短期糖尿病时糖尿病并发症相关物质的研究
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摘要
糖尿病(diabetes mellitus，DM)是以胰岛素相对或绝对不足引起的以糖代谢紊乱为特征的代谢性疾病。若不及时治疗，会出现多种并发症，如疼痛性外周神经病变、损伤性炎症反应、血管通透性发生变化，导致许多心血管并发症，增加高血压和中风的发病率。
背根神经节(dorsal root ganglion，DRG)在糖尿病状态下十分脆弱，对微血管病具有特殊的易感性，尤其在长期糖尿病时DRG中肽能初级感觉神经系统退化，说明DRG是糖尿病的直接靶位，参与糖尿病多种神经病的发生发展。DRG神经元中含有CGRP，COX，SP等神经递质，当内环境发生变化时，通过改变这些递质的合成和释放而影响神经血管的功能。
降钙素基因相关肽(calcitonin gene-related peptide，CGRP)广泛分布于脑、脊髓，常与其他神经递质共存于一个神经元中。在外周，CGRP主要存在于感觉神经节，如三叉神经节(trigeminal ganglion，TG)和DRG中，常与P物质(substance P)和环氧化酶-2(cyclooxygenase，COX-2)共存，在内环境发生变化时，通过血浓度的变化，共同参与疼痛和炎症等有害刺激反应。糖尿病时，SP和CGRP从DRG的顺行转运受阻，感觉神经元和血浆中的CGRP会发生改变，同时血管对CGRP的反应性减弱，CGRP释放减少。CGRP的这些改变是糖尿病时疼痛增加和发生损伤性炎症的重要原因。
COX-2基因是8.3kb的即早基因，是几年前发现的COX-1的异形体，主要与组织损伤性炎症前列腺素(prostaglandins，PGs)的合成有关，COX-2诱导(基因表达增加)的直接结果是以PGE_2为主的炎性PGs产物合成增加。COX-2表达升高不仅是糖尿病的危险因子之一，而且，在糖尿病时，血糖升高，COX-2 mRNA合成的量及其活性增加，增加PG产物的合成，引起糖尿病有关的痛觉过敏和异常疼痛以及血管并发症。
当正常生理状态下，循环灌注压降低时，血管壁感觉神经元释放CGRP，CGRP反过来激活腺苷酸环化酶，使cAMP增加，激活ATP敏感性钾通道(K_(ATP))，扩张动脉，以保持稳定的血流。K_(ATP)是一类受细胞内ATP浓度
(【ATp〕i)控制的内向整流钾通道。目前已经在许多细胞上发现有KATP的
存在，发挥着重要的生理和病理生理功能。许多内源性扩血管物质部分是
通过激活动脉平滑肌的K^TP而发挥作用。糖尿病时，血管舒张功能的损伤，
即K人二的开放功能减弱。这种变化促使糖尿病血管并发症得以发展，如动
脉硬化，冠心病，高血压等。
因此研究糖尿病时，DRG中CGRP和COX一2以及血管平滑肌K，功
能的变化，对阐明糖尿病神经血管并发症发生机制非常重要。本研究的目
的是研究在短期糖尿病(<2 months)的不同阶段，(l)通过对DRG中
CGRP mRNA、DRG和主动脉COX一2 mRNA的合成以及血中相应产物
CGRP和PGE:的研究，阐明CGRP和COX一2在短期糖尿病的特异改变;
(2)通过对血管平滑肌细胞KATP电生理特性的研究，进一步揭示糖尿病血
管并发症的发病机理，为糖尿病并发症的早期防治提供理论依据。
方法
将雄性Wistar大鼠随机分为对照组和糖尿病模型组。链佐霉素(strep一’
tozotoein，S咒)溶于冰冷的0.05M的构椽酸钠溶液中(PH=5.0)，模型组
给予STZ 60m扩kg(1.p.)，对照组等容量注射溶剂。注射STZ一周后，尾
静脉采血，测血糖(blood glueose，BG)。BG>16 rnmol/L者，定为糖尿病模
型成功，将糖尿病大鼠随机分为四组，即糖尿病1周组(lw)、糖尿病2周组
(Zw)，糖尿病3周组(3w)、糖尿病4周组(4w)和糖尿病8周组(sw)。
将大鼠麻醉，直视下心脏采静脉血，4℃，3000甲m离心10 min分离血
浆，一70℃保存。
心脏取血后，剪下并分离胸腹主动脉，于脊柱两侧取胸腰段DRG约10
只，迅速置于液氮中，之后再转移至一70℃低温冰箱中贮存。
肠系膜动脉急性分离，选用细胞膜完整，有立体感，胞体呈梭形的细胞
进行全细胞记录ATp敏感性钾电流(IKATP)。用膜片电极进行千兆封接。
全细胞状态形成后，用Pelamps .5 .1软件程序发放刺激并收集记录信号。
设保持电位为一40 mv，指令电位为一100一+50mv，步阶脉冲10mv，波宽
300 ms，刺激间隔65的方波钳制方案进行刺激。
根据异硫氢酸肌一酚:氯仿(T班201)抽提一步法提取大鼠DRG和主动
脉总RNA，首先进行逆转录，以p一肌动蛋白(p一actin)作为本实验的内参
照物，总RNA经逆转录后以CGRP，COx一2和p一ac血引物进行PCR扩
增，扩增后取适量PCR扩增产物进行2%琼脂糖电泳，嗅化乙锭(EB)染色，
在自动凝胶成像分析仪上，以Cox一2扩增条带与p一ac血扩增条带的荧
光强度比评定其mRNA水平。CGRP，COX一2和p一actin扩增长度分别为
gobp，584bp和422bp。
按照放免药盒说明书所介绍的方法对样品进行处理，检测血中CGRP
和PGE:的浓度。
结果
STZ腹腔注射次日血糖升高，一周后即可出现糖尿病。在短期糖尿病，
以CGR]刀p一actin为半定量指标，DRG中CGRP mRNA有先降后升的趋
势，对照组为1.15土0.19，糖尿病lw，3w，4w，sw组分别为0.934土0.10，
0.822土0.083，1‘272土0.18，1.47土0.18;血中CGRP的变化趋势与DRG
中CGRp mRNA的变化是反向的。以COx一2/p一actin为半定量指?
Aim
Diabetes mellitus ( DM) is a metabolic disease characterized by disorder of glucose metabolism induced by absolute or relative deficiency of insulin. Kinds of complications will be induced if DM cannot be controlled effectively on time, such as pain peripheral neuropathy and inflammatory reaction. The change in vascular permeability induced by DM results in cardiovascular complications, which increase morbidity of hypertension and stroke.
Dorsal root ganglion ( DRG) is very vulnerable in DM, and particularly susceptible to microangiopathy. DRG is a direct target of diabetes damage. In long - term diabetes, degeneration of peptidergic primary afferent nerve system accounts for the early sensory abnormalities in diabetes and the development of neuropathy complication.
Calcitonin gene - related peptide ( CGRP) distributes in neurons of brain tissue and spinal cord. In peripheral system, CGRP mainly exists in sensory ganglions, such as trigeminal ganglion ( TG) and DRG, and usually concomitant with other neural transmitters, such as substance P (SP). Cyclooxygenase - 2 ( COX - 2) also coexists with it. In long - term DM, blockade of direct transportation of CGRP and SP results in the decrease of CGRP in blood and sensory neuron and attenuation of response to CGRP of vessels. All of these changes participate in increasing of nociceptive reaction and allodynia in diabetes.
COX-2 gene is a small (8. 3kb) immediate early gene, an isoform of COX - 1, participates in synthesis of prostaglandins ( PG) that related to inflammatory process. In DM the induction of COX - 2 results in increased gene expression, which followed by increased PGs synthesis, especially PGE2. Increased activity of COX - 2 in smooth muscle cell induces over production of
PGs in blood. So the induction of COX - 2 is a risk factor of diabetes.
ATP - sensitive potassium channel ( KATP) is a member of inward rectifying potassium channel, and is controlled by concentration intracellular of ATP. So far, it was found many tissues and cells containing KATP. KATP plays a key role in physiological and pathological functions. Many endogenous vasoactive agents come into effect partially by activating KATP on arterial smooth muscle cells. In normal condition, when the pressure of circulation decreases, sensory neurons on arterial adventitia release CGRP, in turn, CGRP activates adenosine cyclase and increases cAMP, activation of KATP that followed by cAMP increase dilates artery to maintain enough and steady blood flow. In diabetes, the reasons of decreased vasodilation comprises two: one is synthesis and release of vasodilators decrease; another is attenuation of dilating function, which opening function of KATP decrease. It was reported that diabetes usually affected relaxation capacity of vascular complication in diabetes, such as atherosclerosis, coronary heart disease , hypertension, etc .
The objectives of the present study were (1) to determine the change in synthesis of CGRP mRNA in DRG, COX -2 mRNA in DRG and aorta, as well as corresponding products of them in different period of short - term diabetes ( <2 months) ; (2) to claim the mechanism of diabetes vascular complication further, and to provide theoretical basis of prevention and treatment of diabetes complications.
Methods
Male Wistar rats were divided into control and experimental diabetes groups. Streptozotocin (STZ) was dissolved in 0.05M ice trisodium citrate buffer (pH 5.0). Experimental diabetes were induced by STZ (60mg/kg) by single intraperitoneal injection. The controls received STZ citrate buffer carrier a-lone. Rats were accepted as diabetic if the whole - blood glucose level was higher than 16mmol/L. Glucose was measured with a glucometer in whole blood taken from tail vein. Then diabetes rats were divided into four groups, i. e. 1-week - diabetes (1w) , 3 - week - diabetes ( 3w) , 4 - week - diabetes (4w) ,
8 - week - diabete (8w) rats.
Rats were anesthetized, and vein blood was drawn from right heart, then centrifuged at 3000 rpm, 4℃. Plasma was removed, and
背根神经节(dorsal root ganglion，DRG)在糖尿病状态下十分脆弱，对微血管病具有特殊的易感性，尤其在长期糖尿病时DRG中肽能初级感觉神经系统退化，说明DRG是糖尿病的直接靶位，参与糖尿病多种神经病的发生发展。DRG神经元中含有CGRP，COX，SP等神经递质，当内环境发生变化时，通过改变这些递质的合成和释放而影响神经血管的功能。
降钙素基因相关肽(calcitonin gene-related peptide，CGRP)广泛分布于脑、脊髓，常与其他神经递质共存于一个神经元中。在外周，CGRP主要存在于感觉神经节，如三叉神经节(trigeminal ganglion，TG)和DRG中，常与P物质(substance P)和环氧化酶-2(cyclooxygenase，COX-2)共存，在内环境发生变化时，通过血浓度的变化，共同参与疼痛和炎症等有害刺激反应。糖尿病时，SP和CGRP从DRG的顺行转运受阻，感觉神经元和血浆中的CGRP会发生改变，同时血管对CGRP的反应性减弱，CGRP释放减少。CGRP的这些改变是糖尿病时疼痛增加和发生损伤性炎症的重要原因。
COX-2基因是8.3kb的即早基因，是几年前发现的COX-1的异形体，主要与组织损伤性炎症前列腺素(prostaglandins，PGs)的合成有关，COX-2诱导(基因表达增加)的直接结果是以PGE_2为主的炎性PGs产物合成增加。COX-2表达升高不仅是糖尿病的危险因子之一，而且，在糖尿病时，血糖升高，COX-2 mRNA合成的量及其活性增加，增加PG产物的合成，引起糖尿病有关的痛觉过敏和异常疼痛以及血管并发症。
当正常生理状态下，循环灌注压降低时，血管壁感觉神经元释放CGRP，CGRP反过来激活腺苷酸环化酶，使cAMP增加，激活ATP敏感性钾通道(K_(ATP))，扩张动脉，以保持稳定的血流。K_(ATP)是一类受细胞内ATP浓度
(【ATp〕i)控制的内向整流钾通道。目前已经在许多细胞上发现有KATP的
存在，发挥着重要的生理和病理生理功能。许多内源性扩血管物质部分是
通过激活动脉平滑肌的K^TP而发挥作用。糖尿病时，血管舒张功能的损伤，
即K人二的开放功能减弱。这种变化促使糖尿病血管并发症得以发展，如动
脉硬化，冠心病，高血压等。
因此研究糖尿病时，DRG中CGRP和COX一2以及血管平滑肌K，功
能的变化，对阐明糖尿病神经血管并发症发生机制非常重要。本研究的目
的是研究在短期糖尿病(<2 months)的不同阶段，(l)通过对DRG中
CGRP mRNA、DRG和主动脉COX一2 mRNA的合成以及血中相应产物
CGRP和PGE:的研究，阐明CGRP和COX一2在短期糖尿病的特异改变;
(2)通过对血管平滑肌细胞KATP电生理特性的研究，进一步揭示糖尿病血
管并发症的发病机理，为糖尿病并发症的早期防治提供理论依据。
方法
将雄性Wistar大鼠随机分为对照组和糖尿病模型组。链佐霉素(strep一’
tozotoein，S咒)溶于冰冷的0.05M的构椽酸钠溶液中(PH=5.0)，模型组
给予STZ 60m扩kg(1.p.)，对照组等容量注射溶剂。注射STZ一周后，尾
静脉采血，测血糖(blood glueose，BG)。BG>16 rnmol/L者，定为糖尿病模
型成功，将糖尿病大鼠随机分为四组，即糖尿病1周组(lw)、糖尿病2周组
(Zw)，糖尿病3周组(3w)、糖尿病4周组(4w)和糖尿病8周组(sw)。
将大鼠麻醉，直视下心脏采静脉血，4℃，3000甲m离心10 min分离血
浆，一70℃保存。
心脏取血后，剪下并分离胸腹主动脉，于脊柱两侧取胸腰段DRG约10
只，迅速置于液氮中，之后再转移至一70℃低温冰箱中贮存。
肠系膜动脉急性分离，选用细胞膜完整，有立体感，胞体呈梭形的细胞
进行全细胞记录ATp敏感性钾电流(IKATP)。用膜片电极进行千兆封接。
全细胞状态形成后，用Pelamps .5 .1软件程序发放刺激并收集记录信号。
设保持电位为一40 mv，指令电位为一100一+50mv，步阶脉冲10mv，波宽
300 ms，刺激间隔65的方波钳制方案进行刺激。
根据异硫氢酸肌一酚:氯仿(T班201)抽提一步法提取大鼠DRG和主动
脉总RNA，首先进行逆转录，以p一肌动蛋白(p一actin)作为本实验的内参
照物，总RNA经逆转录后以CGRP，COx一2和p一ac血引物进行PCR扩
增，扩增后取适量PCR扩增产物进行2%琼脂糖电泳，嗅化乙锭(EB)染色，
在自动凝胶成像分析仪上，以Cox一2扩增条带与p一ac血扩增条带的荧
光强度比评定其mRNA水平。CGRP，COX一2和p一actin扩增长度分别为
gobp，584bp和422bp。
按照放免药盒说明书所介绍的方法对样品进行处理，检测血中CGRP
和PGE:的浓度。
结果
STZ腹腔注射次日血糖升高，一周后即可出现糖尿病。在短期糖尿病，
以CGR]刀p一actin为半定量指标，DRG中CGRP mRNA有先降后升的趋
势，对照组为1.15土0.19，糖尿病lw，3w，4w，sw组分别为0.934土0.10，
0.822土0.083，1‘272土0.18，1.47土0.18;血中CGRP的变化趋势与DRG
中CGRp mRNA的变化是反向的。以COx一2/p一actin为半定量指?
Aim
Diabetes mellitus ( DM) is a metabolic disease characterized by disorder of glucose metabolism induced by absolute or relative deficiency of insulin. Kinds of complications will be induced if DM cannot be controlled effectively on time, such as pain peripheral neuropathy and inflammatory reaction. The change in vascular permeability induced by DM results in cardiovascular complications, which increase morbidity of hypertension and stroke.
Dorsal root ganglion ( DRG) is very vulnerable in DM, and particularly susceptible to microangiopathy. DRG is a direct target of diabetes damage. In long - term diabetes, degeneration of peptidergic primary afferent nerve system accounts for the early sensory abnormalities in diabetes and the development of neuropathy complication.
Calcitonin gene - related peptide ( CGRP) distributes in neurons of brain tissue and spinal cord. In peripheral system, CGRP mainly exists in sensory ganglions, such as trigeminal ganglion ( TG) and DRG, and usually concomitant with other neural transmitters, such as substance P (SP). Cyclooxygenase - 2 ( COX - 2) also coexists with it. In long - term DM, blockade of direct transportation of CGRP and SP results in the decrease of CGRP in blood and sensory neuron and attenuation of response to CGRP of vessels. All of these changes participate in increasing of nociceptive reaction and allodynia in diabetes.
COX-2 gene is a small (8. 3kb) immediate early gene, an isoform of COX - 1, participates in synthesis of prostaglandins ( PG) that related to inflammatory process. In DM the induction of COX - 2 results in increased gene expression, which followed by increased PGs synthesis, especially PGE2. Increased activity of COX - 2 in smooth muscle cell induces over production of
PGs in blood. So the induction of COX - 2 is a risk factor of diabetes.
ATP - sensitive potassium channel ( KATP) is a member of inward rectifying potassium channel, and is controlled by concentration intracellular of ATP. So far, it was found many tissues and cells containing KATP. KATP plays a key role in physiological and pathological functions. Many endogenous vasoactive agents come into effect partially by activating KATP on arterial smooth muscle cells. In normal condition, when the pressure of circulation decreases, sensory neurons on arterial adventitia release CGRP, in turn, CGRP activates adenosine cyclase and increases cAMP, activation of KATP that followed by cAMP increase dilates artery to maintain enough and steady blood flow. In diabetes, the reasons of decreased vasodilation comprises two: one is synthesis and release of vasodilators decrease; another is attenuation of dilating function, which opening function of KATP decrease. It was reported that diabetes usually affected relaxation capacity of vascular complication in diabetes, such as atherosclerosis, coronary heart disease , hypertension, etc .
The objectives of the present study were (1) to determine the change in synthesis of CGRP mRNA in DRG, COX -2 mRNA in DRG and aorta, as well as corresponding products of them in different period of short - term diabetes ( <2 months) ; (2) to claim the mechanism of diabetes vascular complication further, and to provide theoretical basis of prevention and treatment of diabetes complications.
Methods
Male Wistar rats were divided into control and experimental diabetes groups. Streptozotocin (STZ) was dissolved in 0.05M ice trisodium citrate buffer (pH 5.0). Experimental diabetes were induced by STZ (60mg/kg) by single intraperitoneal injection. The controls received STZ citrate buffer carrier a-lone. Rats were accepted as diabetic if the whole - blood glucose level was higher than 16mmol/L. Glucose was measured with a glucometer in whole blood taken from tail vein. Then diabetes rats were divided into four groups, i. e. 1-week - diabetes (1w) , 3 - week - diabetes ( 3w) , 4 - week - diabetes (4w) ,
8 - week - diabete (8w) rats.
Rats were anesthetized, and vein blood was drawn from right heart, then centrifuged at 3000 rpm, 4℃. Plasma was removed, and
引文
1． Majid Sheykhzade, Grethe T. Dalsgaard et al. The effect of long-term streptozotocin-induced diabetes on contractile relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations. Br J pharmacol , 2000 ; 129 : 1212-1218
2． Peter A. Rittenhouse, James E. marchand , Juan chen et al. Streptozotocin-induced diabetes is associated with altered expression of peptide-encoding mRNAs in rat sensory neurons. Peptides, 1996; 17(6) , 1017-1022
3． Douglas A. Green, Angers A. F. Sima, Martin J. Stevens, et al. Complications: neurotrophy, pathogenetic considerations. Diabetes care, 1992; 15,1902-1919
4． Douglas W. Zochodne, Valerie M. K. Verge, Chu Cheng, et al. Does diabetes target ganglion neurons? Progressive sensory neurone involvement in long-term experiment diabetes. Brain, 2001 ; 124,2319-2334
5． Bennett GS, Garrett NE, Diemel LT, et al. Neurogenic cutaneous vasodila-tation and plasma extravasation in diabetic rats : effect of insulin and nerve growth factor. British Journal of pharmacology , 1998 ; 124 ( 7 ) , 1573-1579
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7． Terenghi G, Chen S, Cattington AL, et al. Changes in sensory neuropep-tides in dorsal root ganglion and spinal cord of spontaneously diabetic BB rats, a quantitative immunohistochemical study. Acta diabetology, 1994; 31(4) , 198-204
8． Josef Troger, Christian Humpel, Bernhard Kremser et al. The effect of streptozotocin-induced diabetes mellitus on substance P and calcitonin gene-related peptide expression in the rat trigemmal ganglion. Brain Res ,
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13. T. A. Lutz, T. R. Pieber, B. Walzer et al. Different influence of CGRP_((8-37)), an amylin and CGRP and antagonist, on the anoretic effects of cholecystokinin and bombesin in diabetic and normal rats. Peptides, 1997;17(5),643～649
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15. Manaka Y, Watanabe M, Yamaguchi K, et al. Sequential changes in CGRP-like immunoreactivity in NIDDM model Otsuka Long-Evans Tokushima Fatty (OLETF) rat pancreatic islets. Pancreas, 1998; 17(1), 72～79
16. Van Rossum D, Hanish UK, Quirion R. Neuroanatomical localization, pharmacological characterization and functions of CGRP, related peptides and their receptors. Neuroscience Biobehavior Review, 1997; 21(5), 649-678
17. Khachatryan A, Guerder S, Palluanlt F, et al. Targeted expression of the neuropeptide calcitonin gene-related peptide to beta cells prevents diabetes in NOS mice. Journal of Immunology, 1997; 158(3), 1409～1416
18． Calcutt NA, Chen P, Hua XY. Effects of diabetes on tissue content and e-voked release of calcitonin gene-related peptide-like immunoreactivity from rat sensory nerves. Neuroscience Letter, 1998; 254, 129-132
19． Nemeth J, Szilvassy Z, Than M,et al. Decreased sensory neuropeptide release from trachea of rats with streptozotocin-induced diabetes. European Journal of pharmacology, 1999; 369:221-224
20． Tashima K, Korolkiewcz R, Kuvomi M, et al. Increased susceptibility of gastric mucosa to ulcerogenic stimulation in diabetic rats--role of capsai-cin-sensitive sensory neurons. British Journal of pharmacology, 1998; 124(7) , 1395-1402
21． J. W. Unger, Y. Klitzsch, S, Pera et al. Nerve growth factor (NGF) and diabetic neuropathy in the rat; morphological investigations of the sural nerve, dorsal root ganglion, and spinal cord. Experimental neurology, 1998; 153,23-34
22． D. R. Tomlinson, P. Fernyhough, L, T. Diemel. Role of neurotrophins in diabetic neuropathy and treatment with nerve growth factors. Diabetes, 1997; 46 (suppl 2) ,s43-s49
23． Hindik Mulder, Yanzhen zhang, Nils Danielsen et al. Islet amyloid poly-peptide and calcitonin gene-related peptide expression are down-regulated in dorsal root ganglia upon sciatic nerve transection. Molecular Brain Res, 1997; 47,322-330
24． Torffvifo, Editsson L. Blockade of nitric oxide decreases the renal vasodila-tory effect of neuropeptide Y in the insulin-treated diabetic rat. Pflugers Arch, 1997; 434(4) , 445-450