糖尿病大鼠反复严重低血糖后脑内代谢改变的1H NMR波谱研究

目的 运用基于¹H NMR的代谢组学方法研究糖尿病大鼠反复严重低血糖后脑内代谢的变化，并结合病理学检查探讨糖尿病大鼠反复严重低血糖脑损伤的发病机制。方法 24只7周龄Sparague Dawley大鼠随机分为实验组和对照组，每组12只。实验组大鼠禁食12h，通过腹腔注射链脲佐菌素溶液（65ml/kg）造成糖尿病，此后连续5天每天腹腔注射胰岛素（4~8U/kg）诱导严重低血糖。对照组则在此期间分别给予等量柠檬酸缓冲液及生理盐水。每组6只大鼠的海马和额叶、顶叶、枕叶进行¹H NMR波谱检测，另外6只进行胶质纤维酸性蛋白（GFAP）染色。采用t检验进行统计学处理。结果 实验组大鼠诱导低血糖前血糖值为12.25±2.64mmol/L，注射胰岛素后1h血糖值为1.11±0.45mmol/L，对照组实验期间血糖值6.88±0.59mmol/L。所有实验组大鼠在严重低血糖期间临床表现为精神不振、活动度减弱、体温下降、肢体松软、皮肤苍白，4只严重者出现抽搐。相比对照组，各脑区乳酸浓度升高、谷氨酰胺下降，同时枕叶丙氨酸和琥珀酸含量下降，额、顶叶还存在胆碱含量下降。GFAP染色显示，相比对照组，实验组各脑区的星形胶质细胞数目均显著性增加（P<0.05）。结论 糖尿病反复严重低血糖后脑内代谢改变且存在区域性差异，以能量代谢障碍及谷氨酸-谷氨酰胺循环紊乱为主，糖尿病反复严重低血糖后主要引起神经胶质细胞损伤，代谢改变与神经胶质细胞损伤相关。

CerebralMetabolism in the Diabetic Rats after Recurrent Profound Hypoglycemia:an 1H Nuclear Magnetic Resonance Spectroscopy Study

Objective A high resolution 1H nuclear magnetic resonance spectroscopy(¹H NMR) of metabonomics approach was applied to detect the metabolic changes in the diabetic rats after recurrent profound hypoglycemia, and combined with histopathological examination to explore the mechanism of repeated severe hypoglycemic brain injury in the diabetic rats. Methods Seven-week-old male Sprague-Dawley rats were randomly assigned to normal control group(Con, n=12) and diabetes comorbid recurrent hypoglycemia group(DH, n=12). All the rats were fasted for 12 hours. After that, DH group rats received intraperitoneal injections of streptozotocin(65mg/kg) to induce diabetes, then they were given an insulin injection ip at a dose of 4-8U/kg to induce severe hypoglycemia for 5 consecutive days. Con group received the same amount of sodium citrate buffer and normal saline respectively. Glial fibrillary acidic portein(GFAP) was used to study the changes of glial cells in six rats hippocampus, frontal lobe, parietal lobe and occipital lobe in each group, and high resolution MRS was used to study the metabolic changes in these four brain regions of the others rats. The difference between two groups was compared by t test. Results Changes of plasma glucose concentrations:the glucose levels of DH group before and 1 hour after the injection of insulin were 12.25±2.64mmol/L and 1.11±0.45mmol/L respectively. During the experiment, the blood glucose values of the Con group was 6.88±0.59mmol/L. Clinical manifestations:during severe hypoglycemia, depression, hypokinesia, hypothermia, soft body and pale skin were noted in the DH animals, and four of them had seizure. Metabolic changes:compared to the Con group, significantly increased level of lactate and decreased level of glutamine were found in the four brain regions. Besides, the concentration of alanine and succinate was sharply decreased in the ocipital lobe, and Choline was decreased in the frontal and parietal lobe. GFAP staining:compared to the Con group, the number of astrocytes in the hippocampus, frontal, parietal and occipital lobe of DH group rats were significantly increased (P<0.05), of which the maximum number of GFAP positive cells was in the hippocampus. Conclusion Diabetes comorbid recurrent severe hypoglycemia induces regional specific metabolic changes in the brain, which mainly contain dysfunction of energy metabolism and Glu-Gln cycle. Diabetes comorbid recurrent severe hypoglycemia causes glia cells injury, which may associate with the metabolic changes.