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。
Objective
To study the protective effect and mechanisms of Polygona-polysaccharose(PSP)on experimental diabetic animal models.
Methods
1. 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.
Results
1 Acute toxicity testing
The 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 ALX
2.1 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 STZ
3.1 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.
Conclusions
1 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.
引文
1吕纪凤,王长舟.老年糖尿病的自我保健[J].中国医学理论与实践, 2005(15)1: 71-2.
2张庭廷,夏晓凯,陈传平,等.黄精多糖的生物活性研究[J].中国实验方剂学杂志, 2006(12)7: 42-5.
3 Ding X, Zhang J, Jiang P, et al. Structural features and hypoglycaemic activity of an exopolysaccharide produced by Sorangium cellulosum[J]. Lett Appl Microbiol. 2004;38(3):223-8.
4 Son IS, Kim JH, Sohn HY, et al. Antioxidative and hypolipidemic effects of diosgenin, a steroidal saponin of yam (Dioscorea spp.), on high-cholesterol fed rats[J]. Biosci Biotechnol Biochem. 2007 Dec;71(12):3063-71.
5詹可顺,王华,魏伟.白芍总苷对小鼠化学性肝损伤的保护作用及机制[J].安徽医科大学学报, 2006 (41)6: 664-6.
6 Li Z, Zhang T, Dai H.Liu G, et al. Involvement of Endoplasmic Reticulum Stress in Myocardial Apoptosis of Streptozocin-Induced Diabetic Rats[J]. J Clin Biochem Nutr. 2007,41(1):58-67.
7 Saeed MK, Deng Y, Dai R. Attenuation of Biochemical Parameters in Streptozotocin-induced Diabetic Rats by Oral Administration of Extracts and Fractions of Cephalotaxus sinensis[J].J Clin Biochem Nutr. 2008, 42:21-8.
8董明,侯新国,陈丽,重组瘦素治疗对链脲佐菌素糖尿病大鼠胃促生长素水平的影响[J],山东大学学报(医学版), 2007, 45(5): 486-8.
9 Maiti R, Das UK, Ghosh D, Attenuation of hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats by aqueous extract of seed of Tamarindus indica[J]. Biol Pharm Bull. 2005, 28(7):1172-6.
10曲巍,郝丽萍,于冬,等.长期乙醇摄入对大鼠胰岛形态结构与功能影响[J].中国公共卫生, 2006,22(7): 781-3.
11 Katakam AK, Chipitsyna G, Gong Q, et al. Streptozotocin (STZ) mediates acute upregulation of serum and pancreatic osteopontin (OPN): a novel islet-protective effect of OPN through inhibition of STZ-induced nitric oxide production[J]. J Endocrinol. 2005 Nov;187(2):237-47
12王易棼,穆天慧,陈纪军.等.滇黄精化学成分研究[J].中国中药杂志, 2003, 28(6): 524-526.
13徐世忱,李淑惠,纪耀华,等.黄精炮制前后总多糖含量的比较分析[J].中国中药杂志, 1993, 18(10):600-601.
14 Kun Ho Son, Jae CHul Do. Steroidal Saponins from the Rhizomes of Polygonatum Sibiricum [J]. J. Nat. Prod,1990, 53 (2) : 333 - 333.
15 L i Xing, Cong, Yang Chong, Ren. Steroid Saponins from Po2 lygonatum Kingianum [J]. J. Phytohemistry, 1992, 31(10) : 3559 - 3563.
16袁昌齐.天然药物资源开发利用[M ].南京:江苏科学技术出版社, 2000, 372 - 375
17李友元,邓洪波,张萍,等.黄精多糖对糖尿病模型小鼠糖代谢的影响[J].中国临床康, 2005,9(27):90-91.
18黄敏,王书奎,王小峰,等.不同禁食时间对ALX糖尿病小鼠模型的血清胰岛素和血糖的影响[J].中国药科大学学报, 2001(32)3: 217-20
19 Koncke KD, Fehsel K, Sommer A, et al. Nitric oxide generation during cellular metabolization of the diabetogenic N-methyl-N nitroso-urea strepotozotozin contributes to islet cell DNA damage [J].Biol Chem Hoppe-seyler, 1995, 376 (3):179-185.
20 Castellano JM, Navarro VM, Fernández-Fernández R, et al. Expression of hypothalamic KiSS-1 system and rescue of defective gonadotropic responses by kisspeptin in streptozotocin-induced diabetic male rats[J]. Diabetes. 2006,55(9):2602-10.
21 Matsuoka M, Ogata N, Minamino K, et al. Leukostasis and pigment epithelium-derived factor in rat models of diabetic retinopathy[J]. Mol Vis. 2007,13:1058-65.
22 Yokoyama M, Yagyu H, Hu Y, et al. Apolipoprotein B production reduces lipotoxic cardiomyopathy: studies in heart-specific lipoprotein lipase transgenic mouse[J]. J Biol Chem. 2004 Feb 6;279(6):4204-11.
23 Ishikawa K, Kimura S, Kobayashi A, et al. Increased reactive oxygen species and anti - oxidative response in mitochondrial cardiomyopathy [J]. Circ J, 2005(69)5: 617-20.
24 Luo Q, Cai Y, Yan J, et al. Hypoglycemic and hypolipidemic effects and antioxidant activity offruit extracts from Lycium barbarum.[J]. Life Sci. 2004 Nov 26;76(2):137-49.
25 Anwar MM, Meki AR. Oxidative stress in streptozotocin-induced diabetic rats: effects of garlic oil and melatonin[J]. Comp Biochem Physiol A Mol Integr Physiol. 2003 Aug;135(4):539-47.
26王胜男,徐源梅,李红玉.黄芩提取物对糖尿病小鼠脂代谢和抗氧化作用的影响[J].辽宁医学院学报, 2007, 28(5):29,65.
27王忠山,邹冬辉,赵慧,等.短期糖尿病雄性大鼠前列腺自由基和抗氧化水平变化研究[J]中国病理生理杂志, 2003 ,19(6) : 803 - 805
28龚志刚,胡红霞,朱笃,等.葛根素对糖尿病大鼠血糖和抗氧化能力的影响[J].食品科学2006, 27(11):498-500
29刘冰,刘万顺,韩宝芹等.壳寡糖及其衍生物对实验性糖尿病大鼠调节血脂和抗氧化作用山东大学学报(理学版), 2006,41(4):158-163.
30 Ishikawa K, Kimura S, Kobayashi A, et al. Increased reactive oxygen species and anti - oxidative response in mitochondrial cardiomyopathy [J]. Circ J, 2005(69)5: 617-20.
31 Luo Q, Cai Y, Yan J, Y, et al. Hypoglycemic and hypolipidemic effects and antioxidant activity of fruit extracts from Lycium barbarum.[J]. Life Sci. 2004 Nov 26;76(2):137-49.
32 Anwar MM, Meki AR. Oxidative stress in streptozotocin-induced diabetic rats: effects of garlic oil and melatonin[J]. Comp Biochem Physiol A Mol Integr Physiol. 2003 Aug;135(4):539-47.
33胡丹波,李舜伟.慢性问断性缺氧后一氧化氮和诱生性一氧化氮合酶的表达及维生索E和左旋丁基苯酞对其影响[J].中华神经科学杂志, 2004, 20(2) : 140～4.
34 Vendrame F, Segni M, Grassetti D, et al. Impaired caspase-3 expression by peripheral T cells in chronic autoimmune thyroiditis and in autoimmune polyendocrine syndrome-2[J]. J Clin Endocrinol Metab. 2006,91(12):5064-8.
35 Condorelli G, Roncarati R, Ross J Jr, et al . Heart-targeted overexpression of caspase3 in mice increases infarct size and depresses cardiac function. Proc Natl Acad Sci USA. 2001,98(17):9977-82.
36 Oyadomari S, Takeda K, Takiguchi M, et al. Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[J]. Proc Natl Acad Sci U S A.2001, 98(19): 10845-50.
37 Saldeen J. Cytokines induce both necrosis and apoptosis via a common Bcl-2-inhibitable pathway in rat insulin-producing cells[J]. Endocrinology. 2000, 141(6):2003-10.
38 Deguchi A, Thompson WJ, Weinstein IB. Activation of protein kinase G is sufficient to induce apoptosis and inhibit cell migration in colon cancer cells[J]. Cancer Res. 2004,64(11):3966-7
1叶任高,陆再英.内科学[M]. 6版.北京:人民卫生出版社, 2006: 787.
2 Lally FJ, Ratcliff H, Bone AJ.. Apoptosis and disease progression in the spontaneously diabetic BB/S rat [J]. Diabetologia. 2001, 44(3):320-4.
3 Butler AE, Janson J, Bonner-Weir S, et al. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes[J].Diabetes. 2003,52(1):102-10.
4 Donath MY, St?rling J, Maedler K,,et a1. Inflammatory mediators and islet beta-cell failure: a link between type 1 and type 2 diabetes[J]. J Mol Med. 2003,81(8):455-70.
5 Qin H, Trudeau JD, Reid GS, et a1. Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL[J]. Int Immunol. 2004,16(11):1657-62.
6 Loweth AC, Williams GT, James RF, et a1. Human islets of Langerhans express Fas ligand and undergo apoptosis in response to interleukin-1beta and Fas ligation[J].Diabetes. 1998, 47(5):727-32.
7 Turvey SE, Gonzalez-Nicolini V, Kingsley CI ,et al. Fas ligand-transfected myoblasts and islet cell transplantation [J].Transplantation. 2000,69(9):1972-6.
8 Su X, Hu Q, Kristan JM, Costa C, et al. Significant role for Fas in the pathogenesis of autoimmune diabetes[J]. J Immunol. 2000,164(5):2523-32.
9金光香;宫海民;段文卓,等.蜂贝化瘀胶囊对糖尿病大鼠肾脏细胞Fas和Fas-L表达的影响[J].中医杂志2005, 46(11):851-853.
10 Vincent JA, Mohr S. Inhibition of caspase-1/interleukin-1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia[J]. Diabetes. 2007, 56(1): 224-30.
11 Stephens LA, Thomas HE, Ming L, et al. Tumor necrosis factor-alpha-activated cell death pathways in NIT-1 insulinoma cells and primary pancreatic beta cells. [J] . Endocrinology. 1999, 140(7):3219-27.
12 Ishizuka N, Yagui K, Tokuyama Y, et al . Tumor necrosis factor alpha signaling pathway andapoptosis in pancreatic beta cells[J] . Metabolism. 1999,48(12):1485-92.
13 Suk K, Kim S, Kim YH et al . IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death. [J] J Immunol. 2001,166(7):4481-9.
14 Pierce MA, Chapman HD, Post CM, et al . Adenovirus early region 3 antiapoptotic 10.4K,
14.5K, and 14.7K genes decrease the incidence of autoimmune diabetes in NOD mice. [J] . Diabetes. 2003,52(5):1119-27.
15 Halminen M, Simell O, Knip M, et al . Cytokine expression in unstimulated PBMC of children with type 1 diabetes and subjects positive for diabetes-associated autoantibodies[J]. Scand J Immunol. 2001 May;53(5):510-3.
16 Savinov AY, Wong FS, Chervonsky AV. IFN-gamma affects homing of diabetogenic T cells[J]. J Immunol. 2001,167(11):6637-43.
17 Suk K, Kim S, Kim YH, et al. IFN-gamma/TNF-alpha synergism as the final effector in autoimmune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in pancreatic beta cell death. [J]. J Immunol. 2001,166(7):4481-9.
18 Maedler K, Sergeev P, Ris F. et al. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest. 2002,110(6):851-60.
19 Suarez-Pinzon WL, Strynadka K, Rabinovitch A Destruction of rat pancreatic islet beta-cells by cytokines involves the production of cytotoxic aldehydes[J]. Endocrinology.1996, 137(12):5290-6.
20 Mabley JG, Pacher P, Southan GJ, et al . Nicotine reduces the incidence of type I diabetes in mice[J]. J Pharmacol Exp Ther. 2002, 300(3):876-81.
21 Liu P, Xu B, Cavalieri TA, et al. Age-related difference in myocardial function and inflammation in a rat model of myocardial ischemia-reperfusion[J].Cardiovasc Res, 2002, 56(3): 443-53.
22 Shimabukuro M, Wang MY, Zhou YT, et al. Protection against lipoapoptosis of beta cells through leptin-dependent maintenance of Bcl-2 expression[J]. Proc Natl Acad Sci USA. 1998,95(16):9558-61.
23 Saldeen J .Cytokines induce both necrosis and apoptosis via a common Bcl-2-inhibitable pathway in rat insulin-producing cells[J]. Endocrinology. 2000,141(6):2003-10.
24 Chen Z, Chua CC, Ho YS, et al. Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice[J]. Am J Physiol Heart Circ Physiol. 2001,280(5):H2313-20.
25 Pavlovic D, Chen MC, Bouwens L, et al. Contribution of ductal cells to cytokine responses by human pancreatic islets[J]. Diabetes. 1999, 48(1):29-33.
26 Jeong IK, Oh SH, Chung JH, et al.The stimulatory effect of IL-1beta on the insulin secretion of rat pancreatic islet is not related with iNOS pathway[J]. Exp Mol Med. 2002, 34(1):12-7.
27 Harvey NL , Kumar S. The role of caspases in apoptosis[J]. Adv Biochem Eng Biotechnol. 1998, 62:107-28.
28何琼,张端莲. Caspase-3与糖尿病心肌病变的研究[J].数理医药学杂志, 2007, 20(1):13-15.
29 Golstein P. Controlling cell death[J]. Science. 1997, 275(5303):1081-2.
30 Martin DA, Siegel RM, Zheng L, et al . Membrane oligomerization and cleavage activates the caspase-8 (FLICE/MACHalpha1) death signa[J]l. J Biol Chem. 1998, 273(8):4345-9
31 Maedler K, Spinas GA, Dyntar D et al. Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function[J]. Diabetes. 2001, 50(1):69-76
32 Hui H, Dotta F, Di Mario U , et al. Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death[J]. J Cell Physiol. 2004, 200(2):177-200
33 Evans JL, Goldfine ID, Maddux BA, et al. Are oxidative stress-activated signaling pathways mediators of insulin resistance and beta-cell dysfunction? [J]. Diabetes. 2003;52(1):1-8.
34 Lowell BB, Shulman GI.. Mitochondrial dysfunction and type 2 diabetes [J]. Science. 2005, 307(5708):384-7.