糖尿病大鼠脑能量代谢改变的核磁共振磷谱研究

王娜等
摘要应用链脲佐菌素 (Streptozocin, STZ)制备糖尿病 (Diabetes mellitus, DM)大鼠模型,采用离体的核磁共振磷谱 (31P Magnetic resonance spectroscopy, MRS)方法检测糖尿病大鼠脑组织的生化改变。全脑的31P MRS谱图结果显示,STZ诱导1周后,磷酸单酯和磷酸二酯的含量无明显改变,表明糖尿病大鼠脑中并没有发生膜性结构的改变。二磷酸腺苷峰增高,磷酸肌酸 (Phosphocreatine, PCr)和三磷酸腺苷 (Adenosine triphosphate, ATP)含量无明显改变,但是PCr/ATP降低,说明PCr作为能量缓冲底物维持能量平衡。此外,pH值降低,表明在糖尿病大鼠脑中细胞内环境改变。当糖尿病发展到15周时,磷脂膜代谢和脑能量代谢紊乱。31P MRS 不仅能够无创性提供磷脂代谢情况及能量状况,还可以测得细胞内pH值等方面的生化信息,有助于理解糖尿病脑病的发病机制,并为临床的早期诊断和治疗提供理论依据。
1引言
糖尿病 (Diabetes mellitus, DM)是一种以血糖升高为特征的代谢紊乱综合征,其并发症遍及全身各处\[1\]。其中,糖尿病脑病是糖尿病三大并发症之一,主要表现为认知功能障碍和神经行为缺陷\[2\]。研究表明,糖尿病患者脑中蛋白质结构和功能异常\[3\]以及灰质的体积和密度减少\[4\], 并且,糖尿病患者出现痴呆的危险性增加\[5\],并发抑郁症的几率是正常人的2倍\[6\]。目前,应用各种先进技术研究脑能量代谢与脑病的关系已成为当今脑病研究的热点之一。然而,糖尿病脑病与脑能量代谢变化的机制尚不清楚。
核磁共振波谱 (Magnetic resonance spectroscopy, MRS)是目前唯一可以用作活体无损伤的检测细胞水平能量代谢变化的非侵入性技术,对了解多种疾病的生化、病理生理变化以及疾病的早期诊断都具有极其重要的应用价值。本研究组已利用MRS技术对肝硬化、糖尿病等疾病的发病机制进行了研究[7,8],结果表明,MRS技术能够很好地描述不同疾病状态的代谢模式。31P MRS不仅能够测得生理、病理状态下能量代谢产物,如三磷酸腺苷 (Adenosine triphosphate, ATP)、磷酸肌酸 (Phosphocreatine, PCr)和无机磷 (Inorganic phosphate, Pi),还可根据Pi与PCr化学位移的相对差值计算细胞内的pH值。以此判断细胞目前的能量代谢状况及细胞的受损伤情况。此外,31P MRS 还可以检测到磷酸单酯 (Phosphomonoester, PME)和磷酸二酯 (Phosphodiester, PDE),借以判断细胞膜结构的完整性。本研究应用31P MRS检测不同时期糖尿病大鼠脑代谢的变化,以期进一步了解糖尿病脑损伤的病理机制,为糖尿病脑病的临床早期诊断和治疗提供理论依据。
2实验部分
2.1仪器与试剂
Bruker AVANCE III 600核磁共振谱仪 (Bruker BioSpin International AG);血糖仪 (德国贝朗医疗国际贸易有限公司);冷冻干燥机 (FD1,北京德天佑科技发展有限公司);匀浆机 (上海弗鲁克流体机械制造有限公司)。
甲醇和氯仿 (分析纯,上海国药集团化学试剂有限公司);链脲佐菌素 (Streptozocin, STZ)\, 柠檬酸和柠檬酸钠(美国SigmaAldrich公司);重水 (D2O,99.9%氘代,剑桥同位素实验室);纯水由MilliQ 超纯水系统 (Millipore, Billerica, MA, USA)制得;SpragueDawley (SD)大鼠 (上海斯莱克实验动物有限责任公司)。
2.2I型糖尿病模型的建立
取32只雄性SD大鼠 (180 ± 20) g,分笼饲养,室温(25±3)℃,相对湿度为 50%±10%,12 h交替照明。实验期间大鼠自由饮水、进食。适应喂养1周后,随机分为两组,一组大鼠腹腔注射新鲜配制的STZ柠檬酸钠混悬溶液(70 mg/kg),另一组大鼠注射同体积的柠檬酸钠混悬溶液 (0.10 mol/L,pH 4.5) 作为对照组。STZ注射72 h后测其血糖水平,选取血糖值大于16.70 mmol/L大鼠为糖尿病大鼠。
2.3SD大鼠脑组织的收集和制备
大鼠在STZ诱导1周和15周后断头处死,并将收集的脑组织样本迅速浸入液氮中急冻,做好标记后置于
Symbolm@@ 80 ℃保存。冰冻的脑组织样本称重后,采用甲醇氯仿水提取法提取水溶性的小分子代谢物。在液氮中冷冻后放入真空冻干机中冻干24 h,得到脑代谢物粉末。
2.4脑组织提取物的31P MRS检测
在进行NMR实验前,将得到的脑代谢物粉末再重新溶解于550 μL D2O中,在低温离心机中离心10 min后取上清液,转入NMR样品管中进行测试。使用本实验室的Bruker AVANCE III 600 MHz NMR谱仪采集脑组织样本的31P NMR谱,31P的共振频率为242.9 MHz。实验温度为298 K,累加次数8192,实验中的脉冲翻转角为30°,重复时间为3.5 s,谱宽为20000 Hz,WALTZ16脉冲去耦,采样点数为16 K, 傅里叶变换前FID充零至32 K。所得的31P NMR谱图经傅里叶变换,指定PCr的化学位移为δ
Symbolm@@ 2.33\[9\], 从而确定31P NMR谱上PME、PDE和ATP等信号的化学位移。
实验数据应用SPSS 13.0统计软件处理,以均值±标准差表示,两组间比较采用独立样品t检验。
3结果与讨论
3.1造模结果
STZ诱导的糖尿病大鼠模型是一种与胰岛素水平显著减少相关的I型糖尿病模型,其代谢特征类似于I型糖尿病病人,能较好地模拟人类糖尿病状态下的各种变化\[10,11\]。如表1所示,本实验中对照组大鼠体重随着时间的延长逐渐增加,血糖值与实验前相比无明显变化。相对于年龄匹配的正常组大鼠,1周和15周DM组大鼠体重随糖尿病病程的延长显著下降 (p<0.001),血糖值显著升高 (p<0.001)。在7~8周,有些糖尿病大鼠的眼睛出现晶状体混浊;在11~12周后晶状体完全混浊,发展成白内障,这个结果与文献\[1\]一致。
3.3糖尿病大鼠脑中代谢物含量变化
通过对各组峰面积的积分,得出各种化合物的相对浓度,根据这些化合物相对浓度的比值,可以提供组织代谢的某些信息。表2是年龄匹配的对照组大鼠和糖尿病1周和15周组大鼠t检验统计学分析的结果。
通过对这些物质相对含量的检测以及细胞内pH值的计算,可判断细胞目前的能量代谢状况以及细胞的受损伤情况。本实验结果 (表2)表明,糖尿病1周大鼠脑中pH值降低,说明大鼠患糖尿病1周后,脑中细胞内环境发生了改变。
综上所述,31P NMR避免了对细胞等生物样品的破坏和损伤, 减少复杂的分离纯化等操作,在不影响细胞代谢变化的条件下,给出了细胞内ATP、PCr、PME等含磷化合物的信号。研究结果表明,STZ诱导1周后,患糖尿病大鼠的脑中未发现膜结构改变,PCr作为能量缓冲底物维持脑能量代谢处于平衡状态,而细胞内环境发生改变。当大鼠患糖尿病15周后,磷脂膜代谢和脑能量代谢严重紊乱。因此,31P MRS方法不仅能够无创性提供磷脂代谢情况及能量状况,还可以测得细胞内pH值等方面的生化信息。这些结果有助于理解糖尿病脑病的发病机制,并为临床的早期诊断和治疗提供理论依据。
References
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14RuizCabello J, Cohen J S. NMR Biomed., 1992, 5(5): 226-233
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16Biessels G J, Braun K P, de Graaf R A, van Eijsden P, Gispen W H, Nicolay K. Diabetologia, 2001, 44(3): 346-353
AbstractConsiderable attention has been directed toward studying the impact of diabetes on the central nervous system. The current study investigates the biochemical changes in the brain tissue of streptozotocin (STZ)induced diabetic rat using 31P magnetic resonance spectroscopy (31P MRS). The 31P NMR spectra of the whole brain show no significant changes of phosphomonoesters and phosphodiesters levels one week after STZ induction, suggesting no apparent structural changes in cell membranes. The results identifies the increased level of adenosine diphosphate, negligible changes of phosphocreatine (PCr) and adenosine triphosphate (ATP) , but the decreased ratio of PCr/ATP, indicating that PCr plays a role of balancing the energy. Moreover, the decreased pH value indicates the changes of the intracellular environment in STZdiabetic brains in rats. After 15 weeks of STZ injection, the metabolism of phospholipid membrane and brain energy metabolism has been obviously disturbed. Our study successfully shows that 31P MRS can not only study phospholipid and energy metabolism noninvasively, but also measure intracellular pH and other important biochemical information. All of these spectroscopic characterizations contribute significantly to the understanding of pathogenesis and evolution of diabetes, and provide theoretical basis for early diagnosis and clinical treatment in diabetes.
KeywordsDiabetes mellitus; Brain energy metabolism; 31P magnetic resonance spectroscopy
14RuizCabello J, Cohen J S. NMR Biomed., 1992, 5(5): 226-233
15Boutilier R G. J. Exp. Biol., 2001, 204(18): 3171-3181
16Biessels G J, Braun K P, de Graaf R A, van Eijsden P, Gispen W H, Nicolay K. Diabetologia, 2001, 44(3): 346-353
AbstractConsiderable attention has been directed toward studying the impact of diabetes on the central nervous system. The current study investigates the biochemical changes in the brain tissue of streptozotocin (STZ)induced diabetic rat using 31P magnetic resonance spectroscopy (31P MRS). The 31P NMR spectra of the whole brain show no significant changes of phosphomonoesters and phosphodiesters levels one week after STZ induction, suggesting no apparent structural changes in cell membranes. The results identifies the increased level of adenosine diphosphate, negligible changes of phosphocreatine (PCr) and adenosine triphosphate (ATP) , but the decreased ratio of PCr/ATP, indicating that PCr plays a role of balancing the energy. Moreover, the decreased pH value indicates the changes of the intracellular environment in STZdiabetic brains in rats. After 15 weeks of STZ injection, the metabolism of phospholipid membrane and brain energy metabolism has been obviously disturbed. Our study successfully shows that 31P MRS can not only study phospholipid and energy metabolism noninvasively, but also measure intracellular pH and other important biochemical information. All of these spectroscopic characterizations contribute significantly to the understanding of pathogenesis and evolution of diabetes, and provide theoretical basis for early diagnosis and clinical treatment in diabetes.
KeywordsDiabetes mellitus; Brain energy metabolism; 31P magnetic resonance spectroscopy
14RuizCabello J, Cohen J S. NMR Biomed., 1992, 5(5): 226-233
15Boutilier R G. J. Exp. Biol., 2001, 204(18): 3171-3181
16Biessels G J, Braun K P, de Graaf R A, van Eijsden P, Gispen W H, Nicolay K. Diabetologia, 2001, 44(3): 346-353
AbstractConsiderable attention has been directed toward studying the impact of diabetes on the central nervous system. The current study investigates the biochemical changes in the brain tissue of streptozotocin (STZ)induced diabetic rat using 31P magnetic resonance spectroscopy (31P MRS). The 31P NMR spectra of the whole brain show no significant changes of phosphomonoesters and phosphodiesters levels one week after STZ induction, suggesting no apparent structural changes in cell membranes. The results identifies the increased level of adenosine diphosphate, negligible changes of phosphocreatine (PCr) and adenosine triphosphate (ATP) , but the decreased ratio of PCr/ATP, indicating that PCr plays a role of balancing the energy. Moreover, the decreased pH value indicates the changes of the intracellular environment in STZdiabetic brains in rats. After 15 weeks of STZ injection, the metabolism of phospholipid membrane and brain energy metabolism has been obviously disturbed. Our study successfully shows that 31P MRS can not only study phospholipid and energy metabolism noninvasively, but also measure intracellular pH and other important biochemical information. All of these spectroscopic characterizations contribute significantly to the understanding of pathogenesis and evolution of diabetes, and provide theoretical basis for early diagnosis and clinical treatment in diabetes.
KeywordsDiabetes mellitus; Brain energy metabolism; 31P magnetic resonance spectroscopy
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