淫羊藿素通过抑制Hedgehog信号通路保护终板软骨细胞退变

中国临床药理学与治疗学 ›› 2017, Vol. 22 ›› Issue (4): 373-380.

淫羊藿素通过抑制Hedgehog信号通路保护终板软骨细胞退变

徐永明¹，徐宏光¹，高智¹，张晓玲²

¹皖南医学院第一附属医院弋矶山医院脊柱外科，芜湖 241001，安徽；²中国科学院上海生命科学院骨科细胞与分子生物学实验室，上海 200025

收稿日期:2017-01-06 修回日期:2017-02-14 出版日期:2017-04-26 发布日期:2017-04-26
通讯作者: 徐宏光，男，博士研究生，教授，博士生导师，研究方向：脊柱外科与老年骨病。 Tel: 0553-5739037 E-mail：xuhg@medmail.com.cn
作者简介:徐永明，男，硕士研究生，研究方向：脊柱外科与老年骨病。 Tel: 13866377355 E-mail：xuyongming1986@163.com
基金资助:
国家自然科学基金（81272048）；卫生部公益性行业专项基金（201002018）

Icaritin protects the ICMT-induced degeneration of endplate chondrocytes via inhibiting Hedgehog signaling pathway

XU Yongming ¹, XU Hongguang¹, GAO Zhi¹, ZHANG Xiaoling ²

¹Department of Orthopedic Surgery, Yijishan Hospital, Wannan Medical College, Wuhu 241001, Anhui, China; ² Key Laboratory of Orthopedic Surgery, Shanghai Institutes for Biological Sciences (SBIS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine (SJTUSM), Shanghai 200025, China

Received:2017-01-06 Revised:2017-02-14 Online:2017-04-26 Published:2017-04-26

摘要/Abstract

摘要：

目的: 探讨淫羊藿素药物在体外力学诱导终板软骨细胞退变中的作用机制。方法: 应用FX-5000T flexcell细胞应力加载系统体外建立大鼠终板软骨细胞退变模型，实验分正常对照组（NC组）、力学刺激组（ICMT组）、力学刺激+药物处理组（ICT+ICMT组）。甲苯胺蓝及鬼笔环肽染色观察力学刺激后细胞形态变化，CCK-8法和流式细胞术检测细胞增殖与凋亡，Western blot及RT-qPCR分别检测软骨细胞相关基因及蛋白水平变化。结果: 大鼠终板软骨细胞力学刺激后出现退变，细胞形态由多边形被拉成长梭形，ICMT组、ICT+ICMT组细胞增殖率均高于NC组，但ICMT组与ICT+ICMT组间细胞增殖率比较无统计学差异，ICT+ICMT组细胞凋亡率较ICMT组明显改善，加入淫羊藿素药物处理后终板软骨细胞因力学刺激导致的退变程度有所缓解，Hedgehog信号通路被抑制。结论: 淫羊藿素药物可通过抑制Hedgehog信号通路活性起到保护因力学诱导而引起的终板软骨细胞退变作用。

关键词: 淫羊藿素, 终板软骨细胞, 细胞退变, Hedgehog信号通路

Abstract:

AIM: To investigate the effect of icaritin on degeneration of endplate chondrocytes induced by intermittent cyclic mechanical tension (ICMT) and its mechanism in vitro. METHODS: Rat endplate chondrocytes degeneration model was established by using FX-5000T flexcell stress loading system in vitro. Three groups were erected: normal control group (NC group), ICMT group (10％intermittent cyclic mechanical tension ,10％ICMT, 0.5 Hz, 8 h/d), and ICT+ICMT group. The morphological alteration of the cells was observed with toluidine blue staining and phalloidin staining. Cell proliferation and apoptosis were detected respectively by CCK-8 and flow cytometry. The mRNA levels of chondrocyte-associated genes were detected by qRT-PCR and protein levels were determined by Western blot. RESULTS: Degeneration of endplate chondrocytes was apparently observed after mechanical stimulation. In detail, cell morphology changed from polygon to long spindle after mechanical loading. The cell proliferation rates in ICMT group and ICT+ICMT group were higher than NC group. However, there was no significant difference between ICMT group and ICT+ICMT group. The apoptosis rate of ICT+ICMT group was significantly lower than ICMT group. Interestingly, treatment with icariin effectively alleviated ICMT-induced degeneration of endplate chondrocytes, and significantly suppressed the Hedgehog signaling pathway. CONCLUSION: Icaritin can efficiently protect the ICMT-induced cartilage degeneration by inhibiting the activity of Hedgehog signaling pathway.

Key words: icaritin, endplate chondrocytes, cell degeneration, Hedgehog signaling pathway

中图分类号:

R965.2

徐永明，徐宏光，高智，张晓玲. 淫羊藿素通过抑制Hedgehog信号通路保护终板软骨细胞退变[J]. 中国临床药理学与治疗学, 2017, 22(4): 373-380.

XU Yongming, XU Hongguang, GAO Zhi, ZHANG Xiaoling. Icaritin protects the ICMT-induced degeneration of endplate chondrocytes via inhibiting Hedgehog signaling pathway[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2017, 22(4): 373-380.

参考文献

［1］Manek NJ, Macgregor AJ. Epidemiology of back disorders: prevalence, risk factors, and prognosis［J］. Curr Opin Rheumatol, 2005, 17(2): 134-140.
［2］Mccann MR, Patel P, Pest MA, et al. Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints［J］. Arthritis Rheumatol, 2015, 67(8): 2164-2175.
［3］Risbud MV, Shapiro IM. Role of cytokines in intervertebral disc degeneration: pain and disc content［J］. Nat Rev Rheumatol, 2014, 10(1): 44-56.
［4］Sakai D, Andersson GB. Stem cell therapy for intervertebral disc regeneration: obstacles and solutions［J］. Nat Rev Rheumatol, 2015, 11(4): 243-256.
［5］Livshits G, Popham M, Malkin I, et al. Lumbar disc degeneration and genetic factors are the main risk factors for low back pain in women: the UK Twin Spine Study［J］. Ann Rheum Dis, 2011, 70(10): 1740-1745.
［6］Rodriguez AG, Rodriguez-soto AE, Burghardt AJ, et al. Morphology of the human vertebral endplate［J］. J Orthop Res, 2012, 30(2): 280-287.
［7］Benneker LM, Heini PF, Alini M, et al. 2004 Young Investigator Award Winner: vertebral endplate marrow contact channel occlusions and intervertebral disc degeneration［J］. Spine (Phila Pa 1976), 2005, 30(2): 167-173.
［8］Xu HG, Yu YF, Zheng Q, et al. Autophagy protects end plate chondrocytes from intermittent cyclic mechanical tension induced calcification［J］. Bone, 2014, 66: 232-239.
［9］Peng S, Zhang G, Zhang BT, et al. The beneficial effect of icaritin on osteoporotic bone is dependent on the treatment initiation timing in adult ovariectomized rats［J］. Bone, 2013, 55(1): 230.
［10］张如意, 张丽, 艾厚喜, 等. 淫羊藿苷对线粒体损伤模型大鼠脑内β-淀粉样蛋白和神经营养因子的影响［J］. 中国中药杂志, 2013, 38(9): 1285-1289.
［11］杨晓旭, 王宇, 韩贞爱, 等. 淫羊藿水煎液对小鼠不同免疫水平模型的干预作用［J］. 长春中医药大学学报, 2016, 32(1): 14-17.
［12］徐永明, 徐宏光, 张晓玲, 等. 淫羊藿素药物在终板软骨细胞退变中的保护作用研究［J］. 赤峰学院学报(自然科学版), 2016, 16: 54-57.
［13］Palmer CJ, Galan-caridad JM, Weisberg SP, et al. Zfx facilitates tumorigenesis caused by activation of the Hedgehog pathway ［J］. Cancer Res, 2014, 74(20): 5914-5924.
［14］刘佳, 朱琴梅, 胡弘毅, 等. Hedgehog信号通路在肝细胞癌发生发展中的作用［J］. 临床肝胆病杂志, 2015, 31(2): 300-304.
［15］Razzak M. Osteoarthritis: the hedgehog and the bony spur［J］. Nat Rev Rheumatol, 2012, 8(3): 123.
［16］Zhou J, Chen Q, Lanske B, et al. Disrupting the Indian hedgehog signaling pathway in vivo attenuates surgically induced osteoarthritis progression in Col2a1-CreERT2; Ihhfl/fl mice［J］. Arthritis Res Ther, 2014, 16(1): R11.
［17］Lin AC, Seeto BL, Bartoszko JM, et al. Modulating hedgehog signaling can attenuate the severity of osteoarthritis［J］. Nat Med, 2009, 15(12): 1421-1425.
［18］Kim SY, Im GI. The expressions of the SOX trio, PTHrP (parathyroid hormone-related peptide)/IHH (Indian hedgehog protein) in surgically induced osteoarthritis of the rat ［J］. Cell Biol Int, 2011, 35(5): 529-535.
［19］Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method［J］. Methods (San Diego, Calif), 2001, 25(4): 402-408.
［20］Ferguson SJ, Steffen T. Biomechanics of the aging spine［J］. Eur Spine J, 2003, 12 Suppl 2(2): S97-S103.
［21］Lotz JC, Ulrich JA. Innervation, inflammation, and hypermobility may characterize pathologic disc degeneration: review of animal model data［J］. J Bone Joint Surg Am Vol, 2006, 88 Suppl 2(2): 76-82.
［22］Alkhatib B, Rosenzweig DH, Krock E, et al. Acute mechanical injury of the human intervertebral disc: link to degeneration and pain［J］. Euro Cel Mat, 2014, 28: 98-111.
［23］Xu HG, Zheng Q, Song JX, et al. Intermittent cyclic mechanical tension promotes endplate cartilage degeneration via canonical Wnt signaling pathway and E-cadherin/beta-catenin complex cross-talk［J］. Osteoarthritis Cartilage, 2016, 24(1): 158-168.
［24］Chang CF, Ramaswamy G, Serra R. Depletion of primary cilia in articular chondrocytes results in reduced Gli3 repressor to activator ratio, increased Hedgehog signaling, and symptoms of early osteoarthritis［J］. Osteoarthritis Cartilage, 2012, 20(2): 152-161.
［25］丁婧, 李荣, 胡向阳, 等. 环巴胺对佐剂性关节炎大鼠关节软骨细胞增殖凋亡的影响及其抗凋亡机制［J］. 安徽医科大学学报, 2015, 4: 446-451.

[1]	刘庆生，俞建顺，蔡丹莉，严茂祥，王小奇，桑怡. 大鼠胃癌前病变形成过程中胃黏膜Sonic Hedgehog信号通路的变化[J]. 中国临床药理学与治疗学, 2018, 23(7): 761-769.
[2]	高智，徐宏光，张晓玲，王弘，马明明，肖良，刘祥，徐永明. 中药胡椒碱在终板软骨细胞退变中的保护作用的研究[J]. 中国临床药理学与治疗学, 2016, 21(6): 611-616.