1型糖尿病大鼠血管内皮细胞损伤后血生化学指标变化及锌原卟啉、正铁血红素的干预效应

Changes of blood biochemical indexes after damage of vascular endothelial cells in type 1 diabetic rats and interventional effects of zinc protoporphyrin and ferriheme

目的:观察1型糖尿病血管内皮细胞损伤后内源性一氧化碳、血清总抗氧化能力、丙二醛、游离脂肪酸水平变化,以及锌原卟啉、正铁血红素对这种变化的干预结果。方法:实验于2004-10/2005-06在河北省人民医院老年医学省重点实验室完成。①选用健康雄性SD大鼠89只,8周龄,体质量240～260g。②取17只为对照组:腹腔仅注射柠檬酸缓冲液,4d后隔日腹腔注射生理盐水。将其余72只造成1型糖尿病模型:大鼠按50mg/kg一次性腹腔注射链脲佐菌素(溶于pH4.5的柠檬酸缓冲液中),4d后,有60只大鼠空腹血糖>16mmol/L为1型糖尿病模型复制成功。将其随机分为3组:1型糖尿病组(n=36):造模4d后隔日腹腔注射生理盐水,饲养4,8和12周后处死12,14,10只;正铁血红素+1型糖尿病组(n=12):造模4d后隔日给1型糖尿病大鼠腹腔注射正铁血红素30μmol/kg,连续4周后处死;锌原卟啉+1型糖尿病组(n=12):造模4d后隔日给1型糖尿病大鼠腹腔注射锌原卟啉10μmol/kg,连续4周后处死。③处死前采用Chrion855血气分析仪检测动脉血中碳氧血红蛋白水平,以其反映一氧化碳水平。采用比色法检测血清总抗氧化能力,丙二醛和游离脂肪酸水平。扫描电镜观察股动脉内皮超微结构。④多组均数比较采用单因素方差分析,组间比较采用LSD法。结果:由于造模失败脱失12只,最终进入结果分析77只。①动脉血碳氧血红蛋白水平:1型糖尿病大鼠4周明显低于对照组(P<0.01),正铁血红素+1糖尿病组干预4周后明显高于1型糖尿病组干预4周后(P<0.05),干预8,12周后低于对照组,但差异不明显。②血清总抗氧化能力水平:1型糖尿病大鼠呈时间依赖性降低,均明显低于对照组(P<0.01),锌原卟啉+1型糖尿病组干预4周后明显低于1型糖尿病组干预4周后(P<0.05)。③血清丙二醛水平:1型糖尿病大鼠呈时间依赖性升高,其中干预8,12周后明显高于对照组(P<0.01),锌原卟啉+1型糖尿病组干预4周后明显高于1型糖尿病组干预4周后(P<0.05)。④血清游离脂肪酸水平:锌原卟啉+1型糖尿病组干预4周后明显高于1型糖尿病组干预4周后(P<0.05),1型糖尿病组干预12周后明显高于干预4周后(P<0.05)。⑤股动脉内皮超微形态学观察结果:对照组内皮细胞形态结构正常。1型糖尿病组内皮细胞出现损伤,干预8,12周后可见内皮细胞更严重损伤;应用正铁血红素后1型糖尿病大鼠内皮细胞形态接近对照组;锌原卟啉+1型糖尿病组大鼠内皮细胞形态异常。结论:随病程进展1型糖尿病大鼠内皮细胞抗氧化能力逐渐降低,脂代谢紊乱更加严重,内源性一氧化碳水平下降,并逐渐接近正常。锌原卟啉可加重血管内皮细胞结构损伤,应用正铁血红素可减轻血管内皮细胞结构损伤。

AIM： To observe the changes of the serum total antioxidative capacity, levels of endogenous carbon monoxide, malondialdehyde （MDA）, free fatty acid after vascular endothelial cell injury in type 1 diabetic rats, and the interventional effect of zinc protoporphyrin and ferriheme on these changes. METHODS： The experiment was finished in Geriatric Key Laboratory, Hebei Provincial People＇s Hospital between October 2004 and June 2005. （1）Totally 89 healthy male SD rats of 8 weeks with the body mass of 240- 260 g were selected. （2）Seventccn rats was as control group： Only citrate buffer solution was injected into abdominal cavity, 4 days later saline was injected by intraperitoneal injection every other day. Other 72 rats were established into type 1 diabetic models： The rats received streptozotocin once by intraperitoneal injection based on 50 mg/kg （dissolving in ph 4.5 citrate buffer solution）. Four days later, 60 rats with fasting blood glucose 〉16 mmol/L indicated the success of type 1 diabetic models. The rats were randomly assigned into 3 groups： type 1 diabetic group （n=36）. Four days after establishing models the saline was injected by intraperitoneal injection every other day, and killed 12, 14 and 10 rats after 4, 8 and 12 weeks, respectively. For the ferriheme ＋ type 1 diabetic group （n=12）： animals were treated every other day with abdominal injections of ferriheme （30 μmol/kg） for 4 weeks, and then killed. Zinc protoporphyrin ＋ type 1 diabetic group （n=12）： the rats were treated every other day with abdominal injections of zinc protoporphyrin （10 μmol/kg） for 4 weeks, and then killed. （3）The level of carbonylhemoglobin was determined in arterial blood with Chrion 855 blood gas analyzer before being killed to reflect the level of carbon monoxide. The total antioxidative capacity, the levels of MDA and free fatty acid in sera were assayed with chromatometry. Endothelial ultramicrostructure of arteria femoralis was observed with scanning electron microscope （SEM）. （4）Multiple groups means were compared with single factor analysis of variance, and the comparison among groups was performed with LSD method. RESULTS： Totally 77 rats were involved in the result analysis, because of failure of establishing in 12 models. （1）Level of carbonylhaemoglobin of arterial blood： It was significantly lower at week 4 in type 1 diabetic rats than that in the control group （P 〈 0.01 ）, and it was obviously higher in the ferriheme ＋ type 1 diabetic group than that in the type 1 diabetic group after intervention for 4 weeks （P 〈 0.05）, and lower than that in the control group after intervention for 8 and 12 weeks, but the difference was not marked. （2）Level of serum total antioxidative capacity： It decreased in the type 1 diabetic rats and had time-dependent effect, which was remarkably lower than that in the control group （P 〈 0.01）. It was distinctly lower in the zinc protoporphyrin ＋ type 1 diabetic group than that in the diabetic group after intervention for 4 weeks （P 〈 0.05）. （3） Level of serum MDA： It increased in the type 1 diabetic rats and had timedependent effect, and it was dramatically higher at weeks 8 and 12 than that in the control group （P 〈 0.01 ）. It was distinctly higher in the zinc protoporphyrin ＋ type 1 diabetic group than that in the diabetic group after intervention for 4 weeks （P 〈 0.05）. （4）Level of serum free fatty acids： It was distinctly higher in the zinc protoporphyrin ＋ type 1 diabetic group than that in the diabetic group after intervention for 4 weeks （P 〈 0.05）. It was obviously higher at week 12 than that at week 4 in the type 1 diabetic group （P 〈 0.05）. （5）Observational results of endothelial ultramicromorphology of arteria femoralis： The morphology was normal in the control group. Endothelial cells were damaged in the type 1 diabetic group, and had more severe injury at weeks 8 and 12. The endothelial morphology in type 1 diabetic rats was similar to that of the control group after receiving ferriheme. The endothelial morphology was abnormal in rats of the zinc protoporphyrin ＋ type 1 diabetic group. CONCLUSION： The antioxidative capacity reduces gradually in the type 1 diabetic rat with the progress, disorder of lipid metabolism becomes more severe, and the level of endogenous carbon monoxide is near to the normal gradually. The zinc protoporphyrin can increase the injury of vascular endothelial cell. The ferrriheme can relieve the damage of vascular endothelial cell.