Electroacupuncture delays articular cartilage degeneration in osteoarthritis via Ras-Raf-MEK1/2-ERK1/2 signaling pathway

doi:10.3969/j.issn.2095-4344.2017.24.004

Abstract

Abstract:

BACKGROUND: Previous studies have found that electroacupuncture can delay articular cartilage degeneration mediated by JAK-STAT signaling pathway through upregulating the expression level of transforming growth factor β1 as well as mRNA expression levels of STAT3, Smad3 and LepR. In the meanwhile, electroacupuncture can inhibit the mRNA expression of p38 and Fas mRNA mediated by MAPK signaling pathways, further inhibiting the apoptosis of chondrocytes.

OBJECTIVE: To explore the effect of electroacupuncture on the degeneration of articular cartilage in rats with knee osteoarthritis based on Ras-Raf-MEK1/2-ERK1/2 signaling pathway.

METHODS: 120 male healthy Sprague-Dawley rats aged 2 months olds were selected and randomly divided into normal, model, 15-minite electroacupuncture and 30-minute electroacupuncture groups (n=30 per group). The rats in the latter three groups received the intra-articular injection of 4% papain bilaterally, and the remaining rats received no intervention. At 2 weeks after modeling, the latter two groups were respectively given 15- and 30-minute electroacupuncture, five times weekly for consecutive 12 weeks. The morphology of the cartilage was observed by hematoxylin-eosin staining, the expression level of interleukin-1β in the synovium was detected by ELISA assay, and the protein expression levels of Ras, Raf, MEK1/2, ERK1/2, C-MYC, C-FOS, and C-JUN were detected by western blot analysis.

RESULTS AND CONCLUSION: Hematoxylin-eosin staining showed that: in the model group, the cartilage surface was rough, the cartilage layer became thinner, and the cartilage structure was damaged with incomplete tidal line; in the 15- and 30-minute electroacupuncture groups, the cartilage structure was complete with clear layers and complete tidal line. ELISA showed that the expression level of interleukin-1β in the model group was significantly higher than that in the normal group (P < 0.01), and the level in the 15- and 30-minute electroacupuncture groups was significantly lower than that in the model group (P < 0.05). Western blot assay found that compared with the normal group, the protein expression levels of Ras, Raf, MEK1/2, ERK1/2, C-MYC, C-FOS, and C-JUN were increased in the model group. However, all above protein levels except ERK1/2 in the 15- and 30-minute electroacupuncture groups were significantly lower than those in the model group (P < 0.01, P < 0.05). To conclude, electroacupuncture inhibits the degeneration of articular cartilage in osteoarthritis via Ras-Raf-MEK1/2-ERK1/2 signaling pathway and downregulating the expression level of interleukin-1β.

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: Cartilage, Synovial Membrane, Osteoarthritis, Electroacupuncture, Signal Transduction, Tissue Engineering

CLC Number:

R318

Fu Chang-long, Chen Hou-huang, Zhu Ding-yu, Wu Zhui-le, Xu Xin, Zheng Chun-song, Li Li, Liu Xian-xiang,Li Xi-hai, Wu Ming-xia. Electroacupuncture delays articular cartilage degeneration in osteoarthritis via Ras-Raf-MEK1/2-ERK1/2 signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(24): 3790-3795.

Figures/Tables 2

2.1 实验动物数量分析实验选用大鼠120只，分为4组，实验过程无脱失，全部进入结果分析。 2.2 软骨组织苏木精-伊红染色结果苏木精-伊红染色显示，正常组软骨表层平整，胞质和软骨基质着色均匀并染成粉红色，软骨细胞分布均匀且排列整齐，胞核呈蓝色，4层结构(尤其潮线)完整且清晰可辨(图1A)；模型组关节软骨表面局部粗糙不平，软骨层变薄，软骨细胞裸露、簇集生长且排列絮乱，4层结构(尤其潮线)不完整(图1B)；电针15 min组和电针30 min组浅软骨表层基本完整，软骨层次清晰可辨，潮线相对完整(图1C和1D)。 2.3 ELISA结果电针对骨关节炎大鼠滑膜组织白细胞介素1β表达的影响如图2所示，模型组白细胞介素1β表达明显高于正常组(P < 0.01)，而电针15 min组与电针30 min组的白细胞介素1β表达均低于模型组(P < 0.01) 2.4 Western Blot结果 2.4.1 Ras、Raf、MEK1/2、ERK1/2蛋白表达检测结果Western Blot结果如表1与图3所示，电针作用于骨关节炎大鼠软骨后，可下调Ras、Raf、MEK1/2、ERK1/2蛋白含量表达。与正常组相比，模型组在Ras、Raf、MEK1/2含量均显著升高，差异有显著性意义(P < 0.05或0.01)，在ERK1/2上有较高表达(P > 0.05)，综上提示软骨在一定程度上发生退变；在Ras、Raf、MEK1/2指标上，与模型组相比，电针15 min组与电针30 min组的蛋白含量均显著降低，差异有显著性意义(P < 0.05或P < 0.01)，在ERK1/2指标上表达降低(P > 0.05)，提示电针可通过调控Ras、Raf、MEK1/2、ERK1/2表达，在一定程度上延缓软骨细胞的退变进程。 2.4.2 C-MYC、C-FOS、C-JUN蛋白表达检测结果Western Blot结果如表2与图4所示，电针作用于骨关节炎大鼠软骨后，可下调C-MYC、C-FOS、C-JUN蛋白含量表达。与正常组相比，模型组在C-MYC、C-FOS、C-JUN含量均显著升高，差异有显著性意义(P < 0.01)；与模型组相比，电针15 min组与电针30 min组的蛋白含量表达均显著降低，差异有显著性意义(P < 0.05或P < 0.01)，提示电针可通过调控Ras/Raf/MEK1/2/ERK1/2信号通路的下游信号分子C-MYC、C-FOS、C-JUN蛋白含量表达，延缓软骨细胞的退变进程。"

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