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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。
Objective To study the changes of brain metabolism in rats with recurrent or severe hypoglycemia using ~ 1H NMR-based metabolomics, and to explore the pathogenesis of recurrent severe hypoglycemic brain injury in diabetic rats by pathological examination. Methods 24 7-week-old Sparague Dawley rats were randomly divided into experimental group and control group, with 12 rats in each group. The rats in experimental group were fasted for 12 hours and were given diabetes by intraperitoneal injection of streptozotocin solution (65ml / kg). Serum hypoglycemia was induced by daily intraperitoneal injection of insulin (4-8U / kg) for 5 consecutive days. The control group were given the same amount of citrate buffer and saline during this period. The hippocampus, frontal lobe, parietal lobe and occipital lobe of 6 rats in each group were detected by 1H NMR spectroscopy and the other 6 glial fibrillary acidic protein (GFAP) staining. Using t test for statistical analysis. Results In the experimental group, the blood glucose level before hypoglycemia was 12.25 ± 2.64mmol / L, the blood glucose level at 1h after insulin injection was 1.11 ± 0.45mmol / L, and the control group’s blood glucose level was 6.88 ± 0.59mmol / L during the experiment. All experimental rats in clinical manifestations during severe hypoglycemia, lack of mobility, decreased body temperature, loose limbs, pale skin, convulsions in 4 severe cases. Compared with the control group, the concentration of lactate in each brain area increased and glutamine decreased. Meanwhile, the content of alanine and succinate in occipital lobe decreased. The amount of choline in frontal and parietal lobe decreased. GFAP staining showed that compared with the control group, the number of astrocytes in each brain region in experimental group increased significantly (P <0.05). Conclusions The metabolic changes in the brain after repeated severe hypoglycemia of diabetes mellitus and regional differences exist, with energy metabolism disorders and glutamate - glutamine cycle disorders mainly caused by severe diabetes mellitus after the main hypoglycemia caused by glial injury, metabolic changes and Related to glial injury.