丝胶可保护糖尿病大鼠坐骨神经及相关神经细胞

doi:10.3969/j.issn.1673-5374.2013.06.003

摘要/Abstract

摘要：

在缫丝时被废弃蚕茧中的丝胶，在美容、护肤、营养、抗氧化、抗癌等方面显示了多种作用。实验以小剂量（25mg/kg）链脲佐菌素连续腹腔注射3d建立2型糖尿病大鼠模型。该模型经丝胶灌胃治疗35d后，血糖水平明显下降，且坐骨神经神经微丝蛋白的表达、L4-6脊神经节细胞和L4-6脊髓前角细胞神经生长因子蛋白的表达明显升高，脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达明显降低。证实丝胶可通过上调2型糖尿病大鼠坐骨神经神经微丝蛋白的表达、脊神经节细胞和脊髓前角细胞神经生长因子蛋白的表达，下调脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达，对2型糖尿病时坐骨神经及相关神经细胞损伤具有一定的保护作用。

关键词: 神经再生, 中医药, 糖尿病, 丝胶, 坐骨神经, 脊神经节细胞, 脊髓前角细胞, 神经细胞, 神经微丝蛋白, 神经生长因子, 神经肽, 链脲佐菌素, 图片文章

Abstract:

Sericin from discarded silkworm cocoons of silk reeling has been used in different fields, such as cosmetology, skin care, nutrition, and oncology. The present study established a rat model of type 2 diabetes by consecutive intraperitoneal injections of low-dose (25 mg/kg) streptozotocin. After intragastrical perfusion of sericin for 35 days, blood glucose levels significantly declined, and the expression of neurofilament protein in the sciatic nerve and nerve growth factor in L4–6 spinal ganglion and anterior horn cells significantly increased. However, the expression of neuropeptide Y in spinal ganglion and anterior horn cells significantly decreased in model rats. These findings indicate that sericin protected the sciatic nerve and related nerve cells against injury in a rat type 2 diabetic model by upregulating the expression of neurofilament protein in the sciatic nerve and nerve growth factor in spinal ganglion and anterior horn cells, and downregulating the expression of neuropeptide Y in spinal ganglion and anterior horn cells.

Key words: neural regeneration, traditional Chinese medicine, peripheral nerve injury, diabetes mellitus, sericin, sciatic nerve, spinal ganglion cells, anterior horn cells, nerve cells, neurofilament protein, nerve growth factor, neuropeptide Y, streptozotocin, photographs-containing paper, neuroregeneration

. 丝胶可保护糖尿病大鼠坐骨神经及相关神经细胞[J]. 中国神经再生研究(英文版), 2013, 8(6): 506-513.

Chengjun Song, Zhenjun Yang, Meirong Zhong, Zhihong Chen. Sericin protects against diabetes-induced injuries in sciatic nerve and related nerve cells[J]. Neural Regeneration Research, 2013, 8(6): 506-513.

参考文献

[1] Harati Y. Diabetic peripheral neuropathies. Methodist Debakey Cardiovasc J. 2010;6(2):15-19.

[2] Gebel E. New hope for ending complications. Working together to protect your body from diabetes damage. Diabetes Forecast. 2012;65(4):56, 59-60.

[3] Shiyovich A, Sztarkier I, Nesher L. Toxic hepatitis induced by Gymnema sylvestre, a natural remedy for type 2 diabetes mellitus. Am J Med Sci. 2010;340(6):514-517.

[4] Russell-Jones D. The safety and tolerability of GLP-1 receptor agonists in the treatment of type-2 diabetes. Int J Clin Pract. 2010;64(10):1402-1414.

[5] Kaewkorn W, Limpeanchob N, Tiyaboonchai W, et al. Effects of silk sericin on the proliferation and apoptosis of colon cancer cells. Biol Res. 2012;45(1):45-50.

[6] Nayak S, Talukdar S, Kundu SC. Potential of 2D crosslinked sericin membranes with improved biostability for skin tissue engineering. Cell Tissue Res. 2012;347(3):783-794.

[7] Zhaorigetu S, Yanaka N, Sasaki M, et al. Inhibitory effects of silk protein, sericin on UVB-induced acute damage and tumor promotion by reducing oxidative stress in the skin of hairless mouse. J Photochem Photobiol B. 2003;71(1-3): 11-17.

[8] Fu XM, Zhong MR, Fu WL, et al. Effects of sericine on blood glucose and blood lipid in type 2 diabetes rats. Zhongguo Laonian Xue Zazhi. 2011;31(1):103-105.

[9] Fu XM, Ma HW, Fu WL, et al. Protective effects of sericin on pancreatic islet cells of type II diabetic rat. Jiepou Xue Zazhi. 2010;33(2):161-164.

[10] Liu XY, Fu XM, Gao Y, et al. Protective effects of sericin on islet cells apoptosis of type 2 diabetes rats. Zhongguo Laonian Xue Zazhi. 2012;32(12):2525-2527.

[11] Fu WL, He YQ, Ma HW, et al. Effects of sericine on proliferation and related factors of germ cells in testis of type 2 diabetic rats. Zhongguo Yike Daxue Xuebao. 2010; 39(5):332-335.

[12] Fu WL, Fu XM, Zhong MR, et al. Effects of sericine on growth hormone/insulin-like growth factor-1 axis of testis in type 2 diabetes mellitus rats. Jiepou Xuebao. 2011; 42(1):104-109.

[13] Fu WL, Zhong MR, He YQ, et al. Effects of sericine pretreatment on IGF-1 expression in testes of diabetes mellitus rat model. Zhongguo Zuzhi Huaxue yu Xibao Huaxue Zazhi. 2010;19(4):361-364.

[14] Diolaiti D, Bernardoni R, Trazzi S, et al. Functional cooperation between TrkA and p75(NTR) accelerates neuronal differentiation by increased transcription of GAP-43 and p21(CIP/WAF) genes via ERK1/2 and AP-1 activities. Exp Cell Res. 2007;313(14):2980-2992.

[15] Renton JP, Xu N, Clark JJ, et al. Interaction of neurotrophin signaling with Bcl-2 localized to the mitochondria and endoplasmic reticulum on spiral ganglion neuron survival and neurite growth. J Neurosci Res. 2010;88(10):2239-2251.

[16] Nässel DR, Wegener C. A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling? Peptides. 2011;32(6):1335-1355.

[17] Nguyen AD, Herzog H, Sainsbury A. Neuropeptide Y and peptide YY: important regulators of energy metabolism. Curr Opin Endocrinol Diabetes Obes. 2011;18(1):56-60.

[18] Ackerley S, James PA, Kalli A, et al. A mutation in the small heat-shock protein HSPB1 leading to distal hereditary motor neuronopathy disrupts neurofilament assembly and the axonal transport of specific cellular cargoes. Hum Mol Genet. 2006;15(2):347-354.

[19] Xu G, Pierson CR, Murakawa Y, et al. Altered tubulin and neurofilament expression and impaired axonal growth in diabetic nerve regeneration. J Neuropathol Exp Neurol. 2002;61(2):164-175.

[20] von Wilmowsky C, Stockmann P, Harsch I, et al. Diabetes mellitus negatively affects peri-implant bone formation in the diabetic domestic pig. J Clin Periodontol. 2011;38(8): 771-779.

[21] Pierson CR, Zhang W, Sima AA. Proinsulin C-peptide replacement in type 1 diabetic BB/Wor-rats prevents deficits in nerve fiber regeneration. J Neuropathol Exp Neurol. 2003;62(7):765-779.

[22] Scott JN, Clark AW, Zochodne DW. Neurofilament and tubulin gene expression in progressive experimental diabetes: failure of synthesis and export by sensory neurons. Brain. 1999;122 ( Pt 11):2109-2118.

[23] Li Y, Jung P, Brown A. Axonal transport of neurofilaments: a single population of intermittently moving polymers. J Neurosci. 2012;32(2):746-758.

[24] Shea TB, Lee S. Neurofilament phosphorylation regulates axonal transport by an indirect mechanism: a merging of opposing hypotheses. Cytoskeleton (Hoboken). 2011; 68(11):589-595.

[25] Kanbayashi H, Itoh H, Kashiwaya T, et al. Spatial distribution of nociceptive neuropeptide and nerve growth factor depletion in experimental diabetic peripheral nervous system. J Int Med Res. 2002;30(5): 512-519.

[26] Gezginci-Oktayoglu S, Sacan O, Yanardag R, et al. Exendin-4 improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice. Peptides. 2011;32(2):223-231.

[27] Castro A, Manso MJ, Anadón R. Distribution of neuropeptide Y immunoreactivity in the central and peripheral nervous systems of amphioxus (Branchiostoma lanceolatum Pallas). J Comp Neurol. 2003;461(3): 350-361.

[28] Zhaohui Z, Jingzhu Z, Guipeng D, et al. Role of neuropeptide Y in regulating hypothalamus-pituitary- gonad axis in the rats treated with electro-acupuncture. Neuropeptides. 2012;46(3):133-139.

[29] Férézou I, Hill EL, Cauli B, et al. Extensive overlap of mu-opioid and nicotinic sensitivity in cortical interneurons. Cereb Cortex. 2007;17(8):1948-1957.

[30] Gonsalvez DG, Kerman IA, McAllen RM, et al. Chemical coding for cardiovascular sympathetic preganglionic neurons in rats. J Neurosci. 2010;30(35):11781-11791.

[31] Onuoha GN, Nicholls DP, Alpar EK, et al. Regulatory peptides in the heart and major vessels of man and mammals. Neuropeptides. 1999;33(2):165-172.

[32] Baldassano S, Wang GD, Mulè F, et al. Glucagon-like peptide-1 modulates neurally evoked mucosal chloride secretion in guinea pig small intestine in vitro. Am J Physiol Gastrointest Liver Physiol. 2012;302(3): G352-358.

[33] Jacques D, Sader S, El-Bizri N, et al. Neuropeptide Y induced increase of cytosolic and nuclear Ca2+ in heart and vascular smooth muscle cells. Can J Physiol Pharmacol. 2000;78(2):162-172.

[34] Jacques D, Abdel-Samad D. Neuropeptide Y (NPY) and NPY receptors in the cardiovascular system: implication in the regulation of intracellular calcium. Can J Physiol Pharmacol. 2007;85(1):43-53.

[35] The Ministry of Science and Technology of the People’s Republic of China. Guidance Suggestions for the Care and Use of Laboratory Animals. 2006-09-30.

[36] Xiang X, Wang Z, Zhu Y, et al. Dosage of streptozocin in inducing rat model of type 2 diabetes mellitus. Wei Sheng Yan Jiu. 2010;39(2):138-142.

[37] Li X, Cui X, Sun X, et al. Mangiferin prevents diabetic nephropathy progression in streptozotocin-induced diabetic rats. Phytother Res. 2010;24(6):893-899.

[38] Zhan YL, Huang CF, Chen GL. Hypoglycemic activity of the decoction of cocoon shell on alloxan-diabetic model mice. Canye Kexue. 2003;29(4):446-448.

[39] Chen ZH, He YQ, Fu WL, et al. Effects of sericin on heme oxygenase-1 expression in the hippocampus and cerebral cortex of type 2 diabetes mellitus rats. Neural Regen Res. 2011;6(6):423-427.

[40] Kikuno N, Kawamoto K, Hirata H, et al. Nerve growth factor combined with vascular endothelial growth factor enhances regeneration of bladder acellular matrix graft in spinal cord injury-induced neurogenic rat bladder. BJU Int. 2009;103(10):1424-1428.

[41] Chen ZH, He YQ, Song CJ, et al. Sericin can reduce hippocampal neuronal apoptosis by activating the Akt signal transduction pathway in a rat model of diabetes mellitus. Neural Regen Res. 2012;7(3):197-201.

引言

材料方法

Design

A randomized, controlled, animal study.

Time and setting

The experiments were performed at the Institute of Basic Medicine, Chengde Medical University, China from June 2010 to September 2011.

Materials

Experimental animals

A total of 36 healthy male Sprague-Dawley rats of clean grade, weighing 200–250 g, were provided by the Experimental Animal Center of Hebei Medical University (license No. 712024). They were housed at 20 ± 2°C with a humidity of 40–70%. Animal procedures were conducted in accordance with the Guidance Suggestions for the Care and Use of Laboratory Animals, formulated by the Ministry of Science and Technology of China^[35].

Drugs

Sericin was made from color silkworm cocoons (Sericultural Research Institute, Chengde Medical University) by soaking, water decoction, filtration and condensation.

Methods

Establishment of type 2 diabetic models

The rats were intraperitoneally injected with 2% streptozotocin (25 mg/kg; Sigma, St. Louis, MO, USA; dissolved in citric acid-sodium citrate buffer, pH 4.4) for 3 consecutive days to establish type 2 diabetic models. Rats were included if the fasting blood glucose was ≥ 16.7 mM after 7 days^[36-37]. The control group rats were normally fed and intraperitoneally injected with the same volume of citric acid-sodium citrate buffer.

Sericin treatment

Following model establishment, sericin group rats were intragastrically perfused with sericin (2.4 g/kg per day), once a day for 35 consecutive days^[38-39]. The control and model groups were intragastrically perfused with the same volume of normal saline for 35 days.

Blood glucose detection

All rats were deprived of food for 12 hours, and the rats were anesthetized by intraperitoneal injection with 4% chloral hydrate. 3 mL of blood was harvested from the posterior orbital venous plexus, centrifuged at 3 000 r/min for 20 minutes, and serum was stored at –20°C. Blood glucose levels were detected using the glucose oxidase method (Blood Glucose Detection Kit, No. 20071030; Baoding Great Wall Clinical Reagents Co., Ltd., Baoding, China) in a Boehringer Mannheim/ Hitachi 717 automatic clinical biochemical analyzer (Hitachi, Tokyo, Japan)^[8].

Immunohistochemistry detection for neurofilament protein, nerve growth factor and neuropeptide Y expression

The rats were sacrificed after the blood sampling, and the sciatic nerve (4 cm above the knee joint), spinal cord at L_4–6levels and corresponding spinal ganglia were harvested, fixed in Bouin’s solution, paraffin embedded, and sectioned into 5-μm-thick blocks. The expression level of neurofilament protein in sciatic nerve cells, and of nerve growth factor and neuropeptide Y in spinal ganglion and anterior horn cells was determined using streptavidin-peroxidase immunohistochemistry^{[12, 40]}. The sections were incubated with mouse anti-neurofilament protein polyclonal antibody (1:50; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-nerve growth factor polyclonal antibody (1:50; Santa Cruz Biotechnology) and rabbit anti-neuropeptide Y polyclonal antibody (1:50; Wuhan Boster, Wuhan, China) overnight at 4°C, followed by biotin-labeled goat anti-rabbit/mouse IgG (undiluted; Beijing Zhongshan Goldenbridge Biotechnology, Beijing, China) at 37°C for 30 minutes. The sections were visualized by diaminobenzidine, and the reaction was terminated by tap water. The nuclei were counterstained with hematoxylin. Negative controls were treated with PBS instead of the primary antibody. A positive neurofilament protein reaction was documented by brown yellow particles in the neurites of sciatic nerves; positive reactions of nerve growth factor and neuropeptide Y were represented by brown yellow particles in the nuclei or cytoplasm of spinal ganglion and anterior horn cells. Six rats from each group and six sections from each rat were randomly selected. Three fields of view were randomly selected from each section and quantitatively analyzed by 200 × light microscope (Olympus, Tokyo, Japan) using the MiVnt image analysis system (Echung Electronics, Shandong, China). Relative expression levels were represented by the mean value of the area ratio of positive products to field of view^[41].

Statistical analysis

Data were analyzed using SPSS version 15.0 (SPSS, Chicago, IL, USA) and were expressed as mean ± SD. Multiple sample comparison was made by one-way analysis of variance, and intergroup differences were compared by q-test.

讨论

文章快阅

延伸阅读

1 中文内容介绍
采用小剂量链脲佐菌素（2%，25mg/kg）连续（3d）腹腔注射的方法建立2型糖尿病大鼠模型，以空腹血糖≥16.7mM作为成模标准。待模型成功建立后，给予糖尿病大鼠丝胶（蚕茧水提物，2.4g/kg/d，1次/d）灌胃治疗35d。采用免疫组织化学染色方法观察各组大鼠坐骨神经神经微丝蛋白，脊神经节和脊髓前角细胞神经生长因子和神经肽Y蛋白的表达，探讨丝胶对糖尿病周围神经病变的保护作用。结果发现糖尿病模型组大鼠坐骨神经神经微丝蛋白的表达、脊神经节细胞和脊髓前角细胞神经生长因子的表达明显低于正常对照组大鼠，脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达明显高于正常对照组大鼠；丝胶灌胃治疗35d后，丝胶治疗组大鼠坐骨神经神经微丝蛋白蛋白的表达、脊神经节细胞和脊髓前角细胞神经生长因子蛋白的表达明显高于模型组大鼠，脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达明显低于模型组大鼠。因此表明，丝胶可通过上调2型糖尿病大鼠坐骨神经神经微丝蛋白的表达、脊神经节细胞和脊髓前角细胞神经生长因子蛋白的表达，下调脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达，对2型糖尿病时坐骨神经及相关神经细胞损伤具有一定的保护保护作用。

2 实验设计及文章构思
鉴于民间有蚕茧泡水降血糖的验方，而降血糖的西药长期应用存在毒副作用，因此考虑通过动物实验的方法探讨丝胶是否具有确切的降糖作用以及丝胶是否对糖尿病的多种并发症具有治疗作用。作者所在课题组的前期研究发现，丝胶可有效降低血糖和改善糖尿病时血脂代谢紊乱，并具有预防作用；进一步的研究表明，丝胶可保护糖尿病时胰岛β细胞损伤、肾脏改变及雄性生殖功能障碍。鉴于以往的研究结果，实验探讨了丝胶对糖尿病并发的周围神经病变的保护作用。

3 课题设计中区别于以往相关研究的特点
(1)首次将丝胶用于治疗糖尿病并发的周围神经病变，为临床治疗糖尿病及其并发症提供了新思路。(2)采用免疫组织化学染色方法，系统观察了坐骨神经神经微丝蛋白蛋白的表达，对应脊神经节神经细胞及脊髓节段（L4-6）前角细胞神经微丝蛋白和神经肽Y蛋白的表达，首次发现丝胶可通过上调2型糖尿病大鼠坐骨神经神经微丝蛋白的表达、脊神经节细胞和脊髓前角细胞神经生长因子蛋白的表达，下调脊神经节细胞和脊髓前角细胞神经肽Y蛋白的表达，对2型糖尿病时坐骨神经及相关神经细胞损伤具有一定的保护保护作用。