Minocycline affects the expression of glial cell derived neurotrophic factor family in a rat model of Parkinson’s disease

doi:10.3969/j.issn.2095-4344.2016.27.010

Abstract

Abstract:

BACKGROUND: Researches have found that minocycline plays a neuroprotective effect by inhibiting the microglia cell proliferation and activation and suppressing glial cells to release cytokines and chemokines.
OBJECTIVE: To investigate the influence of minocycline on glial cell line derived neurotrophic factor, NTN and gene expression in substantia nigra and corpus striatum in Parkinson’s disease model rats.
METHODS: 144 rats were randomly divided into four groups, with 36 rats in each group. In the normal control group, no intervention was given. In the model and experimental groups, 6-hydroxydopamine was injected in the right substantia nigra pars compacta and ventral tegmental area to establish Parkinson’s disease models. In the sham surgery group, vitamin C was injected in the two points. In the experimental group, after model establishment, rats were intragastrically given 4.5 g/L minocycline 45 mg/kg. From then on, additional 22.5 mg/kg minocycline was added every 12 hours. The last group was normal control group. Immediately, 12 hours, 1, 7, 14 and 21 days after model induction, SP immunohistochemistry was used to detect the expression of glial cell line derived neurotrophic factor and NTN expression in the substantia nigra and corpus striatum. RT-PCR was used to identify glial cell line derived neurotrophic factor and NTN mRNA expression in the substantia nigra and corpus striatum.
RESULTS AND CONCLUSION: Both in the substantia nigra and corpus striatum, the positive cell number and relative gene expression of glial cell line derived neurotrophic factor and NTN were lower in the model group than in the normal control and sham surgery groups (P < 0.05). Glial cell line derived neurotrophic factor- and NTN-positive cell number and relative expression were higher in the experimental group than in the model group (P < 0.05). These findings suggest that minocycline can delay the process of Parkinson’s disease pathogenesis by promoting glial cell line derived neurotrophic factor protein and gene expression.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Parkinson Disease, Minocycline, Animals, Laboratory, Tissue Engineering

CLC Number:

R318

Xing Hong-xia, Jiang Jian-kai, Qin Li-yuan, Wang Yu-mei . Minocycline affects the expression of glial cell derived neurotrophic factor family in a rat model of Parkinson’s disease[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(27): 4020-4028.

Figures/Tables 3

2.1 一般情况正常对照组和假手术组72只大鼠无神经功能缺损及死亡，其余的72只用于造模，造模成功54只，其中模型组死亡5只，实验组死亡3只，造模成功率为75%，造模未成功的大鼠给予随机补充并重新造模，最终使其符合造模成功要求。造模成功后的大鼠表现出神经功能缺损，包括旋转时以健侧前肢或后肢为支点、身体环曲、首尾相接的原地旋转，伴觅食样动作。 2.2 行为学测定术后大鼠出现震颤、僵直、运动缓慢等典型的帕金森病表现，并出现拒捕行为的减弱、竖毛、毛色变黄变脏、弓背等症状。mNSS评分结果发现，模型组mNSS评分大都在13-18分之间，属重度损害；实验组大鼠mNSS评分大都在7-12分之间，属中度损害；正常对照组和假手术组大鼠无异常行为，mNSS评分均为0分。 2.3 免疫组织化学检测结果大鼠脑黑质GDNF表达：①从同一时间点来看，模型组大鼠黑质GDNF阳性细胞数低于正常对照组和假手术组(P < 0.05)；与模型组相比，实验组GDNF的阳性细胞数增多(P < 0.05)。②从不同时间点来看，随着时间的延长，模型组GDNF表达呈不断减少趋势，但在各时间点差异无显著性意义(P > 0.05)，实验组GDNF表达呈不断增加趋势，在各时间点比较差异无显著性意义(P > 0.05)，见表1，图1。大鼠脑纹状体GDNF表达：正常对照组和假手术组两组相比无明显差异；模型组GDNF阳性细胞数呈递减趋势，其中14 d最低，与正常对照组和假手术组相比差异显著(P < 0.05)；实验组GDNF阳性细胞数呈不断增加趋势，治疗21 d达到高峰，与模型组相比差异显著(P < 0.05)，见表2，图2。大鼠脑黑质NTN表达：①从同一时间点来看，正常对照组和假手术组两组相比无明显差异(P > 0.05)，模型组NTN阳性细胞数低于正常对照组和假手术组(P < 0.05)；实验组NTN阳性细胞数高于模型组(P < 0.05)。②从不同时间点来看，各组NTN阳性细胞数均在1 d达到低峰期，随着时间的延长，模型组NTN表达呈不断减少趋势，但各时间点间差异无显著性意义(P > 0.05)；实验组NTN表达呈不断增加趋势，在各时间点比较差异无显著性意义(P > 0.05)，见表3，图3。大鼠脑纹状体NTN表达：①从同一时间点来看，正常对照组和假手术组相比无明显差异，模型组、实验组NTN阳性细胞数低于正常对照组和假手术组(P < 0.05)；实验组NTN阳性细胞数高于模型组(P < 0.05)。②从不同时间点来看，各组NTN阳性细胞数均在24 h达到低峰期，随着时间的延长，模型组NTN表达呈不断减少趋势，但各时间点比较无差异(P > 0.05)；实验组NTN表达呈不断增加趋势，各时间点比较无差异(P > 0.05)，见表4，图4。 2.4 荧光定量PCR结果大鼠黑质GDNF mRNA半定量表达：用ImageJ定量分析DNA电泳条带得到相对mRNA量结果显示：①从同一时间点来看，模型组大鼠黑质GDNF mRNA相对表达量低于正常对照组和假手术组(P < 0.05)，实验组GDNF mRNA的相对表达量多于模型组(P < 0.05)。②从不同时间点来看，随着时间的延长，模型组GDNF mRNA表达呈不断减少趋势，但各时间点比较无差异(P > 0.05)；实验组GDNF mRNA表达呈不断增加趋势，治疗7 d、14 d、21 d与治疗即刻、12 h，24 h比较差异显著(P < 0.05)，见表5，图5。大鼠脑纹状体GDNFmRNA半定量表达：纹状体是黑质多巴胺能神经元的主要投射部位，是黑质多巴胺能神经元发挥作用的重要环节。正常对照组和假手术组比较无明显差异，模型组GDNF mRNA相对表达量呈递减趋势，其中14 d最低，与正常对照组和假手术组相比差异明显(P < 0.05)。实验组GDNF mRNA相对表达量呈不断增加趋势，治疗21 d达到高峰，与模型组相比差异明显(P < 0.05)，见表6，图5。大鼠脑黑质NTN mRNA半定量表达：NTN在腹侧中脑表达逐渐下降，而在纹状体中表达增加，结果显示：①从同一时间点来看，模型组NTN mRNA相对表达量低于正常对照组和假手术组(P < 0.05)；实验组NTN mRNA相对表达量高于模型组(P < 0.05)。②从不同时间点来看，随着时间的延长，模型组NTN mRNA表达呈不断减少趋势，但各时间点比较无差异(P > 0.05)；实验组NTN mRNA表达呈不断增加趋势，在14 d达到高峰期，见表7，图6。大鼠脑纹状体NTN mRNA半定量表达：结果显示：①从同一时间点来看，模型组NTN mRNA相对表达量低于正常对照组和假手术组(P < 0.05)；实验组NTN mRNA的相对表达量高于模型组(P < 0.05)。②从不同时间点来看，随着时间的延长，模型组NTN mRNA表达呈不断减少趋势，但各时间点比较无差异(P > 0.05)；实验组NTN mRNA表达呈不断增加趋势，在14 d达到高峰期，见表8，图6。"

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