Electroacupuncture inhibits the ultrastructural degeneration of osteoarthritic chondrocytes mediated by Ras/Raf/MEK1/2/ERK1/2 signaling pathways

doi:10.3969/j.issn.2095-4344.2017.32.009

Abstract

Abstract:

BACKGROUND: Previous studies have found that electroacupuncture (EA) could effectively inhibit the expression of p38 and Fas mRNA mediated by MAPK signaling pathways, to further inhibit the apoptosis of chondrocytes. Meanwhile, EA delays the degeneration of articular cartilage mediated by JAK-STAT signaling pathway through upregulating the expression levels of transforming growth factor β1 as well as the mRNA expression levels of STAT3, Smad3 and LepR.
OBJECTIVE: To explore the effect of EA on the cartilage ultrastructure and mRNA expression levels of Ras, Raf, MEK1/2 and ERK1/2 in the rat with knee osteoarthritis.
METHODS: The rat models of knee osteoarthritis were established, and randomized into four groups at 2 weeks after modeling: model group received no interventions; 15- and 30-minute EA groups were given EA at the Hsiyen (medical, extra) of bilateral knee joints for 15 and 30 minutes, respectively; PD98059 group was given the intravenous injection of extracellular signal-regulated kinase inhibitor PD98059. The intervention time was 3 months. Those rats received normal feeding served as blank control group.
RESULTS AND CONCLUSION: Transmission electron microscopy showed that compared with the model group, the chondrocytes in the 15- and 30-minute EA and PD98059 groups changed little, the nucleus was larger, partial endoplasmic reticulum cisterna expanded, and the mitochondria structure was clear. ELISA results showed that the 15- and 30-minute EA and PD98059 groups had a significant decrease in the level of tumor necrosis factor α compared with the model group (P < 0.01). RT-PCR revealed that the mRNA expression levels of Ras, Raf, MEK1/2 and ERK1/2 in the 15- and 30-minute EA and PD98059 groups were significantly downregulated (P < 0.05 or P < 0.01). These results indicate that EA can alleviate chondrocyte injury in the rat osteoarthritic model, reduce the level of tumor necrosis factor α in the synovium, and downregulated the expression levels of Ras, Raf, MEK1/2 and ERK1/2 mRNA, further delaying the chondrocyte degeneration in osteoarthritis.

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: Tissue Engineering, Knee Joint, Osteoarthritis

CLC Number:

R318

Fu Chang-long1, Lin Jie1, Zhao Zhong-sheng1, Wu Guang-wen1, Hong Xiu-e2, Zheng Chun-song1, Lin Qiu-ying3, Dai Yi-chen3, Wu Ming-xia2. Electroacupuncture inhibits the ultrastructural degeneration of osteoarthritic chondrocytes mediated by Ras/Raf/MEK1/2/ERK1/2 signaling pathways[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(32): 5134-5139.

Figures/Tables 2

2.1 实验动物数量分析经治疗干预3个月后，正常组大鼠在饲养过程中出现1只死亡，剩余29只。实验组经造模后，由于个体差异等原因，出现4只大鼠因麻醉死亡，此外因于造模后2周随机抽取4只大鼠进行模型鉴定，故实验组共剩余112只大鼠，在此基础上经随机分为4组并进行相关治疗后，模型组出现2只死亡，剩余26只；电针15，30 min组在实验过程中分别出现3只和5只死亡，分别剩余25及23只；PD98059组出现6只大鼠死亡剩余22只。最终125只大鼠进入结果分析。 2.2 电针后各组关节组织超微结构变化透射电镜超微结构结果示，正常组软骨细胞外形规则，近球形，表面敷着较丰富的微绒毛样突起，细胞核大，常染色质为主，少有异染色质，细胞间链接较紧密，线粒体数个呈现球星，胞质中有较为发达的粗面内质网且附着有核糖体，胞外基质见胶原原纤维(图1A)；模型组关节软骨细胞发生变性，形态结构改变，异染色质丰富、浓染，细胞器结构紊乱，内质网核糖体脱落，部分糖原聚集，呈现凋亡现象(图1B)；电针15，30 min组的软骨细胞形态变化较小，细胞核较大，模型结构不清，部分内质网池扩张，线粒体结构仍较清晰，糖原散在分布(图1C，D)；PD98059抑制剂组细胞外形近椭圆，表面有突起，胞核近椭圆，异染色质散在，糖原颗粒较多且散在分布，粗面内质网较发达，线粒体呈椭圆形散在分布(图1E)。 2.3 电针对骨关节炎大鼠膝关节部滑膜组织中肿瘤坏死因子α水平的影响 ELISA检测显示如表1示，与正常组比，模型组肿瘤坏死因子α水平表达明显升高(P < 0.01)，与模型组比，PD98059抑制剂组、电针15，30 min组肿瘤坏死因子α水平表达降低(P < 0.01)。见表1。 2.4 各组关节组织Ras，Raf，MEK1/2，ERK1/2 mRNA表达 RT-PCR结果如图2与表1所示，电针干预骨关节炎大鼠软骨后，可降低关节软骨中Ras及Raf、MEK1/2及ERK1/2 mRNA表达。模型组Ras及Raf，MEK1/2及ERK1/2 mRNA表达较正常组显著升高(P < 0.05或P < 0.01)，提示大鼠关节软骨受损；而PD98059抑制剂组、电针15，30 min组的mRNA表达较模型组均显著降低(P < 0.05或P < 0.01)。进而提示电针可缓解4%木瓜蛋白酶所致大鼠软骨退变，其治疗保护机制为降低退变软骨组织中Ras，Raf，MEK1/2及ERK1/2 mRNA表达。"

References

[1] Chen S, Huang Y, Chen W, et al. Protective effects of Tougu Xiaotong capsule on morphology and osteoprotegrin/nuclear factor-κB ligand expression in rabbits with knee osteoarthritis. Mol Med Rep. 2016;13(1):419-425.

[2] Li X, Zhen Z, Tang G, et al. MiR-29a and MiR-140 Protect Chondrocytes against the anti-proliferation and cell matrix signaling changes by IL-1β. Mol Cells. 2016;39(2):103-110.

[3] 刘献祥.基于陈可冀学术思想之骨性关节炎研究[J].康复学报, 2016,26(1):2-5.

[4] 林洁,付长龙,李俐,等.电针治疗膝骨性关节炎疗效的系统评价[J].康复学报,2016,26(4):52-58.

[5] Wu MX, Li XH, Lin MN, et al. Clinical study on the treatment of knee osteoarthritis of Shen-Sui insufficiency syndrome type by electroacupuncture. Chin J Integr Med. 2010;16(4): 291-297.

[6] Ye J, Wu G, Li X, et al. Millimeter wave treatment inhibits apoptosis of chondrocytes via regulation dynamic equilibrium of intracellular free Ca2+. Evid Based Complement Alternat Med. 2015:464161.

[7] 吴明霞,李西海,吴广文,等.电针后血清对肿瘤坏死因子α诱导软骨细胞凋亡的影响[J].中国组织工程研究与临床康复, 2011, 15(46):8551-8555.

[8] Wang X, Li F, Fan C, et al. Effects and relationship of ERK 1 and ERK 2 ininterleukin-1-induced alterations in MMP3, MMP13, typeII collagen and aggrecan expression in human chondrocytes. Int J Mol Med. 2011;27(4):583-589.

[9] Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol. 2008;9(7):517-531.

[10] Roskoski R. ERK1/2 MAP kinases: structure,function,and regulation. Pharmacol Res. 2012; 66(2):105-143.

[11] 李西海,梁文娜,党传鹏,等.补肾壮筋汤抑制炎性细胞因子表达延缓骨关节炎软骨退变的研究[J].风湿病与关节炎, 2014,3(5): 20-25.

[12] 陈后煌,邵翔,李俐,等.肿瘤坏死因子α诱导大鼠软骨细胞凋亡模型的建立及鉴定[J].中国组织工程研究,2017,21(4):527-531.

[13] Fu CL, Zheng CS, Lin J, et al. Cibotium barometz polysaccharides stimulate chondrocyte proliferation in vitro by promoting G1/S cell cycle transition. Mol Med Rep. 2017;15(5): 3027-3034.

[14] 林木南,林艳红,刘献祥,等.温热疏密波对膝骨关节炎模型大鼠软骨细胞RAS/丝裂原活化蛋白激酶信号通路的影响[J].中医正骨, 2016,28(2):81-86.

[15] 高世超,殷海波,刘宏潇,等.MAPK信号通路在骨关节炎发病机制中的研究进展[J].中国骨伤,2014,27(5):441-444.

[16] O'Neill E, Kolch W. Conferring specificity on the ubiquitous Raf/MEK signalling pathway. Br J Cancer. 2004;90(2): 283-288.

[17] Liu L, Cao Y, Chen C, et al. Sorafenib blocks the raf/mek/erk pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res. 2006;66(24):11851-11858.

[18] 蒋成英,戴广海.Ras/Raf/Mek/Erk信号传导通路在肝细胞癌发生中的作用机制及在靶向治疗中的应用[J].中国肿瘤临床, 2008, 35(23):1377-1380.

[19] Karnoub AE, Weinberg RA. Ras oncogenes: split personalities. Nat Rev Mol Cell Biol. 2008;9(7):517-531.

[20] Roskoski R. ERK1 /2 MAP kinases: structure, function, and regulation. Pharmacol Res. 2012;66(2):105-143.

[21] Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol. 2007;213:626-634.

[22] Wang X, Li F, Fan C, et al. Effects and relationship of ERK 1 and ERK 2 in interleukin-1β-induced alterations in MMP3,MMP13, type II collagen and aggrecan expression in human chondrocytes. Int J Mol Med. 2011;27(4):583-589.

[23] 李冠,甘丽娇,钟妙容.TNF-α与β1,4- GalT-I在骨关节炎滑膜炎症过程中的关系研究[J].临床和实验医学杂志, 2016,15(2): 137-139.

[24] 朱芳晓,周润华,石宇红,等.小白菊内酯对兔膝骨关节炎血清IL-1β及TNF-α的影响[J].中国中西医结合杂志, 2013,33(10): 1382-1384.

[25] 李迎雪,陈丹丹,张茹,等.双歧杆菌脂磷壁酸通过激活小鼠腹腔巨噬细胞MEK/ERK通路促进TNF-α表达[J].海南医学院学报, 2015,21(4):442-448.

[26] 包飞,张燕,吴志宏,等.电针治疗膝骨关节炎疗效观察及对软骨磁共振T2图的影响[J].中国针灸,2013,33(3):193-197.

[27] 刚嘉鸿,宓轶群,王华敏.电针与美洛昔康治疗早中期膝骨关节炎临床疗效比较随机对照研究[J].中国针灸,2016,36(5):467-470.

[28] 吴明霞,李西海,李俐,等.电针后血清对TNF-α诱导凋亡软骨细胞MAPK信号通路的影响[J].福建中医药,2011,42(6):43-45.

[29] 吴明霞,李西海,李俐,等.电针对骨性关节炎软骨细胞JAK-STAT信号通路表达的影响[J].福建中医药大学学报, 2011,21(6): 21-23.

[30] Yang X, He H, Zhou Y, et al. Pulsed electromagnetic field at different stages of knee osteoarthritis in rats induced by low-dose monosodium iodoacetate: Effect on subchondral trabecular bone microarchitecture and cartilage degradation. Bioelectromagnetics. 2017;38(3):227-238.

[31] Baudi P, Catani F, Rebuzzi M, et al. Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post-Traumatic Shoulder Instability. Anat Rec (Hoboken). 2017;300(7): 1208-1218.

[32] Atik O?, Erdo?an D, Seymen CM, et al. Is there crosstalk between subchondral bone, cartilage, and meniscus in the pathogenesis of osteoarthritis? Eklem Hastalik Cerrahisi. 2016;27(2):62-67.

[33] Iijima H, Aoyama T, Tajino J, et al. Subchondral plate porosity colocalizes with the point of mechanical load during ambulation in a rat knee model of post-traumatic osteoarthritis. Osteoarthritis Cartilage. 2016;24(2):354-363.

[34] Kaderli S, Viguier E, Watrelot-Virieux D, et al. Efficacy study of two novel hyaluronic acid-based formulations for viscosupplementation therapy in an early osteoarthrosic rabbit model. Eur J Pharm Biopharm. 2015;96:388-395.

[35] Prieto-Potin I, Largo R, Roman-Blas JA, et al. Characterization of multinucleated giant cells in synovium and subchondral bone in knee osteoarthritis and rheumatoid arthritis. BMC Musculoskelet Disord. 2015;16:226.

[36] Tomatsu S, Alméciga-Díaz CJ, Montaño AM, et al. Therapies for the bone in mucopolysaccharidoses. Mol Genet Metab. 2015;114(2):94-109.

[37] Asakawa-Tanne Y, Su S, Kunimatsu R, et al. Effects of enzymatic degradation after loading in temporomandibular joint. J Dent Res. 2015;94(2):337-343.

[38] Cheleschi S, Cantarini L, Pascarelli NA, et al. Possible chondroprotective effect of canakinumab: an in vitro study on human osteoarthritic chondrocytes. Cytokine. 2015;71(2): 165-172.

[39] Neu CP, Novak T, Gilliland KF, et al. Optical clearing in collagen- and proteoglycan-rich osteochondral tissues. Osteoarthritis Cartilage. 2015;23(3):405-413.

[40] Prokopovich P. Interactions between mammalian cells and nano- or micro-sized wear particles: physico-chemical views against biological approaches. Adv Colloid Interface Sci. 2014;213:36-47.

[41] Yan JY, Tian FM, Wang WY, et al. Age dependent changes in cartilage matrix, subchondral bone mass, and estradiol levels in blood serum, in naturally occurring osteoarthritis in Guinea pigs. Int J Mol Sci. 2014;15(8):13578-12595.

[42] Salzmann GM, Erdle B, Porichis S, et al. Long-term T2 and Qualitative MRI Morphology After First-Generation Knee Autologous Chondrocyte Implantation: Cartilage Ultrastructure Is Not Correlated to Clinical or Qualitative MRI Outcome. Am J Sports Med. 2014;42(8):1832-1840.

[43] Espino DM, Shepherd DE, Hukins DW. Viscoelastic properties of bovine knee joint articular cartilage: dependency on thickness and loading frequency. BMC Musculoskelet Disord. 2014;15:205.

[44] Speirs AD, Beaulé PE, Ferguson SJ, et al. Stress distribution and consolidation in cartilage constituents is influenced by cyclic loading and osteoarthritic degeneration. J Biomech. 2014;47(10):2348-2353.

[45] Roemer FW, Eckstein F, Hayashi D, et al. The role of imaging in osteoarthritis. Best Pract Res Clin Rheumatol. 2014;28(1): 31-60.