Expression and effects of matrix metalloproteinase 2 and 9 and tissue inhibitor of metalloproteinase 1 and 2 in the denervated skeletal muscle of rats

doi:10.3969/j.issn.2095-4344.0083

Abstract

Abstract:

BACKGROUND: Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMP) are involved in the remodeling of extracellular matrix under normal or pathological conditions, and the positive expression of MMP-2 and MMP-9 has been shown to play important physiological and pathological roles in the denervated skeletal muscle.
OBJECTIVE: To investigate the expression and effect of MMP-2 and MMP-9 and their tissue inhibitors (TIMP-1 and TIMP-2) in the denervated skeletal muscle.
METHODS: The right sciatic nerve of rats was cut off to establish the model of denervated skeletal muscle, and the right skeletal muscle of rats was harvested after different intervals. The morphologic changes of the denervated skeletal muscle were detected through hematoxylin-eosin staining, immunohistochemical staining, and RT-PCR. The expression and change of MMP-2 and MMP-9 and TIMP-1 and TIMP-2 were compared with the sham operation group.
RESULTS AND CONCLUSION: Atrophy and fibrosis were observed in the denervated skeletal muscle. There was a rapid increase of MMP-2 and TIMP-2 with double peaks at the 3rd and 56th days after modeling; at the 3rd and 70th days, the expression of MMP-9 increased significantly and reached the peak respectively; mRNA level of TIMP-1 only increased in the early stage, reached the peak at the 3rd day and gradual declined to normal level, without the second peak. To conclude, MMPs and TIMPs are involved in the tissue changes following denervation. TIMP-1 and TIMP-2 have a protective role in the remodeling progression. The altered balance between MMPs and TIMPs in the late stage of denervation may be responsible for extracellular matrix degradation leading to the atrophy and fibrosis progression.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Muscle, Skeletal, Matrix Metalloproteinase 2, Matrix Metalloproteinase 9, Tissue Engineering

CLC Number:

R318

Chen Bo1, Liang Jie1, Chen Zhen-bing2. Expression and effects of matrix metalloproteinase 2 and 9 and tissue inhibitor of metalloproteinase 1 and 2 in the denervated skeletal muscle of rats[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(4): 516-522.

Figures/Tables 4

2.1 实验动物数量分析纳入58只大鼠，按骨骼肌失神经支配天数设置11个实验组(n=5)和1个假手术对照组(n=3)，全部进入结果分析，无脱失。 2.2 苏木精-伊红染色结果正常及假手术组的大鼠腓肠肌苏木精-伊红染色后呈现出一整齐的外观：肌纤维呈红色，肌纤维外膜清晰，细胞核小而规则，位于肌纤维周边；细胞外的纤维成分及其他细胞成分少；肌纤维间隔间有淡红丝状的结缔组织分割；血管边界清晰，血管周围红色的结缔组织排列有序，肌束膜红染，肌肉内的神经纤维排列整齐(图1A)。坐骨神经切断后，肌纤维呈现出一个溶解、增生、重构的过程(图1B-F)：部分细胞细胞质透明样坏死变性，血管周围的结缔组织中含有大量的蓝染较深且体积较小的炎性细胞；肌纤维周径变小，肌纤维变性数目较前明显增多，细胞淡染，肌纤维间质中红染的纤维成分增多；肌纤维间结缔组织成分逐渐增多，血管管腔变小，周围结缔组织增多增厚，肌纤维被间质成分分割。 2.3 免疫组织化学染色结果正常的肌肉组织中(图2A)，MMP-2、MMP-9肌纤维组织中表达零星出现，表达很少，而血管壁周围则表达较多。神经切断后的早期(1，2 d)，MMP-2在肌纤维和肌束间的几乎没有表达；在神经切断后的第3，4天(图2B，C)，发现大量的MMP-2出现在肌纤维膜中。肌纤维膜结构分辨不清，肌纤维间距增宽，黄染的MMP-2颗粒沿其间的纤维性的基质分布；血管壁及周围的间质也有大量的黄染颗粒沉积。坐骨神经切断术后第7天，MMP-2黄染颗粒少见，主要集中在血管周围。坐骨神经切断术后第21-42天期间(图2D)，MMP-2表达出现一个缓慢变化过程，主要在肌纤维之间的细胞外基质以及肌纤维膜周围分布，肌纤维膜之间还有较多的含黄染颗粒的细胞，肌束间的纤维成分逐渐增加，黄染颗粒沿纤维成分分布，其中亦含有大量的炎性细胞。术后的第56-70天(图2E，F)，细胞外的MMP-2逐渐减少，肌纤维间的MMP2黄染颗粒减少，肌束间的黄染颗粒亦减少。 MMP-9表达变化也呈现一个大致相同过程(图3)，术后14 d内呈现出一个先增加后减少的趋势。术后第4天(图3C)，纤维细胞结构不清，肌纤维间隔中未见黄染颗粒的出现，肌束膜表面呈现出黄染的颗粒样物质，血管壁及周围纤维组织亦含有黄染的颗粒物质；术后7 d内，肌纤维细胞间与肌纤维边缘可见淡染的黄色颗粒沉积；14 d时，肌纤维间少量黄染颗粒沉积，肌纤维间的纤维及细胞成分明显，肌纤维细胞变小，密集明显，肌束上面有较少黄染颗粒沉积；术后21 d(图3D)，肌纤维间的炎性细胞成分含量明显增多，MMP-9黄染颗粒也出现在血管周围及其肌束间的纤维膜，肌纤维间未发现黄染颗粒存在。通过统计学分析MMP-2和MMP-9的蛋白阳性表达率发现，术后呈现出先增加后减少的趋势，差异有显著性意义(P < 0.01)，见表1。 2.4 MMP-2，MMP-9及TIMP-1，TIMP-2的RT-PCR结果分析 2.4.1 MMP-2，MMP-9 mRNA表达正常的肌肉组织中MMP-2、MMP-9表达很少。在神经切断后的3 d，MMP-2、MMP-9 mRNA表达显著增高达到第1个高峰，差异有显著性意义(P < 0.01)；在神经切断后的4-42 d，MMP-2 mRNA 表达逐渐降低，但仍然达正常肌肉组织的2倍以上，MMP-9 mRNA则降低至正常肌肉的水平。在神经切断后的56， 70 d ，MMP-2，MMP-9 mRNA表达分别出现第2个高峰，差异有显著性意义(P < 0.01)，见表2及图4，5。 2.4.2 TIMP-1，TIMP-2 mRNA的表达与MMP-2，MMP-9 mRNA类似，TIMP-1，TIMP-2 mRNA表达在神经切断后的3 d达到10倍于正常肌肉组织水平(P < 0.01)；但随后二者出现分化，TIMP-1 mRNA表达在神经切断后的4-70 d降低至正常水平(P > 0.05)，没有第2个高峰出现；TIMP-2 mRNA表达在神经切断后的4-42 d逐渐降低至正常水平(P > 0.05)，56 d出现第2个高峰，达到正常肌肉组织中12倍的水平，然后下降，70 d仍为正常肌肉组织中2倍的水平(P < 0.01)，见图6，7。"

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