased, and it was given for 1 weeks. The grasping force value of 4 weeks was higher than that of model group (P0.05), and the grasping force value of GGQL2 group increased. After 4 weeks and 5 weeks, the grasping force value of group GGQL2 was higher than that of model group (P0.05), and it was higher than that of GGQL1 group at 5 weeks after administration.
6. anus temperature: in group GGQL1, the value of anus temperature in group GGQL2 decreased. At 5 weeks of administration, the value of Anal temperature in group GGQL1 was lower than that of model group (P0.05).
7. insulin tolerance: the rate of blood sugar decline was higher in each group, and the AUC value of blood sugar was significantly lower than that of the model group (P0.05), but there was no significant difference between the group and the normal group.
8. glucose tolerance: the rate of blood glucose change in each group was higher than that in the model group, and the AUC value of blood glucose was higher than that in the model group, and the GGQL2 group was significantly higher than that in the model group (P0.05).
9. TG, TC:GGQL1 group TG, TC values were lower than the model group, but there was no statistically significant difference in GGQL2 group TG value (P0.05) was lower than the model group, TC value difference was not statistically significant difference.
10.HE staining: compared with the model group, GGQL1 group, GGQL2 group of islet cells were improved islet more rules, shrinkage islet rare, vacuolar degeneration of cells decreased islet cells, increased islet cell; ileum staining increased slightly by the circular muscularis mucosa, submucosa, muscular layer ring layer increases. The ring is regular, fold distribution closely, the internal cavity is relatively reduced
11. immunohistochemistry and apoptotic results: in group GGQL1, the apoptosis rate in pancreas of group GGQL2 was significantly lower than that of model group (P0.05), and insulin IOD/Area was higher than that of model group (P0.05). The mean GLP-1 optical density of intestinal GLP-1 was higher than that of model group (P0.05), and GIP average optical density was lower than that of model group (P0.05).
conclusion
The characterization of 1 Gegen Qin Lian Decoction on type 2 diabetic rats
Gegenqinlian decoction can be maintained in type 2 diabetic rats weight, reduce weight reduce symptoms; reduce in type 2 diabetic rats fasting blood glucose, reduce hyperglycemia in type 2 diabetic rats; reduce food intake, reduce the polyphagia; increase in type 2 diabetic rats light sound rise rate, improve dark eyes dull, dry lips, dry hair, claw dry run, improve the activity of rats decreased, tongue less fluid, less shiny nose grab resistance, not run, improve fatigue, body fluid deficiency symptoms; increased in type 2 diabetic rats grip, improve the fatigue symptoms; improve type 2 diabetes in anus the temperature rise stage, improve Fanre symptoms. Therefore, Gegenqinlian decoction can improve from the different degree of symptoms of type 2 diabetes.
Biological basis of 2 Gegen Qin Lian Decoction related to GLP-1
Gegenqinlian decoction can improve insulin resistance, increase insulin 30min in type 2 diabetic rats after glucose decreased rate of blood glucose change tendency of increased oral glucose in rats with type 2 diabetes mellitus; regulate blood lipids, reduce TG, TC value trend; improve islet, ileum form; increased islet insulin secreting islet B cell area and insulin secretion; increase the level of GLP-1 in the ileum, reduce the level of GIP; decrease apoptosis of pancreatic islet cells. The rate of Gegenqinlian Decoction in addition to increase the level of GLP-1, reduce the level of GIP, the function of regulating blood fat, which he was consistent with the biological mechanism of GLP-1 and that of Gegenqinlian decoction may through the relevant mechanism of GLP-1 to treat type 2 diabetes.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285.5

【参考文献】