电针“胰俞”穴对2型糖尿病大鼠肝损伤以及肝脏GLP-1受体影响的研究

发布时间：2018-03-29 10:12

  本文选题：2型糖尿病　切入点：肝损伤　出处：《北京中医药大学》2017年硕士论文

【摘要】：[目的]1.通过高糖、高脂饲料,饲养大鼠,结合腹腔注射:链脲菌素(streptozotocin STZ),对大鼠血糖、血脂、肝脏相关转氨酶、肝脏形态、结构、功能进行检测,验证糖尿病肝损伤大鼠的模型是否能够成功建立。通过观察电针"胰俞'"、电针"非穴'"、"格列美脲给药"对糖尿病肝损伤大鼠血糖、血脂、体重、肝脏相关转氨酶、以及肝脏形态、结构、功能的影响;并进行三种干预方式对比,初步判断,电针"胰俞"穴是否对糖尿病大鼠肝脏具有保护作用。2.检测糖尿病大鼠肝脏中胰高血糖素样肽-1(glucagon-like peptide-1;GLP-1)蛋白表达位置,以及对比空白组与模型组基因和蛋白表达量的差异,各组干预后表达量的差异,检测电针"胰俞"穴是否能增加肝脏GLP-1基因及蛋白表达,探讨电针"胰俞"穴是否能够对糖尿病肝损伤具有保护作用。[方法]实验动物40只雄性大鼠,适应性饲养1周后,随机取8只为空白组,给予普通饲料喂养。其余32只大鼠作为造模组,以高糖高脂饲料喂养50d后,一次性腹腔注射STZ溶液72h后测随机血糖,成模标准:随机血糖≥16.7mmol/L。动物分为空白组、模型组、电针"胰俞"组、电针"非穴'"组、"格列美脲药物"组,所有动物8只/笼饲养。其中,空白组于适应性饲养1周结束后机抽取,其余动物于适应性饲养后经造模并评估成模后,随机抽取。各组干预均为1次/d,6d/周,共4周。电针参数:连续波,5Hz,3-5mA,20min。将所有动物,在干预每隔一周时,对大鼠进行尾部取血,检测大鼠空腹血糖、体重。末次干预结束后测定血脂,转氨酶:丙氨酸转氨酶(alanine transaminase;ALT)、天冬氨酸转氨酶(aspartatetransaminase;AST)、γ-谷氨酰转移酶(γ-glutamyltransferase;GGT)。并对肝脏进行HE染色观察肝细胞形态变化。以实时聚合酶链反应(Real-time PCR;RT-PCR)法检测肝脏GLP-1基因表达,以蛋白质印迹法(Western blot;West-ern)检测大鼠肝脏中GLP-1蛋白水平。[结果]实验1显示:采取高糖、高脂饲料饮食,结合了 STZ溶液腹腔注射,创建2型糖尿病(type2diabetesmellitus;T2DM)大鼠模型,成模标准:随机血糖多16.7mmol/L。统计学显示,各时间点模型组血糖,均高于空白组(P0.05),差异有统计学意义;模型组大鼠的总胆固醇(total cholesterol;TC)、低密度脂蛋白胆固醇(low density lipoprotein-cholesterol;LDL-C)、甘油三酯(triglyce:ride;TG)水平均显著高于空白组(P0.05),高密度脂蛋白胆固醇(high density lipoprotein cholesterin;HDL-C)显著低于空白组(P0.05),差异有统计学意义;模型组大鼠的AST、ALT、GGT水平均显著高于空白组(P0.05),差异有统计学意义;且HE染色肝组织切片可见:肝细胞显示广泛肿胀,窦间隙明显变窄,出现炎细胞浸润以及纤维组织增生,可见,大鼠的肝脏出现了明显的损伤现象。电针胰俞穴,可快速调节2型糖尿病大鼠升高的血糖,其长期疗效也较为可靠,且优于电针非穴组。同时,电针胰俞穴,也可快速抑制2型糖尿病大鼠体重下降,但随着病程的进展,其调节体重的作用并不显著。然而,对于血脂的各项指标所言,电针胰俞穴,可有效的降低2型糖尿病大鼠所升高的TC、TG、LDL-C水平,且在调节TG、LDL-C上,优于格列美脲灌胃;而在调节TC、LDL-C上,优于电针非穴。此外,电针胰俞穴,可有效降低2型糖尿病大鼠血清中升高的AST、ALT、GGT水平,且其作用优于电针非穴。电针胰俞穴,可有效降低2型糖尿病大鼠血清中升高的AST、ALT、GGT水平,且其作用优于电针非穴。肝脏病理切片:电针胰俞穴,能够有效的降低肝细胞胞质红染,核固缩,细胞凋亡,炎症细胞浸润,小胆管增生,小泡性脂滴的现象,改善脂质代谢紊乱,且改善效果,优于电针非穴组和格列美脲组,电针非穴组和格列美脲组,均能很好的改善炎症细胞浸润和小胆管增生,而格列美脲改善小泡性脂滴现象,优于电针非穴组。实验2显示:1.在大鼠肝脏中显示存在GLP-1受体蛋白的表达,并在53kD处出现明显蛋白条带,与其他实验证明相同。对肝脏GLP-1受体蛋白进行灰度值检测,统计学分析后,各组之间具有可比性(P0.05),以空白组为标准对象,模型组、胰俞组、格列美脲组、非穴组的GLP-1受体蛋白灰度值,均呈下降状态(p0.05);与模型组比较,胰俞组、格列美脲组、非穴组依然呈显著上升状态(P0.05);与胰俞组比较,格列美脲组与非穴组GLP-1受体蛋白灰度值,差异无统计学意义,虽然全部低于胰俞组,但均无显著性差异;与格列美脲组比较,非穴组GLP-1受体蛋白灰度值,高于格列美脲组,并与胰俞组基本一致。2.在基因层面表达中,与空白组相比较,模型组、胰俞组、非穴组、格列美脲组的基因表达量均呈显著下降状态,差异有统计学意义(P0.05),与模型组相比较,胰俞组基因表达量显著升高(P0.05),格列美脲组与非穴组也均有升高,但无统计学意义;与胰俞穴相比,格列美脲组与非穴组均降低,其中非穴组与之有统计学意义(P0.05);格列美脲组与非穴组基本一致。[结论]提示:通过高糖、高脂饲料饮食,结合STZ溶液腹腔注射,验证了糖尿病肝损伤大鼠的模型成功建立,符合相关文献报道,可完成后续实验部分。电针胰俞穴,能够有效的调节2型糖尿病大鼠血糖,在短期内抑制糖尿病患者的体重下降,显著降低TC、TG、LDL-C水平,并且有效改善了脂质代谢紊乱,并进一步保护了肝细胞。电针"胰俞"穴,可以有效的增加GLP-1受体蛋白及基因表达。保护肝脏可能是通过降低血糖、血脂、改善糖脂代谢紊乱,产生的作用,从而增加GLP-1受体蛋白和基因表达,最终改善糖尿病肝损伤。
[Abstract]:To]1. by high glucose and high fat diet fed rats, combined with intraperitoneal injection of streptozotocin (streptozotocin, STZ), blood lipids of rats, blood glucose, liver transaminase, liver morphology, structure and function were detected in rats of diabetic liver injury model verify whether can be successfully established. Through the observation of electroacupuncture "the pancreas Yu '", "non acupoint acupuncture", "glimepiride medicine" blood glucose in rats of diabetic liver injury, blood lipid, body weight, liver transaminase, and liver morphology, structure, function and effect; comparison, three kinds of intervention methods preliminary judgment, whether Electroacupuncture of "pancreatic Yu" point with protection the role of.2. in detection of diabetic rat liver glucagon like peptide -1 on the liver of diabetic rats (glucagon-like peptide-1; GLP-1) the expression of position, and the comparison of the control group and model group gene and protein expression differences, differences in the amount of expression groups,. Measurement of Electroacupuncture of "pancreatic Yu" point whether can increase the expression of hepatic GLP-1 mRNA and protein, to investigate whether electroacupuncture "pancreatic Yu" point to have protective effects. Methods: animal experiment 40 male rats of diabetic liver injury, after 1 week adaptive feeding, 8 rats were randomly selected as control group, given ordinary feed. The remaining 32 rats as model by high-fat diet and 50D after intraperitoneal injection of STZ 72h solution after blood glucose was measured, become the standard model: random blood glucose than 16.7mmol/L. animal were divided into blank group, model group, electroacupuncture group, electroacupuncture "Yi Shu" "non acupoint '" group, "drug glimepiride" group, all 8 animal per cage. Among them, the blank group in the 1 week of adaptive feeding after extraction machine, the remaining animal in adaptive feeding after modeling and evaluation model, randomly selected. Each intervention was 1 times /d, 6d/ week for 4 weeks. The electric needle parameters: continuous wave 5Hz, 3-5mA, 20min., the The animal, in the intervention every week, the rat tail blood, fasting plasma glucose, lipids were determined by weight. At last, after the intervention of transaminase (alanine: alanine aminotransferase transaminase; ALT), aspartate aminotransferase (aspartatetransaminase; AST), gamma glutamyl transferase (gamma -glutamyltransferase; GGT) and the liver were observed. Liver cell morphology HE staining. By real-time polymerase chain reaction (Real-time, PCR; RT-PCR) was used to detect the expression of GLP-1 gene in liver, by Western blotting (Western blot; West-ern) to detect GLP-1 protein levels in the livers of rats. The results showed: 1] experiment of high carbohydrate diet. High fat diet combined with intraperitoneal injection of STZ solution, creating a type 2 diabetes mellitus (type2diabetesmellitus; T2DM) rat model, become the standard model: random blood glucose 16.7mmol/L. statistics, each time point in model group were higher than those of control group. (P0.05), the difference was statistically significant; the total cholesterol of rats in model group (total cholesterol; TC), low density lipoprotein cholesterol (low density lipoprotein-cholesterol; LDL-C), triglycerides (triglyce:ride; TG) were significantly higher than the control group (P0.05), high density lipoprotein cholesterol (high density lipoprotein cholesterin; HDL-C) was significantly lower than that of the control group (P0.05), the difference was statistically significant; the rats in the model group AST, ALT, GGT levels were significantly higher than the control group (P0.05), the difference was statistically significant; and HE staining of liver tissue visible: liver cells showed extensive swelling, sinus gap narrowed significantly, inflammatory cells infiltration and fiber tissue, visible, rat liver injury appeared obviously. Electroacupuncture acupoints can quickly adjust the pancreas, type 2 diabetic rats elevated blood glucose, its long-term efficacy is more reliable, and better than EA non acupoint group. 鏃，

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