【作者】 公惠玲；

【导师】 李卫平； 尹艳艳；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 硕士

【摘要】 目的:探讨黄精多糖(Polygona-polysaccharose,PSP)对实验性糖尿病模型动物的保护作用及其可能的作用机制。方法:1.在预实验未能测出LD50的基础上,采用最大耐受量的实验方法,观察PSP对小鼠的急性毒性作用。2.采用四氧嘧啶(Alloxan,ALX)腹腔一次注射法(220 mg/kg)建立糖尿病小鼠模型;将成功模型随机分为模型组、二甲双胍(DMBG,250mg/kg)和PSP (250、500、1 000 mg/kg)组,与对照组一起连续ig给药14d;末次给药24h后,用葡萄糖氧化酶法测定各组小鼠的空腹血糖(FBG);称胸腺、肝脏、脾脏和肾脏的重量,计算脏器指数;采用化学比色法分别检测血清和肝脏中的T-SOD、GSH-Px活性及MDA含量变化;常规HE染色法观察胰腺病理学组织学变化。3.采用链脲菌素(Streptozotocin,STZ)腹腔一次注射法(60 mg/kg)建立糖尿病大鼠模型;将成功模型随机分为模型组、二甲双胍(DMBG,233mg/kg)和PSP (195、390、780 mg/kg)组,与对照组一起连续ig给药28d;造模后第30d测定大鼠体重、进食量、进水量和尿量;末次给药24h后,腹主动脉取血,分离血清待用;采用葡萄糖氧化酶法测定FBG、采用放射免疫法检测血清胰岛素(INS)水平、采用化学比色法测定糖化血清蛋白(GSP)、甘油三脂(TG)、总胆固醇(TC)和高密度脂蛋白(HDL)含量变化。4.采用常规HE染色法观察STZ糖尿病大鼠胰腺组织的胰岛结构;采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(Terminal -deoxynucleotidyltransferase mediated nick end labeling,TUNEL)检测STZ糖尿病大鼠胰岛凋亡细胞情况;采用免疫组化染色法观察STZ糖尿病大鼠胰岛中INS和Caspase-3蛋白表达情况;采用硝酸还原酶法检测血清一氧化氮(NO)含量;采用逆转录-聚合酶链反应法(Reverse transcription- Polymerase chain reaction, RT-PCR)检测大鼠胰腺组织诱导型一氧化氮合酶(iNOS)mRNA的表达水平。结果:1急性毒性实验PSP的最大耐受量为31.25 g/kg,提示PSP治疗剂量几乎无毒副作用。2 PSP对ALX糖尿病小鼠的保护作用2.1 PSP能明显降低糖尿病小鼠血糖,显著提高糖尿病小鼠的胸腺指数、脾脏指数和肝脏指数。2.2 PSP明显提高ALX糖尿病小鼠血清和肝脏中降低的T-SOD和GSH-Px活性,降低血清和肝脏中升高的MDA含量,提示PSP可能一定程度上改善ALX糖尿病小鼠血清和肝脏的抗氧化能力。2.3常规HE染色结果显示模型组小鼠胰腺几乎找不到完整的胰岛,胰岛结构破坏严重,各用药组小鼠胰岛形态相对清晰,边界清楚,胰岛细胞排列整齐,说明PSP对STZ糖尿病小鼠胰腺的损伤有一定的保护作用。3 PSP对STZ糖尿病大鼠的保护作用3.1 PSP各剂量组大鼠“三多一少”的症状得到了显著的改善,PSP (195、390、780 mg/kg)组和盐酸二甲双胍(Metformin Hydrochloride Tablets,DMBG)组FBG、GSP含量显著降低,血清INS水平升高,胰岛中INS阳性表达率升高。3.2模型组TG及TC含量显著升高,HDL含量降低;PSP(390、780 mg/kg)组和DMBG组TG及TC含量有明显降低,同时HDL有一定程度的升高。3.3 PSP组和DMBG组血清NO含量显著性降低;免疫组化结果显示,模型组胰岛中Caspase-3表达率明显升高,PSP组胰岛中Caspase-3表达率降低;TUNEL法检测结果显示,模型中胰岛细胞凋亡率明显提高,PSP组胰岛细胞凋亡率下降。3.4 RT-PCR结果显示,正常对照组胰腺iNOS mRNA的几乎不表达或表达量很少,模型组胰腺iNOS mRNA表达量显著升高,PSP (390、780 mg/kg )组和DMBG组不同程度的下调胰腺iNOS mRNA的表达。结论:1. PSP对ALX糖尿病小鼠有一定的保护作用,其机制可能与其提高ALX糖尿病小鼠血清和肝脏的抗氧化能力有关。2. PSP能够降低STZ糖尿病大鼠升高的血糖,调节脂代谢,对STZ糖尿病大鼠具有一定的保护作用,其机制可能与其抑制胰岛细胞凋亡,下调Caspase-3和iNOSmRNA表达有关。更多还原

【Abstract】 ObjectiveTo study the protective effect and mechanisms of Polygona-polysaccharose(PSP)on experimental diabetic animal models.Methods1. The experiment of the maximally tolerated dose (MTD) was used to observe the acute toxicity of PSP in mice.2. The mice model of diabetes were established by injecting Alloxan (ALX) (220 mg/kg) into its abdominal cavity. Diabetic mice were randomly divided into model group, DMBG(250mg/kg) and PSP(250、500、1000 mg/kg).After 14d administration of PSP, FBG, organ weight, MDA, T-SOD and GSH-Px levels were determined. Pancreatic pathology was studied by morphological method.3. The rats model of diabetes were established by injecting Streptozotocin (STZ) (60 mg/kg) into its abdominal cavity. Diabetic rats were randomly divided into model group, DMBG(233mg/kg) and PSP(195,390,780mg/kg).The amount of water drinking, food intake, urinary volume and body weight were measured at the fourth week after the treatment. The blood samples were drawn to determine the indexes of FBG, GSP, INS, TG, TC and LDL. 4. Pancreatic pathology was studied by morphological method and immunohistochemical method. The distribution of apoptotic cells and the expression of Caspase-3 were observed by Terminal -deoxynucleotidyl transferase mediated nick end labeling (TUNEL) and immunohistochemistry. Using the method of nitrate reductase to detect the index of NO. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect iNOS mRNA expression.Results1 Acute toxicity testingThe MTD of PSP in mice is 31.25 g/kg, which prompted that PSP almost had non-toxic side effects of treatment dose.2 Protective effects of PSP on diabetic mice induced by ALX2.1 PSP could decrease the blood glucose level and increase thymus index, spleen index and liver index in diabetic mice induced by ALX.2.2 PSP could decrease the content of MDA and enhance the T-SOD, GSH-Px in blood serum and liver tissue of diabetic mice induced by ALX.2.3 The results of HE showed that, the pancrea in the model group almost could not find complete islet, the islet structural disorderd and the edge was not clear. The islet in PSP groups were relativly regularly. The result of HE showed that the PSP had some protective effect of pancreatic islet. 3 Protective effect of PSP on diabetic rats induced by STZ3.1 Levels of FBG, GSP and the amount of water drinking, food intake, urinary volume in the PSP treated groups were obviously lower than those in the model group while INS increased.3.2 Compared with model group, PSP decreased the rate of apoptotic cells, the levels of Caspase-3 and NO in blood serum. While the iNOSmRNA expression up-regulated in the diabetes model group, the iNOSmRNA expression in PSP treated groups down-regulated.Conclusions1 PSP may have protective effect on diabetic mice induced by ALX and the mechanism might be related to its antioxidant activity.2 PSP can effectivly decrease blood glucose and have some protection of diabetic rats. The mechanism may be related with inhibiting islet cell apoptosis, lowering Caspase-3 and suppressing iNOSmRNA.更多还原