γ-氨基丁酸强化米缓解2型糖尿病模型小鼠胰腺损伤
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摘要
目的探究γ-氨基丁酸(γ-aminobutyric acid,GABA)强化米饲料干预对2型糖尿病(type 2 diabetes mellitus,T2DM)模型小鼠氧化应激和胰腺损伤的影响。方法 70只雄性ICR小鼠,按体重随机选取10只作空白对照组,始终饲喂正常精白米饲料;其余60只小鼠饲喂高脂精白米饲料9周后,禁食12 h,连续两天腹腔注射50 mg/kg链脲佐菌素(streptozocin,STZ),对照组注射等体积生理盐水。随后将造模成功的50只T2DM小鼠按照血糖随机分为5组,每组10只,分别为:T2DM模型对照组,发芽糙米阳性对照组(GABA含量为每公斤饲料0.2 g),GABA强化米低、中、高剂量组,GABA含量分别为每公斤饲料0.02、0.1和0.2 g,饲喂6周。实验结束前一周口服葡萄糖耐量实验观察不同剂量GABA强化米降糖效果;实验结束后HE染色观察胰腺组织形态;同时检测血浆和胰腺氧化还原指标:活性氧自由基(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、总抗氧化能力(total antioxidantcapacity,T-AOC)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、超氧化物歧化酶(superoxide dismutase,SOD);实时荧光定量PCR(real-time fluorescent quantitative PCR)测定胰腺氧化应激相关基因:糖原合成激酶-3β(glycogen synthase kinase,GSK-3β)、核转录因子(nuclear transcription factor 2,Nrf2)、血红素氧合酶(heme oxygenase 1,HO-1)、醌氧化还原酶[NAD(P)H:quinoneoxidoreductase,NQO1],胰岛素分泌相关基因:胰十二指肠同源框因子-1(pancreatic and duodenal homeobox 1,PDX-1)、肌腱膜纤维肉瘤肿瘤基因同系物A(mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family protein A,MafA)、葡萄糖激酶(glucokinase,GCK)、葡萄糖转运体2(glucose transporter 2,GLUT2),以及细胞凋亡相关基因:B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关的X蛋白(Bcl-2-associated X protein,Bax)、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)的基因相对表达量。结果 GABA强化米能够改善T2DM小鼠血糖水平上升和胰岛素分泌不足,缓解了血浆和胰腺氧化应激,上调胰岛素分泌相关基因PDX-1、GCK、GLUT2的表达,抑制促凋亡基因caspase-3的上调,促进抗凋亡基因Bcl-2的表达。以上结果存在剂量-效应关系,以0.2 g/kg剂量组最为显著,达到和发芽糙米类似的结果。结论 GABA强化米能够显著改善STZ诱导的T2DM小鼠血浆和胰腺氧化还原状态,调节氧化应激以及凋亡相关基因的表达水平,进而保护胰腺组织形态,改善胰腺胰岛素分泌,从而发挥改善糖代谢的作用。
OBJECTIVE To investigate the effects of gamma aminobutyric acid(GABA) fortified rice diet intervention on oxidative stress and pancreatic injury in type 2 diabetes mellitus(T2 DM) mice. METHODS Of the 70 male ICR mice, 10 were randomly selected as blank control group and they were always fed with the normal white rice feed. The remaining 60 mice were fed with high-fat white rice for 9 weeks. They were fasted for 12 h and injected intraperitoneally with streptozocin(STZ) at a dose of 50 mg/kg body weigh for two consecutive days. The control group was injected with the corresponding volume of normal saline. Subsequently, 50 T2 DM mice with successful modeling were randomly divided into 5 groups according to blood glucose, 10 in each group: T2 DM model control group, germinated brown rice positive control group(GABA content is 0.2 g/kg feed), GABA-fortified rice low, medium and high dose group(GABA content was 0.02, 0.1 and 0.2 g/kg feed respectively) and each target diet was fed for 6 weeks. Oral glucose tolerance test was performed one week before the end of the experiment to observe the hypoglycemic effect of different doses of GABA fortified rice. After the end of the experiment, HE staining was used to observe the morphology of pancreas. At the same time, the redox indicators from plasma and pancreas of reactive oxygen species(ROS), malondialdehyde(MDA), total antioxidant capacity(T-AOC), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD) were examined in each group; The mRNA expressions of oxidative stress-related genes including glycogen synthase kinase-3β(GSK-3β), nuclear transcription factor 2(Nrf2), heme oxygenase 1(HO-1) and NAD(P)H: quinone oxidoreductase1(NQO1), insulin secretion related genes including pancreatic and duodenal homeobox 1(PDX-1), mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A(MafA), glucokinase(GCK), glucose transporter 2(GLUT2) and the apoptosis associated genes including b-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax) and caspase-3 in pancreas were assayed by real-time fluorescent quantitative PCR. RESULTS The intervention of GABA fortified rice could alleviate the improvement of the blood glucose level and the lack of insulin secretion in T2 DM mice and relieve plasma and pancreatic oxidative stress. besides, The intervention of GABA fortified rice could up-regulate the expression of insulin secretion-related genes PDX-1, GCK, GLUT2, inhibit the expression of pro-apoptotic gene caspase-3 and promote the expression of anti-apoptosis gene Bcl-2. There was a dose-response relationship between the above result and the 0.2 g/kg dose group was the most significant, which achieved similar result to germinated brown rice. CONCLUSION GABA-fortified rice can significantly improve the plasma and pancreatic redox status of STZ-induced T2 DM mice, regulate the expression levels of oxidative stress-related genes and apoptosis-related genes, thereby protect pancreatic tissue morphology, improve pancreatic insulin secretion and thereby alleviate glucose metabolism.
OBJECTIVE To investigate the effects of gamma aminobutyric acid(GABA) fortified rice diet intervention on oxidative stress and pancreatic injury in type 2 diabetes mellitus(T2 DM) mice. METHODS Of the 70 male ICR mice, 10 were randomly selected as blank control group and they were always fed with the normal white rice feed. The remaining 60 mice were fed with high-fat white rice for 9 weeks. They were fasted for 12 h and injected intraperitoneally with streptozocin(STZ) at a dose of 50 mg/kg body weigh for two consecutive days. The control group was injected with the corresponding volume of normal saline. Subsequently, 50 T2 DM mice with successful modeling were randomly divided into 5 groups according to blood glucose, 10 in each group: T2 DM model control group, germinated brown rice positive control group(GABA content is 0.2 g/kg feed), GABA-fortified rice low, medium and high dose group(GABA content was 0.02, 0.1 and 0.2 g/kg feed respectively) and each target diet was fed for 6 weeks. Oral glucose tolerance test was performed one week before the end of the experiment to observe the hypoglycemic effect of different doses of GABA fortified rice. After the end of the experiment, HE staining was used to observe the morphology of pancreas. At the same time, the redox indicators from plasma and pancreas of reactive oxygen species(ROS), malondialdehyde(MDA), total antioxidant capacity(T-AOC), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD) were examined in each group; The mRNA expressions of oxidative stress-related genes including glycogen synthase kinase-3β(GSK-3β), nuclear transcription factor 2(Nrf2), heme oxygenase 1(HO-1) and NAD(P)H: quinone oxidoreductase1(NQO1), insulin secretion related genes including pancreatic and duodenal homeobox 1(PDX-1), mus musculus v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A(MafA), glucokinase(GCK), glucose transporter 2(GLUT2) and the apoptosis associated genes including b-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax) and caspase-3 in pancreas were assayed by real-time fluorescent quantitative PCR. RESULTS The intervention of GABA fortified rice could alleviate the improvement of the blood glucose level and the lack of insulin secretion in T2 DM mice and relieve plasma and pancreatic oxidative stress. besides, The intervention of GABA fortified rice could up-regulate the expression of insulin secretion-related genes PDX-1, GCK, GLUT2, inhibit the expression of pro-apoptotic gene caspase-3 and promote the expression of anti-apoptosis gene Bcl-2. There was a dose-response relationship between the above result and the 0.2 g/kg dose group was the most significant, which achieved similar result to germinated brown rice. CONCLUSION GABA-fortified rice can significantly improve the plasma and pancreatic redox status of STZ-induced T2 DM mice, regulate the expression levels of oxidative stress-related genes and apoptosis-related genes, thereby protect pancreatic tissue morphology, improve pancreatic insulin secretion and thereby alleviate glucose metabolism.
引文
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[12] 吴群励,张宏,朴元林,等.筋脉通胶囊通过增强背根神经节Nrf2和HO-1的表达改善糖尿病大鼠的周围神经痛[J].基础医学与临床,2014,34 (2):179-184.
[13] 李文松,崔璨,李强.磷脂酰肌醇3激酶/蛋白激酶B/糖原合成酶激酶3β信号通路对胰岛β细胞凋亡调控作用的研究进展 [J].中国糖尿病杂志,2014,22 (12):1138-1140.
[14] 蔺忆,高翠翠,陈立立,等.GABA对高糖诱导氧化损伤的RIN-m5f细胞的保护作用和机制 [J].食品与生物技术学报,2015 (2):195-200.
[15] WAN Y,WANG Q H,PRUD′ HOMME G J.GABAergic system in the endocrine pancreas:a new target for diabetes treatment[J].Diabetes Metab Syndr Obes,2015,8:79-87.
[2] 李飞,隋新,毕丽娜,等.发芽糙米降血糖作用的研究[J].中国食品添加剂,2016 (5):168-171.
[3] CHO D H,LIM S T.Germinated brown rice and its bio-functional compounds [J].Food Chem,2016,196 (8):259-271.
[4] PRUD′ HOMME G J,GLINKA Y.GABA protects human islet cells against the deleterious effects of immunosuppressive drugs and exerts immunoinhibitory effects alone [J].Transplantation,2013,96 (7):616-623.
[5] 梁彦,张传军,吕艳荣.马齿苋多糖对高脂饮食联合链脲佐菌素诱导的糖尿病小鼠的作用及机制[J].食品科学,2014,35(3):217-220.
[6] 许芳芳,王楠,李刚强,等.2型糖尿病小鼠模型的建立与评价[J].中国医学科学院学报,2017,39 (3):324-329.
[7] ROSENGREN A H,BRAUN M,MANDI T,et al.Reduced insulin exocytosis in human pancreatic beta-cells with gene variants linked to type 2 diabetes[J].Diabetes,2012,61 (7):1726-1733.
[8] LIU X D,RUAN J X,XIA J H,et al.The study of regulatory effects of Pdx-1,MafA and NeuroD1 on the activity of porcine insulin promoter and the expression of human islet amyloid polypeptide[J].Mol Cell Biochem,2014,394 (1-2):59-66.
[9] PARK J H,STOFFERS D A,NICHOLLS R D,et al.Development of type 2 diabetes following intrauterine growth retardation in rats is associated with progressive epigenetic silencing of Pdx1[J].Appl Magn Reson,2014,45 (8):723-730.
[10] KASSAB A,PIWOWAR A.Cell oxidant stress delivery and cell dysfunction onset in type 2 diabetes[J].Biochimie,2012,94 (9):1837-1848.
[11] ZOU Y,HONG B,FAN L,et al.Protective effect of puerarin against beta-amyloid-induced oxidative stress in neuronal cultures from rat hippocampus:involvement of the GSK-3β/Nrf2 signaling pathway[J].Free Radic Res,2013,47 (1):55-63.
[12] 吴群励,张宏,朴元林,等.筋脉通胶囊通过增强背根神经节Nrf2和HO-1的表达改善糖尿病大鼠的周围神经痛[J].基础医学与临床,2014,34 (2):179-184.
[13] 李文松,崔璨,李强.磷脂酰肌醇3激酶/蛋白激酶B/糖原合成酶激酶3β信号通路对胰岛β细胞凋亡调控作用的研究进展 [J].中国糖尿病杂志,2014,22 (12):1138-1140.
[14] 蔺忆,高翠翠,陈立立,等.GABA对高糖诱导氧化损伤的RIN-m5f细胞的保护作用和机制 [J].食品与生物技术学报,2015 (2):195-200.
[15] WAN Y,WANG Q H,PRUD′ HOMME G J.GABAergic system in the endocrine pancreas:a new target for diabetes treatment[J].Diabetes Metab Syndr Obes,2015,8:79-87.