过表达降钙素基因相关肽(CGRP)改善糖尿病小鼠周围神经病变
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摘要
目的观察降钙素基因相关肽(CGRP)对糖尿病小鼠周围神经病变的影响,探讨过表达CGRP能否改善糖尿病小鼠周围神经病变。方法建立糖尿病小鼠模型,根据小鼠体质量和血糖水平随机分为模型组、 CGRP过表达(Ad-CGRP)组、空载体腺病毒(Ad)阴性对照组,另设正常对照组。Ad-CGRP组以及Ad阴性对照组小鼠左侧后肢肌肉注射Ad-CGRP或者阴性对照Ad,正常组及模型组小鼠注射同等体积的生理盐水。给药4周后,实时定量PCR检测CGRP mRNA水平, Western blot法检测CGRP蛋白水平;智能热板仪检测疼痛潜伏期,生理信号采集处理系统检测神经传导速度,激光多普勒血流仪检测神经血流情况。结果与正常组比较,模型组小鼠的CGRP mRNA和蛋白水平均降低、血糖明显升高,疼痛潜伏期延长,运动神经传导速度降低,神经血流量减少。与模型组比较, Ad-CGRP组小鼠的CGRP mRNA和蛋白水平均显著增加,疼痛潜伏期缩短,神经传导速度增快,神经血流量增加; Ad阴性对照组均无明显改变。结论过表达CGRP促进神经血管扩张,增加血流,改善糖尿病周围神经病变。
Objective To observe the effect of calcitonin gene-related peptide(CGRP) on peripheral neuropathy in diabetic mice, and to explore whether overexpressed CGRP improves peripheral neuropathy in diabetic mice. MethodsDiabetic mouse model was induced by intraperitoneal injection of streptozotocin(STZ). The mice were randomly divided into 4 groups according to their body mass and blood glucose: normal control group, model group, Ad-CGRP group and Ad negative control group. The mice in the Ad-CGRP group and the Ad-negative control group were intramuscularly injected with adenovirus in the muscles of left hind limb. The mice in the normal group and the model group were injected with equal volume of normal saline. After 4 weeks of administration, the expression of CGRP mRNA was detected by real-time quantitative PCR. The expression of CGRP protein was detected by Western blot analysis. The latency of pain was measured by hot plate test. The motor nerve conduction velocity(MNCV) was measured by physiological signal acquiring system. The nerve blood flow was monitored with Laser Doppler Flowmetry(LDF). Results Compared with the normal group, the mRNA and protein levels of CGRP in the model group were reduced, the level of glucose was significantly raised, the latency of pain was extended, the MNCV and the nerve blood flow were decreased. Compared with the model group, the mRNA and protein levels of CGRP in the Ad-CGRP group were significantly increased, the latency of pain was shortened, the MNCV and the nerve blood flow was increased. There was no significant change in the Ad-negative control group. Conclusion Overexpression of CGRP may ameliorate diabetic peripheral neuropathy by expanding blood vessel and increasing blood flow.
Objective To observe the effect of calcitonin gene-related peptide(CGRP) on peripheral neuropathy in diabetic mice, and to explore whether overexpressed CGRP improves peripheral neuropathy in diabetic mice. MethodsDiabetic mouse model was induced by intraperitoneal injection of streptozotocin(STZ). The mice were randomly divided into 4 groups according to their body mass and blood glucose: normal control group, model group, Ad-CGRP group and Ad negative control group. The mice in the Ad-CGRP group and the Ad-negative control group were intramuscularly injected with adenovirus in the muscles of left hind limb. The mice in the normal group and the model group were injected with equal volume of normal saline. After 4 weeks of administration, the expression of CGRP mRNA was detected by real-time quantitative PCR. The expression of CGRP protein was detected by Western blot analysis. The latency of pain was measured by hot plate test. The motor nerve conduction velocity(MNCV) was measured by physiological signal acquiring system. The nerve blood flow was monitored with Laser Doppler Flowmetry(LDF). Results Compared with the normal group, the mRNA and protein levels of CGRP in the model group were reduced, the level of glucose was significantly raised, the latency of pain was extended, the MNCV and the nerve blood flow were decreased. Compared with the model group, the mRNA and protein levels of CGRP in the Ad-CGRP group were significantly increased, the latency of pain was shortened, the MNCV and the nerve blood flow was increased. There was no significant change in the Ad-negative control group. Conclusion Overexpression of CGRP may ameliorate diabetic peripheral neuropathy by expanding blood vessel and increasing blood flow.
引文
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[21] Goadsby P J,Holland P R,Martins-Oliveira M,et al.Pathophysiology of migraine:a disorder of sensory processing[J].Physiol Rev,2017,97(2):553-622.
[22] Tepper D E.Calcitonin gene-related protein(CGRP)-targeted treatments for migraine prevention[J].Headache,2019,59(3):477-480.
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[25] Gonkowski S,Kamińska B,Bossowska A,et al.The influence of experimental Bacteroides fragilis infection on substance P and somatostatin-immunoreactive neural elements in the porcine ascending colon-a preliminary report[J].Folia Morphol (Warsz),2003,62(4):455-457.
[26] 徐派的,张红星,杨云,等.电针对功能性消化不良模型大鼠胃肠动力及VIP和CGRP水平的影响[J].中国中西医结合杂志,2017,37(3):360-364.Xu P,Zhang H,Yang Y,et al.Effects of electroacupuncture on gastrointestinal motility and expressions of VIP and CGRP in functional dyspepsia model rats[J].Zhong Guo Zhong Xi Yi Jie He Za Zhi,2017,37(3):360-364.
[27] González-Hernández A,Marichal-Cancino B A,Lozano-Cuenca J,et al.Functional characterization of the prejunctional receptors mediating the inhibition by ergotamine of the rat perivascular sensory peptidergic drive[J].ACS Chem Neurosci,2019 Feb 13.DOI:10.1021/acschemneuro.8b00611.[Epub ahead of print].
[28] Afroz S,Arakaki R,Iwasa T,et al.CGRP induces differential regulation of cytokines from satellite glial cells in trigeminal ganglia and orofacial nociception[J].Int J Mol Sci,2019,20(3):E711.DOI:10.3390/ijms20030711.
[29] Zheng S,Li W,Xu M,et al.Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase[J].Am J Physiol Cell Physiol,2010,299(6):C1485-C1492.
[2] Costa Y M,Karlsson P,Bonjardim L R,et al.Trigeminal nociceptive function and oral somatosensory functional and structural assessment in patients with diabetic peripheral neuropathy[J/OL].Sci Rep,2019,9(1):169.DOI:10.1038/s41598-018-37041-4.
[3] Han Y,Wang M,Shen J,et al.Differential efficacy of methylcobalamin and alpha-lipoic acid treatment on symptoms of diabetic peripheral neuropathy[J].Minerva Endocrinol,2018,43(1):11-18.
[4] You Z,Zhang Z,Blagg B S J,et al.KU-596 decreases mitochondrial superoxide and improves bioenergetics following downregulation of manganese superoxide dismutase in diabetic sensory neurons[J].Exp Neurol,2019,313:88-97.
[5] Liu X,Xu Y,An M,et al.The risk factors for diabetic peripheral neuropathy:A meta-analysis[J/OL].PLoS One,2019,14(2):e0212574.DOI:10.1371/journal.pone.0212574.eCollection 2019.
[6] Simpson D M,Robinson-papp J,Van J,et al.Capsaicin 8% patch in painful diabetic peripheral neuropathy:a randomized,double-blind,placebo-controlled study[J].J Pain,2017,18(1):42-53.
[7] 任秋月,张硕,闫田田,等.黄芪桂枝五物汤干预糖尿病周围神经病变疗效观察[J].天津中医药,2017,34(3):155-158.Ren Q,Zhang S,Yan T,et al.Clinical observation of Huangqi Guizhi Wuwu decoction intervention on diabetic peripheral neuropathy[J].Tian Jin Zhong Yi Yao,2017,34(3):155-158.
[8] Mao H,Wei W,Fu X L,et al.Efficacy of autologous bone marrow mononuclear cell transplantation therapy in patients with refractory diabetic peripheral neuropathy[J].Chin Med J (Engl),2019,132(1):11-16.
[9] Iyengar S,Ossipov M H,Johnson K W.The role of calcitonin gene-related peptide in peripheral and central pain mechanisms including migraine[J].Pain,2017,158(4):543-559.
[10] Godlewski J,Kaleczyc J.Somatostatin,substance P and calcitonin gene-related peptide-positive intramural nerve structures of the human large intestine affected by carcinoma[J].Folia Histochem Cytobiol,2010,48(3):475-483.
[11] Hay D L,Garelja M L,Poyner D R,et al.Update on the pharmacology of calcitonin/CGRP family of peptides:IUPHAR review 25[J].Br J Pharmacol,2018,175(1):3-17.
[12] Adeghate E,Ponery A.Diabetes mellitus influences the degree of colocalization of calcitonin gene-related peptide with insulin and somatostatin in the rat pancreas[J].Pancreas,2004,29(4):311-319.
[13] Al-Salam S,Hameed R,Parvez H S,et al.Diabetes mellitus decresses the expression of calcitonin-gene related peptide,gamma-amion butyric acid and glutamic acid decarboxylase in human pancreatic islet cells[J].Neuro Endocrinol Lett,2009,30(4):506-510.
[14] Ko M H,Hu M E,Hsieh Y L,et al.Peptidergic intraepidermal nerve fibers in the skin contribute to the neuropathic pain in paclitaxel-induced peripheral neuropathy[J].Neuropeptides,2014,48(3):109-117.
[15] Wang D,Gao Y,Ji H,et al.Topical and systemic administrations of ketanserin attenuate hypersensitivity and expression of CGRP in rats with spinal nerve ligation[J].Eur J Pharmacol,2010,627(1/2/3):124-130.
[16] Wang L H,Zhou S X,Li R C,et al.Serum levels of calcitonin gene-related peptide and substance P are decreased in patients with diabetes mellitus and coronary artery disease[J].J Int Med Res,2012,40(1):134-140.
[17] Sun T,Guo Z,Liu C J,et al.Preservation of CGRP in myocardium attenuates development of cardiac dysfunction in diabetic rats[J].Int J Cardiol,2016,220:226-234.
[18] Li T P,Guo Z,Liu C J,et al.Association of down-regulation of calcitonin gene-related peptide and substance P with increase of myocardial vulnerability in diabetic neuropathic rats[J].Peptides,2017,96:1-7.
[19] 韩梅,苏俊平,刘欣荣,等.高压氧联合硫辛酸注射液治疗糖尿病足的临床疗效及对血管生成因子的影响[J].河北医学,2018,24(11):1829-1833.Han M,Su J,Liu X,et al.Curative efficacy of hyperbaric oxygen combined with thiocic acid injection in treatment of diabetic foot and its effects on angiogenesis factors[J].He Bei Yi Xue,2018,24(11):1829-1833.
[20] Tarighi N,Menger D,Pierre S,et al.Thromboxane-induced α-CGRP release from peripheral neurons is an essential positive feedback loop in capsaicin-induced neurogenic inflammation[J].J Invest Dermatol,2019 ,139(3):656-664.
[21] Goadsby P J,Holland P R,Martins-Oliveira M,et al.Pathophysiology of migraine:a disorder of sensory processing[J].Physiol Rev,2017,97(2):553-622.
[22] Tepper D E.Calcitonin gene-related protein(CGRP)-targeted treatments for migraine prevention[J].Headache,2019,59(3):477-480.
[23] Li X Q,Verge V M,Johnston J M,et al.CGRP peptide and regenerating sensory axons[J].J Neuropathol Exp Neurol,2004,63(10):1092-1103.
[24] MaassenVanDenBrink A,Meijer J,Villalón C M,et al.Wiping out CGRP:potential cardiovascular risks[J].Trends Pharmacol Sci,2016,37(9):779-788.
[25] Gonkowski S,Kamińska B,Bossowska A,et al.The influence of experimental Bacteroides fragilis infection on substance P and somatostatin-immunoreactive neural elements in the porcine ascending colon-a preliminary report[J].Folia Morphol (Warsz),2003,62(4):455-457.
[26] 徐派的,张红星,杨云,等.电针对功能性消化不良模型大鼠胃肠动力及VIP和CGRP水平的影响[J].中国中西医结合杂志,2017,37(3):360-364.Xu P,Zhang H,Yang Y,et al.Effects of electroacupuncture on gastrointestinal motility and expressions of VIP and CGRP in functional dyspepsia model rats[J].Zhong Guo Zhong Xi Yi Jie He Za Zhi,2017,37(3):360-364.
[27] González-Hernández A,Marichal-Cancino B A,Lozano-Cuenca J,et al.Functional characterization of the prejunctional receptors mediating the inhibition by ergotamine of the rat perivascular sensory peptidergic drive[J].ACS Chem Neurosci,2019 Feb 13.DOI:10.1021/acschemneuro.8b00611.[Epub ahead of print].
[28] Afroz S,Arakaki R,Iwasa T,et al.CGRP induces differential regulation of cytokines from satellite glial cells in trigeminal ganglia and orofacial nociception[J].Int J Mol Sci,2019,20(3):E711.DOI:10.3390/ijms20030711.
[29] Zheng S,Li W,Xu M,et al.Calcitonin gene-related peptide promotes angiogenesis via AMP-activated protein kinase[J].Am J Physiol Cell Physiol,2010,299(6):C1485-C1492.