实验性低血糖对糖尿病大鼠血管内皮功能及氧化应激的影响
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摘要
目的：探讨实验性低血糖对糖尿病大鼠血管内皮功能和氧化应激的影响。
方法：健康雄性wistar大鼠随机分为正常组和糖尿病组,适应性饲养一周后,正常组继续饲以普通饲料,糖尿病组饲以高脂饲料。8周后在高脂饲料喂养的大鼠中选取体重大于340克的大鼠,给予链脲佐菌素(STZ) 25mg/Kg体重腹腔注射。一周后尾静脉取血测定禁食6小时非空腹血糖,以GLU≥16.7mmol/L为造模成功。正常大鼠随机分为对照组(NC)和低血糖组(NL),糖尿病大鼠随机分为对照组(DC)、单纯低血糖组(DL)、低血糖后高血糖组(DLH)和糖尿病治疗组(DT)。禁食12h后,低血糖组给予生物合成人胰岛素腹腔注射。注射后1.5h,NLH组给予50%葡萄糖灌胃。除DT组外其余各组分别于胰岛素或生理盐水注射前、注射后1.5h、4.5h、6.5h心脏取血0.6ml,测定血糖、血清丙二醛(MDA)、超氧化物歧化酶(SOD)和内皮素(ET-1)含量。DT组给予精蛋白生物合成人胰岛素3U、4U于6am、6pm皮下注射控制血糖2周,于治疗前、后心脏取血0.6ml,测定上述指标。
结果：①血糖：各低血糖组血糖在注射胰岛素后1.5h达到最低点,NL组较DL、DLH组降低明显。之后NL、DL组血糖略有上升,DLH组血糖上升明显。DT组在治疗2天后血糖下降并平稳控制至2周。②MDA:NL组MDA含量在注射胰岛素后4.5h达到最高点,后逐渐下降。DL、DLH组MDA含量逐渐下降,在注射胰岛素后4.5h达到最低点,后逐渐回升。DT组治疗后MDA含量较治疗前降低。③ET-1:NL组在注射胰岛素后1.5h达最高点,后逐渐下降。DL、DLH组在注射胰岛素后6.5h明显上升,且DL组6.5h较NL组1.5h最高点升高明显,DLH组6.5h较DL组6.5h升高明显(P均<0.05)。DT组治疗后ET-1水平较治疗前下降。④SOD：各组SOD变化均无显著性差异。
结论：(1)2型糖尿病大鼠体内存在血管内皮损伤和氧化应激,应用胰岛素降低2型糖尿病大鼠血糖可以减轻其内皮的损伤和氧化应激。(2)低血糖可以损伤血管内皮细胞,糖尿病大鼠血管内皮细胞较正常大鼠更易受到低血糖损伤。(3)血糖的剧烈波动对血管内皮细胞造成的损伤比持续高血糖和低血糖更严重。(4)严重低血糖可以引起正常大鼠氧化应激。
Objective:To ascertain the influence of insulin-induced acute hypoglycemia on vascular endothelium cells(VEC) and oxidative stress of Diabetic Mice.
Method:Healthy male Wistar mice were divided randomly into normal group and diabetic group,one week after adaptability,the normal group was fed with standard laboratory diet and the diabetic group was fed with high-fat diet for 8 weeks. Then the mice in diabetic group whose body wight were above 340g were chosen and injected intraperitoneally with streptozotocin (Sigma) at a dose of 25 mg/kg body weight. One week later, the glucose concentration was determined in tail blood samples, and only those with blood glucose above 16.7 mmol/L were considered as diabetic mice.Normal mice were divided randomly into control group(NC) and hypoglycemia group(NL),streptozotocin induced type 2 diabetic male Wistar mice were divided randomly into control group(DC),hypoglycemia group(DL),hypoglycemia followed by hyperglycemia group(DLH) and treating group(DT).After 12h fasted, hypoglycemia groups were injected with Biosynthesis of human insulin.1.5h after injection,the NLH group was given 50% glucose solution 3ml intragastric administration,other groups were given normal sodium 3ml intragastric administration.Blood samples were obtained from heart at the time before injection and 1.5h,4.5h,6.5h after injection.DT group was treated with Biosynthesis of human insulin Protamine 3u,4u subcutaneous injection at 6am and 6pm for 2 weeks. Blood samples were obtained from heart before and after treatment.After detected by glucometer, the blood serum was separated and used to detected MDA,SOD and ET-1.
Result:①Blood glucose:The blood glucose of insuline-injection groups reached the minimum at 1.5h after injection,and then the NL,DL groups'blood glucose increased slightly,DLH group'blood glucose increased significantly.The blood glucose of DT group lowered down 2 days after treatment and kept stable for 2 weeks.②MDA:The MDA level of NL group reached the maximum 4.5h after injection,then decreased.DL,DLH groups' MDA level decreased and reached the minimum at the time 4.5h after injection,then increased. DT group'MDA level lowered down after treatment.③ET-1:The ET-1 level of NL group reached the maximum at 1.5h after injection,and then decreased.DL and the ET-1 level of DLH groups' elevation was more obvious at the time 6.5h after injection,and the DLH group' ET-1 level increased more higher than the DL group'.DT group'ET-1 level lowered down after treatment.④SOD:The activity of SOD in each group didn't changed significantly.
Conclusion:(1)VEC injuiry and oxidative stress exist in type 2 diabetic mice,insulin treatment can reduce the VEC injuiry and oxidative stress of Type 2 diabetic mice.(2)Hypoglycemia can lead VEC injuiry,and the diabetic endothelium cells can be injuried more easily by hypoglycemia than health groups.(3)Glucose fluctuation induces endothelial dysfunction more obviously than uncontrolled hypoglycemia and hyperglycemia.(4)Sever hypoglycemia can lead to oxidative stress.
方法：健康雄性wistar大鼠随机分为正常组和糖尿病组,适应性饲养一周后,正常组继续饲以普通饲料,糖尿病组饲以高脂饲料。8周后在高脂饲料喂养的大鼠中选取体重大于340克的大鼠,给予链脲佐菌素(STZ) 25mg/Kg体重腹腔注射。一周后尾静脉取血测定禁食6小时非空腹血糖,以GLU≥16.7mmol/L为造模成功。正常大鼠随机分为对照组(NC)和低血糖组(NL),糖尿病大鼠随机分为对照组(DC)、单纯低血糖组(DL)、低血糖后高血糖组(DLH)和糖尿病治疗组(DT)。禁食12h后,低血糖组给予生物合成人胰岛素腹腔注射。注射后1.5h,NLH组给予50%葡萄糖灌胃。除DT组外其余各组分别于胰岛素或生理盐水注射前、注射后1.5h、4.5h、6.5h心脏取血0.6ml,测定血糖、血清丙二醛(MDA)、超氧化物歧化酶(SOD)和内皮素(ET-1)含量。DT组给予精蛋白生物合成人胰岛素3U、4U于6am、6pm皮下注射控制血糖2周,于治疗前、后心脏取血0.6ml,测定上述指标。
结果：①血糖：各低血糖组血糖在注射胰岛素后1.5h达到最低点,NL组较DL、DLH组降低明显。之后NL、DL组血糖略有上升,DLH组血糖上升明显。DT组在治疗2天后血糖下降并平稳控制至2周。②MDA:NL组MDA含量在注射胰岛素后4.5h达到最高点,后逐渐下降。DL、DLH组MDA含量逐渐下降,在注射胰岛素后4.5h达到最低点,后逐渐回升。DT组治疗后MDA含量较治疗前降低。③ET-1:NL组在注射胰岛素后1.5h达最高点,后逐渐下降。DL、DLH组在注射胰岛素后6.5h明显上升,且DL组6.5h较NL组1.5h最高点升高明显,DLH组6.5h较DL组6.5h升高明显(P均<0.05)。DT组治疗后ET-1水平较治疗前下降。④SOD：各组SOD变化均无显著性差异。
结论：(1)2型糖尿病大鼠体内存在血管内皮损伤和氧化应激,应用胰岛素降低2型糖尿病大鼠血糖可以减轻其内皮的损伤和氧化应激。(2)低血糖可以损伤血管内皮细胞,糖尿病大鼠血管内皮细胞较正常大鼠更易受到低血糖损伤。(3)血糖的剧烈波动对血管内皮细胞造成的损伤比持续高血糖和低血糖更严重。(4)严重低血糖可以引起正常大鼠氧化应激。
Objective:To ascertain the influence of insulin-induced acute hypoglycemia on vascular endothelium cells(VEC) and oxidative stress of Diabetic Mice.
Method:Healthy male Wistar mice were divided randomly into normal group and diabetic group,one week after adaptability,the normal group was fed with standard laboratory diet and the diabetic group was fed with high-fat diet for 8 weeks. Then the mice in diabetic group whose body wight were above 340g were chosen and injected intraperitoneally with streptozotocin (Sigma) at a dose of 25 mg/kg body weight. One week later, the glucose concentration was determined in tail blood samples, and only those with blood glucose above 16.7 mmol/L were considered as diabetic mice.Normal mice were divided randomly into control group(NC) and hypoglycemia group(NL),streptozotocin induced type 2 diabetic male Wistar mice were divided randomly into control group(DC),hypoglycemia group(DL),hypoglycemia followed by hyperglycemia group(DLH) and treating group(DT).After 12h fasted, hypoglycemia groups were injected with Biosynthesis of human insulin.1.5h after injection,the NLH group was given 50% glucose solution 3ml intragastric administration,other groups were given normal sodium 3ml intragastric administration.Blood samples were obtained from heart at the time before injection and 1.5h,4.5h,6.5h after injection.DT group was treated with Biosynthesis of human insulin Protamine 3u,4u subcutaneous injection at 6am and 6pm for 2 weeks. Blood samples were obtained from heart before and after treatment.After detected by glucometer, the blood serum was separated and used to detected MDA,SOD and ET-1.
Result:①Blood glucose:The blood glucose of insuline-injection groups reached the minimum at 1.5h after injection,and then the NL,DL groups'blood glucose increased slightly,DLH group'blood glucose increased significantly.The blood glucose of DT group lowered down 2 days after treatment and kept stable for 2 weeks.②MDA:The MDA level of NL group reached the maximum 4.5h after injection,then decreased.DL,DLH groups' MDA level decreased and reached the minimum at the time 4.5h after injection,then increased. DT group'MDA level lowered down after treatment.③ET-1:The ET-1 level of NL group reached the maximum at 1.5h after injection,and then decreased.DL and the ET-1 level of DLH groups' elevation was more obvious at the time 6.5h after injection,and the DLH group' ET-1 level increased more higher than the DL group'.DT group'ET-1 level lowered down after treatment.④SOD:The activity of SOD in each group didn't changed significantly.
Conclusion:(1)VEC injuiry and oxidative stress exist in type 2 diabetic mice,insulin treatment can reduce the VEC injuiry and oxidative stress of Type 2 diabetic mice.(2)Hypoglycemia can lead VEC injuiry,and the diabetic endothelium cells can be injuried more easily by hypoglycemia than health groups.(3)Glucose fluctuation induces endothelial dysfunction more obviously than uncontrolled hypoglycemia and hyperglycemia.(4)Sever hypoglycemia can lead to oxidative stress.
引文
1陈名道.波动性高血糖与糖尿病并发症[J].国际内分泌代谢杂志,2006,26(5)：312.
2 Holman RR, Paul SK, Bethel MA, Matthews DR,Neil HAW.10-Year Follow-up of Intensive Glucose Control in Type 2 Diabetes[J]. The New England Journal of Medicine,2008,359(15):1577-1589.
3 The Diabetes Control and Complication Trial Research Group. The relationship of glycemic exposure (HbAlc) to the risk of development and progression of retinopathy in the diabetes control and complications trial[J]. Diabetes,1995,44(9):968.
4 quagliaro I, picomi I, Assokni R, et al. Intermittent high glucose enhances apoptosis relaxer to axilatira stress in human umbilical vein endorthelial cells.The role of protein kinase C and NAD(P)H-oxilase activation[J]. Diabets,2003,52(11):2785.
5 Monnier L, Colette C, Owens DR. Integrating glycaemic variabilityin the glycaemic disorders of type 2 diabetes:a move towards a unified glucose tetrad concept[J]. Diabetes Metab Res Rev.2009,:5.
6张汝学,贾正平,王娟等.实验性2型糖尿病大鼠模型的建立和评价(Ⅰ)-体重、血糖和肝糖原的变化[J].西北国防医学杂志,2007,28(3)：161-163.
7郑玉宝,徐希岳,王启之.一氧化氮及内皮素对肝硬化患者动脉血氧分压的影响[J].实用全科医学,2006,4(2)：140-141.
8 BUSE MG. Hexosamines, insulin resistance, and the complications of diabetes: current status[J]. Am J Physiol Endocrinol Metab.2006 Jan;290(1):E1-E8.
9 BODEN G,SHE P,MOZZ OL I M, et al. Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factor-kappaB pathway in rat liver[J]. Diabetes,2005,54(12):34582-3465.
10 Kim F et al. Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta[J]. Arterioscler Thromb Vasc Biol,2005,25(5):9892-9941.
11 The Action to Control Cardiovascular Risk in Diabetes Study Group. Effects of Intensive Glucose Lowering in Type 2 Diabetes[J].The NEW ENGLAND JOURNAL of MEDICINE,2008,358(24):2545-2559.
12 The ADVANCE Collaborative Group. Intensive Blood Glucose Control and Vascular Outcomes in Patients with Type 2 Diabetes [J].The NEW ENGLAND JOURNAL of MEDICINE,2008,358(24):2560-2572.
13 Duckworth W, Abraira C,Moritz T, Reda D, Emanuele N, Reaven PD, Zieve FJ, Marks J, Davis SN, Hayward R, Warren SR, Goldman S,McCarren M, Vitek ME, Henderson WG,Huang GD. Glucose Control and Vascular Complications in Veterans with Type 2 Diabetes[J]. The NEW ENGLAND JOURNAL of MEDICINE,2009,360(2):129-139.
14 Philip E. Cryer, Stephen N. Davis, Happy Shamoon. Hypoglycemia in Diabetes[J]. Diabetes Care,2003,26:1902-1912.
15 El-Osta A, Brasacchio D, Yao D, et al. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia[J].Exp Med,2008,205:2409-2417.
16范立群,韦龙祥,彭鹰.糖尿病和氧化应激的研究进展[J].右江民族医学院学报,2007，(4)：629-631.
17徐贵森,刘合年,杨淑霞,等.缺血再灌注脊髓丙二醛含量和超氧化物歧化酶活性的变化[J].西南军医,2006,8(2)：10-11.
18 Signorini AM, Fondelli C, Renzeni E et al. Antioxidant effects of gliciazide, glibenclamide, and metformin in patients with type 2 diabete mellitus[J]. Current Theraputic Res,2002,63 (7):411-420.
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2 Holman RR, Paul SK, Bethel MA, Matthews DR,Neil HAW.10-Year Follow-up of Intensive Glucose Control in Type 2 Diabetes[J]. The New England Journal of Medicine,2008,359(15):1577-1589.
3 The Diabetes Control and Complication Trial Research Group. The relationship of glycemic exposure (HbAlc) to the risk of development and progression of retinopathy in the diabetes control and complications trial[J]. Diabetes,1995,44(9):968.
4 quagliaro I, picomi I, Assokni R, et al. Intermittent high glucose enhances apoptosis relaxer to axilatira stress in human umbilical vein endorthelial cells.The role of protein kinase C and NAD(P)H-oxilase activation[J]. Diabets,2003,52(11):2785.
5 Monnier L, Colette C, Owens DR. Integrating glycaemic variabilityin the glycaemic disorders of type 2 diabetes:a move towards a unified glucose tetrad concept[J]. Diabetes Metab Res Rev.2009,:5.
6张汝学,贾正平,王娟等.实验性2型糖尿病大鼠模型的建立和评价(Ⅰ)-体重、血糖和肝糖原的变化[J].西北国防医学杂志,2007,28(3)：161-163.
7郑玉宝,徐希岳,王启之.一氧化氮及内皮素对肝硬化患者动脉血氧分压的影响[J].实用全科医学,2006,4(2)：140-141.
8 BUSE MG. Hexosamines, insulin resistance, and the complications of diabetes: current status[J]. Am J Physiol Endocrinol Metab.2006 Jan;290(1):E1-E8.
9 BODEN G,SHE P,MOZZ OL I M, et al. Free fatty acids produce insulin resistance and activate the proinflammatory nuclear factor-kappaB pathway in rat liver[J]. Diabetes,2005,54(12):34582-3465.
10 Kim F et al. Free fatty acid impairment of nitric oxide production in endothelial cells is mediated by IKKbeta[J]. Arterioscler Thromb Vasc Biol,2005,25(5):9892-9941.
11 The Action to Control Cardiovascular Risk in Diabetes Study Group. Effects of Intensive Glucose Lowering in Type 2 Diabetes[J].The NEW ENGLAND JOURNAL of MEDICINE,2008,358(24):2545-2559.
12 The ADVANCE Collaborative Group. Intensive Blood Glucose Control and Vascular Outcomes in Patients with Type 2 Diabetes [J].The NEW ENGLAND JOURNAL of MEDICINE,2008,358(24):2560-2572.
13 Duckworth W, Abraira C,Moritz T, Reda D, Emanuele N, Reaven PD, Zieve FJ, Marks J, Davis SN, Hayward R, Warren SR, Goldman S,McCarren M, Vitek ME, Henderson WG,Huang GD. Glucose Control and Vascular Complications in Veterans with Type 2 Diabetes[J]. The NEW ENGLAND JOURNAL of MEDICINE,2009,360(2):129-139.
14 Philip E. Cryer, Stephen N. Davis, Happy Shamoon. Hypoglycemia in Diabetes[J]. Diabetes Care,2003,26:1902-1912.
15 El-Osta A, Brasacchio D, Yao D, et al. Transient high glucose causes persistent epigenetic changes and altered gene expression during subsequent normoglycemia[J].Exp Med,2008,205:2409-2417.
16范立群,韦龙祥,彭鹰.糖尿病和氧化应激的研究进展[J].右江民族医学院学报,2007，(4)：629-631.
17徐贵森,刘合年,杨淑霞,等.缺血再灌注脊髓丙二醛含量和超氧化物歧化酶活性的变化[J].西南军医,2006,8(2)：10-11.
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