温肾方对甲状腺功能减退症大鼠神经信号传导及基因表达的影响
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摘要
目的 甲状腺功能减退症是一种常见的内分泌综合症侯群，可导致全身多个系统病变，引起各种合并症。甲状腺功能减退症合并脑功能损害临床较为常见，这种甲减性脑损害，随着病情加重，几乎所有病例都会出现，表现为不同程度神经精神症状。目前，临床对甲减性脑损害并无专门的治疗，现代医学仅是针对甲状腺功能减退本身，采用甲状腺素替代治疗，忽视对脑功能损害自身的治疗，疗效不如人意。发挥中医优势，运用中医药治疗甲减合并症，有利于提高本病的疗效。温肾方临床效果良好，不仅可改善甲减本身的征状，而且对神经精神症状改善明显。我们在温肾方治疗甲减性脑损害的临床的基础上，以甲状腺功能减退症动物模型为实验对象，运用现代医学的理论和试验方法，对温肾方治疗甲减性脑损害的作用及其机理进行了研究，从而为临床提供了理论与实验依据。
方法 将受试大鼠平均分为五组：正常对照组、模型组、甲状腺片组、中药小剂量组、中药大剂量组。后四组予以造模，模型大鼠按1.35ml／200g体重／只，每日灌服0.04％他巴唑混悬液，共计造模45天。造模完成后开始灌服相应药物，连续15天。然后各实验组分别进行如下指标的检测：采用放射免疫法测定血清T_3、T_4、FT_3、FT_4、TSH水平；选用水迷宫法、穿梭箱法观察药物对模型大鼠行为学障碍的影响；运用逆转录-多聚酶联反应(RT-PCR)、蛋白免疫印迹法、免疫组织化学的方法、Lowery法及改良Takai法，分别检测模型大鼠海马NO、nNOS含量及nNOSmRNA表达、Goa蛋白含量及GoamRNA表达、PKC活性测定。
结果 1．模型组大鼠造模后出现肾阳虚的症状，反映甲减程度的临床指标血清T_3、T_4、FT_3、FT_4的含量显著低于正常对照组，血清TSH的含量显著高于正常对照组。中药组尤其中药大剂量组不仅能明显改善试验大鼠甲减症状，而且能明显升高T_3、T_4、FT_3、FT_4水平，降低TSH含量，但不强于甲状腺片组。2．温肾方能显著改善模型大鼠行为学障碍，Morris水迷宫实验表现为游泳逃避潜伏期及游泳路径总长明显缩短，同时穿梭箱实验也表现为电击次数(被动逃避次数)及电击时间明显减少，且中药大剂量组优于甲状腺片组。3．温肾方能明显升高模型大鼠NO生成和释放的含量，调高NO信号系统的基因表达；能明显下调模型大鼠GoamRNA表达水平，降低Goa蛋白含量；能明显下调模型大鼠PKC活性，其作用强于甲状腺片组。
结论 温肾方对甲减模型大鼠不仅能明显改善实验大鼠甲减症状，而且能明显升高T_3、T_4、FT_3、FT_4水平，降低TSH含量，但不强于甲状腺片组。温肾方能显著改善模型大鼠行为学障碍，能明显升高模型大鼠NO生成和释放的含量，调高NO信号系统的基因表达；能明显下调模型大鼠GoamRNA表达水平，降低Goa蛋白含量；能明显下调模型大鼠PKC活性，其作用均强于甲状腺片组。表明温肾方对甲减性脑损害治疗作用显著。温肾方大剂量组对模型大鼠行为学障碍改善优于甲状腺片组，进一步表明温肾方对甲减性脑损害的治疗作用，除增加内源性甲状腺素分泌外，还能直接作用于脑，如改善脑信号传导系统，是通过多途经发挥作用的。
Objective Hypothyroidism is a common disease of endocrine system, which could to lead to pathological changes and syndromes of many systems. It is common in clinical that hypothyroidism merging the impairment of brain and with the development of hypothyroidism, every patient have the impairment of brain, showing neurotic and psychiatric symptoms. Now only thyroxine sodium is used in treatment of hypothyroidism itself and has no specially therapeutic methods for the impairment of brain caused by hypothyroidism in clinic, and the effect is dissatisfaction. So it is therapeutic current with Chinese medicine for prevention and treatment of the impairment of brain caused by hypothyroidism by giving full play to the advantages of Traditional Chinese Medicine. It was confirmed that the therapeutic effect of Wenshentang in clinic is well. It could improve the symptoms not only hypothyroidism itself but also the impairment of brain caused by hypothyroidism. We study the function of Wenshentang on the impairment of br
ain caused by hypothyroidism, and its mechanism under the basic theory and experimental methods of modern medicine with the hypothyroidism animal model in order to provide a theory and experimental base for the clinical practice.
Methods The rats were randomly divided into 5 groups, such as normal control group, model group, thyroxin group, small dosage group of Wenshentang and the large dosage of Wenshentang. The rats of the latter 4 groups were made model by infusing 0.04% tapazole suspension 1.35%/200g/day for 45 days. After that, the rats were treated with corresponding medicines for 15 days. Then the following detection were taken out: radioimmunoassay on serum FT3, FT4, T3, T4 and TSH; water labyrinth and shuttling box methods on the behavior changes; RT-PCR, Western blot, Lowery, improved Takai and immunohistochemical method on the content or expression of NOmRNA, Go a mRNA and the activity of protein kinase C (PKC) in the hippocampus of hypothyroidism rats.
Results (1) In the rats of model group, the behavior changes of kidney yang-insuf f iciency in hypothyroidism were appeared, and the indexes which may imply the severe degree of hypothyroidism presented the decreasing of FT3, FT4, T3, T4 and the increasing of TSH compared with the normal control group. Wenshentang, especially with large dosage had not only improved the symptoms of hypothyroidism, but also increased the content of FT3, FT4, T3, T4 and decreased the content of TSH, but the effect was not better than that of thyroxin group. (2) Wenshentang significantly improved the behavior disorder of the hypothyroidsm rats. The evasive latent period and length of swimming were significantly
shorten in the experiment of Morris water labyrinth, at the same time the times and the hours of get electric shock were significantly decreased in the experiment of shuttling box. The effect of large dosage group was better than that of thyroxin group. (3) Wenshentang significantly increased the content or expression of NOmRNA, decreased the content or expression of Go a mRNA and downregulation the activity of PKC of the hypothyroidsm rats, and the effect was better than that of thyroxin group.
Conclusion In the rats of model group, the behavior changes of kidney yang-insufficiency in hypothyroidism were appeared , and the indexes which may imply the severe degree of hypothyroidism presented the decreasing of FT3, FT4, T3, T4 and the increasing of TSH compared with the normal control group. Wenshentang had not only improved the symptoms of hypothyroidism rats, but also increased the content of FT3, FT4, T3, T4 and decreased the content of TSH, but the effect were not excel the thyroxin group. Wenshentang significantly increased the content or expression of NOmRNA, decreased the content or expression of Go a mRNA and down regulation the activity of PKC of the hypothyroidism rats, and the effect were excel the thyroxin group. Wenshentang significantly improved the behavior disorder of the hypothyroidism rats which implied that Wenshentang have signific
方法 将受试大鼠平均分为五组：正常对照组、模型组、甲状腺片组、中药小剂量组、中药大剂量组。后四组予以造模，模型大鼠按1.35ml／200g体重／只，每日灌服0.04％他巴唑混悬液，共计造模45天。造模完成后开始灌服相应药物，连续15天。然后各实验组分别进行如下指标的检测：采用放射免疫法测定血清T_3、T_4、FT_3、FT_4、TSH水平；选用水迷宫法、穿梭箱法观察药物对模型大鼠行为学障碍的影响；运用逆转录-多聚酶联反应(RT-PCR)、蛋白免疫印迹法、免疫组织化学的方法、Lowery法及改良Takai法，分别检测模型大鼠海马NO、nNOS含量及nNOSmRNA表达、Goa蛋白含量及GoamRNA表达、PKC活性测定。
结果 1．模型组大鼠造模后出现肾阳虚的症状，反映甲减程度的临床指标血清T_3、T_4、FT_3、FT_4的含量显著低于正常对照组，血清TSH的含量显著高于正常对照组。中药组尤其中药大剂量组不仅能明显改善试验大鼠甲减症状，而且能明显升高T_3、T_4、FT_3、FT_4水平，降低TSH含量，但不强于甲状腺片组。2．温肾方能显著改善模型大鼠行为学障碍，Morris水迷宫实验表现为游泳逃避潜伏期及游泳路径总长明显缩短，同时穿梭箱实验也表现为电击次数(被动逃避次数)及电击时间明显减少，且中药大剂量组优于甲状腺片组。3．温肾方能明显升高模型大鼠NO生成和释放的含量，调高NO信号系统的基因表达；能明显下调模型大鼠GoamRNA表达水平，降低Goa蛋白含量；能明显下调模型大鼠PKC活性，其作用强于甲状腺片组。
结论 温肾方对甲减模型大鼠不仅能明显改善实验大鼠甲减症状，而且能明显升高T_3、T_4、FT_3、FT_4水平，降低TSH含量，但不强于甲状腺片组。温肾方能显著改善模型大鼠行为学障碍，能明显升高模型大鼠NO生成和释放的含量，调高NO信号系统的基因表达；能明显下调模型大鼠GoamRNA表达水平，降低Goa蛋白含量；能明显下调模型大鼠PKC活性，其作用均强于甲状腺片组。表明温肾方对甲减性脑损害治疗作用显著。温肾方大剂量组对模型大鼠行为学障碍改善优于甲状腺片组，进一步表明温肾方对甲减性脑损害的治疗作用，除增加内源性甲状腺素分泌外，还能直接作用于脑，如改善脑信号传导系统，是通过多途经发挥作用的。
Objective Hypothyroidism is a common disease of endocrine system, which could to lead to pathological changes and syndromes of many systems. It is common in clinical that hypothyroidism merging the impairment of brain and with the development of hypothyroidism, every patient have the impairment of brain, showing neurotic and psychiatric symptoms. Now only thyroxine sodium is used in treatment of hypothyroidism itself and has no specially therapeutic methods for the impairment of brain caused by hypothyroidism in clinic, and the effect is dissatisfaction. So it is therapeutic current with Chinese medicine for prevention and treatment of the impairment of brain caused by hypothyroidism by giving full play to the advantages of Traditional Chinese Medicine. It was confirmed that the therapeutic effect of Wenshentang in clinic is well. It could improve the symptoms not only hypothyroidism itself but also the impairment of brain caused by hypothyroidism. We study the function of Wenshentang on the impairment of br
ain caused by hypothyroidism, and its mechanism under the basic theory and experimental methods of modern medicine with the hypothyroidism animal model in order to provide a theory and experimental base for the clinical practice.
Methods The rats were randomly divided into 5 groups, such as normal control group, model group, thyroxin group, small dosage group of Wenshentang and the large dosage of Wenshentang. The rats of the latter 4 groups were made model by infusing 0.04% tapazole suspension 1.35%/200g/day for 45 days. After that, the rats were treated with corresponding medicines for 15 days. Then the following detection were taken out: radioimmunoassay on serum FT3, FT4, T3, T4 and TSH; water labyrinth and shuttling box methods on the behavior changes; RT-PCR, Western blot, Lowery, improved Takai and immunohistochemical method on the content or expression of NOmRNA, Go a mRNA and the activity of protein kinase C (PKC) in the hippocampus of hypothyroidism rats.
Results (1) In the rats of model group, the behavior changes of kidney yang-insuf f iciency in hypothyroidism were appeared, and the indexes which may imply the severe degree of hypothyroidism presented the decreasing of FT3, FT4, T3, T4 and the increasing of TSH compared with the normal control group. Wenshentang, especially with large dosage had not only improved the symptoms of hypothyroidism, but also increased the content of FT3, FT4, T3, T4 and decreased the content of TSH, but the effect was not better than that of thyroxin group. (2) Wenshentang significantly improved the behavior disorder of the hypothyroidsm rats. The evasive latent period and length of swimming were significantly
shorten in the experiment of Morris water labyrinth, at the same time the times and the hours of get electric shock were significantly decreased in the experiment of shuttling box. The effect of large dosage group was better than that of thyroxin group. (3) Wenshentang significantly increased the content or expression of NOmRNA, decreased the content or expression of Go a mRNA and downregulation the activity of PKC of the hypothyroidsm rats, and the effect was better than that of thyroxin group.
Conclusion In the rats of model group, the behavior changes of kidney yang-insufficiency in hypothyroidism were appeared , and the indexes which may imply the severe degree of hypothyroidism presented the decreasing of FT3, FT4, T3, T4 and the increasing of TSH compared with the normal control group. Wenshentang had not only improved the symptoms of hypothyroidism rats, but also increased the content of FT3, FT4, T3, T4 and decreased the content of TSH, but the effect were not excel the thyroxin group. Wenshentang significantly increased the content or expression of NOmRNA, decreased the content or expression of Go a mRNA and down regulation the activity of PKC of the hypothyroidism rats, and the effect were excel the thyroxin group. Wenshentang significantly improved the behavior disorder of the hypothyroidism rats which implied that Wenshentang have signific
引文
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58. Figueiredo BC et al. Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its lowaffinity receptor in cerebellum. Dev Brain Res, 1993;72:237
59. Figueiredo BC et al. Gene expression in the developing oerebellum during perinatal hypo- and hyperthyroidism. Mol Brain Res, 1993; 17:258
60. Van Der Zee CEEM et al. Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice. Science, 1996;274:1729
61. Muller Y et al. Thyroid hormone promotes bc1-2 expression and prev-ents apoptosis of early differentiating cerebellar granule neurone. Int J Dev Neurosci, 1995;13:871
62. Brent GA. The molecular basis of thyroid hormone action. N Eng J Med, 1994;331:847-853
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64. Rivier C. Shen GH. The rat endogenous nitric oxide modulates the re-sponse of the hypothalamic-pituitary-adrenal axis to interieuk in-lvasopressin and oxytocin. J Neurosci, 1994;14:1985-1993
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43. Sarkar S, Biswas s c, Chatterjee O, et al. Protein kinase hlinkde phosphorylation mediates triiodothyronine induced actin gene expression in develoing brain [J].Brain Res Mol Brain Res, 1999,67:158-164
44. Coleman C, Zhao J, Guta M, et al. inhibition of vascular and epithelial differebtiation in murine nitrofen-induced diaphragmatic hernia [J].Am J Physiol, 1998,274(4Pt1):L636-L646
45. Leung F C, Saggerson E D, Clark J B. Signal teansduction processes in the developing brain:perturbations of G protein alpha-subunet abundances by perinatal hypothyroidism [J],Dev Neurosci, 1996,18:153-161
46. Wong C C, Warsh J J, Sibony D, et al. Differetial ontogenetic appear ance and regulation of stimulatory G protein isoforms in rat cerebral cortex by thyroid hormone deficiency [J].Dev Brain Res, 1994,79:136-139
47. Krukoff TL, Gehlen F, Ganten D, et al. Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase hypothalamus and
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56. Segal RA et al. Neurotrophins and programmed cell death during cer-ebellar development. Adv Neurol, 1997;72:79
57. Alvarez-Dolado M et al. Expression of neurotrophins and the trk family of neurotrophin receptors in normal and hypothyroid rat brain. Mol Brain Res, 1994;27:249
58. Figueiredo BC et al. Effects of perinatal hypo- and hyperthyroidism on the levels of nerve growth factor and its lowaffinity receptor in cerebellum. Dev Brain Res, 1993;72:237
59. Figueiredo BC et al. Gene expression in the developing oerebellum during perinatal hypo- and hyperthyroidism. Mol Brain Res, 1993; 17:258
60. Van Der Zee CEEM et al. Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice. Science, 1996;274:1729
61. Muller Y et al. Thyroid hormone promotes bc1-2 expression and prev-ents apoptosis of early differentiating cerebellar granule neurone. Int J Dev Neurosci, 1995;13:871
62. Brent GA. The molecular basis of thyroid hormone action. N Eng J Med, 1994;331:847-853
63. Karanth S. Lyson K. McCann S. Role of nitric oxide interlukm 2-induced corticotropin-releasing factor release from incubated hypothala-mus. Proc Natl Acad USA, 1994;14:3383-3387
64. Rivier C. Shen GH. The rat endogenous nitric oxide modulates the re-sponse of the hypothalamic-pituitary-adrenal axis to interieuk in-lvasopressin and oxytocin. J Neurosci, 1994;14:1985-1993
65. Ueta Y, Levy A. Hypothalamus nitric oxide synthase geneogy is regul-ated by thyroid hormones[J].Endocrinology, 1995,136:4184-4187
66．罗敏，陈源，蔡东升．甲状腺激素对围严期大鼠大脑、嗅脑及小脑一氧化氮合酶的调节[J]．中华内分泌代谢杂志，2000，16：172-174
67. Nishizuka Y,Protein kinase C and lipid signaling for sustained c
e-llular responses. FhSEB J, 1995,9:486-496
68. bixby JL, Harris WA. Molecular mechanisms of axon growth and guidance. Annu Rev Cell boil,1991,7:117-159
69. Battaini F, Pascale A, Paoletti R, et al. The role of anchoring protein RACKI in PKC activation in the aging rat brain[J].Trends Neuros-ci,1997,20:410-415
70. Thanaka C, Nishizuka Y. The protein kinase C family for neuronal si-gnaling. Annu Rev Neurosci, 1994,17:551-567
71. Pavlakovic G, Eyer CI. Isom GE. Neuroprotective effects of PKC inhib-ition against chemical hypoxia. Brain Res, 1995,676:205-211
72. Favaron M, Manev H, Alho H, et al.Gangliosides prevent glutamate and kainite neurotoxicity in primary cultures of neonatal rat cerebellum and cortex. Proc Natl Acad Sci USA, 1988,85:7351-7355.
73. Favaron M, Manev H, Siman R, et al.down-regulation of protein kinase C protecgs cerebellar granule neurons in primary culture from glutamate induced neuronal death. Proc Natl Acad Sci USA, 1990,87: 1983-1987
74. Domanska-Janik K, Zalewsks T. Effect of brain ischemia on protein kinase C.J Neurochem, 1992,58:1432-1439
75．苏青，罗敏，陈源。甲状腺激素缺乏对发育期鼠脑蛋白激酶C活性的影响[J]。中华内分泌代谢杂志，2000，16：106-108。
76. Sarkar S, Biswas s c, Chatterjee O, et al. Protein kinase Alinkde phosphorylation mediates triiodothyronine induced actin gene expression in develoing brain [J].Brain Res Mol Brain Res, 1999,67:158-164
77. Coleman C,Zhao J,Guta M, et al. inhibition of vascular and epithelial differebtiation in murine nitrofen-induced diaphragmatic hernia [J].Am J Physiol, 1998,274(4Pt1):L636-L646
78. Leung F C, Saggerson E D, Clark J B. Signal teansduction processes in the developing brain:perturbations of G protein alpha-subunet abundances by perinatal hypothyroidism [J],Dev Neurosci, 1996,18: 153-161
79. Wong C C,Warsh J J, Sibony D, et al. Differetial ontogenetic appear ance and regulation of stimulatory G protein isoforms in rat cerebral cortex by thyroid hormone deficiency [J].Dev Brain Res, 1994,79:136-139
80. Krukoff TL,Gehlen F, Ganten D, et al.Gene expression of brain nitric oxide synthase and soluble guanylyl cyclase hypothalamus and medulla two-kindney one chip hyperensive rats, Hypertension. 1995, 26:171-176.
81. Th1-Vah1 R. Marquetant R. Bremerich J. et al. Regulation of -adrenergic rsceptors in acute myocardial ischemia:subrype-selective increase of mRNA specific for 1-adrenergic receptors. J Mol Cell Cardiol, 1995,27:437-452
82. Garthwaite J, Charles Sl,Chess-williams R. Endothelium-derived relaxing factor release on activation of NMDA receptor suggests ro
le as intracellular inessenger in the brain. Nature, 1988,336:385-388
83．高尚璞，汝丽娟教授治疗桥本甲状腺炎继发甲状腺功能减退症的经验，上海中医药杂志 2002；1：13
84．梁军，等．补肾填精方治疗甲状腺功能减退症126例，中国中医药科技 2001；4：19
85．胡元奎，中医辨正治甲减，医药与保健 2002；10：17
86．曹金梅，中药内服配合穴位埋线治疗甲状腺功能减退症36例，浙江中医杂志 2003；5：27
87．张宏伟，等．半硫丸对家兔试验性甲减的治疗作用，中医药研究 1993，3：43
88．查良伦，等．甲状腺减退症与肾阳虚关系探讨，中国中西医结合杂志 1993；13(4)：202
89．徐敏，等．温补肾阳对甲减阳虚兔模型心．肾受体的影响，福建中医药杂志 1991；22(2)：30
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