中国修复重建外科杂志

中国修复重建外科杂志

淫羊藿苷治疗小鼠骨关节炎后血清学及组织学改变的实验研究

查看全文

目的通过早期和晚期应用淫羊藿苷(icariin,ICA)治疗小鼠骨关节炎(osteoarthritis,OA),观察 ICA 对小鼠血清骨代谢标志物以及软骨和软骨下骨组织形态学的影响。方法取 80 只 8 周龄雄性 C57BL/6J 小鼠,随机分为 8 组,每组 10 只,分别为假手术/早期生理盐水组(A 组)、假手术/早期 ICA 组(B 组)、前交叉韧带切断术(anterior cruciate ligament transaction,ACLT)/早期生理盐水组(C 组)、ACLT/早期 ICA 组(D 组)、假手术/晚期生理盐水组(E 组)、假手术/晚期 ICA 组(F 组)、ACLT/晚期生理盐水组(G 组)、ACLT/晚期 ICA 组(H 组)。各组小鼠对应给予 ACLT 或单纯打开关节囊处理后,B、D 组于术后第 1 天开始,F、H 组于第 4 周开始,每天采用灌胃方式给予小鼠 ICA(10 mg/kg)至第 8 周;A、C 组及 E、G 组于对应相同时间点给予相同体积生理盐水。于术后第 8 周收集小鼠全血制备血清,采用 ELISA 法测定血清中骨代谢标志物及细胞因子的含量,包括Ⅰ型胶原 C 末端肽(C-telopeptide of type Ⅰ collagen,CTX)、骨钙素(osteocalcin,OC)、IL-6、TNF-α、IL-1β。同时切取膝关节组织行阿辛蓝/苏木精-酸性橙 G 染色,观察软骨及软骨下骨形态改变,并进行骨关节炎国际研究学会(OARSI)评分。结果早期给药组(A、B、C、D 组)间比较:与 A、B 组相比,C 组 CTX、OC 含量显著降低(P<0.05),IL-6、TNF-α、IL-1β 含量升高(P<0.05),OARSI 评分显著升高(P<0.05);与 C 组相比,D 组 CTX、OC 含量升高(P<0.05),IL-6 含量显著降低(P<0.05),TNF-α、IL-1β 含量差异无统计学意义(P>0.05),OARSI 评分降低(P<0.05),组织学观察显示胫骨软骨缺失程度明显改善。晚期给药组(E、F、G、H 组)间比较:与 E、F 组比较,G 组 CTX、OC 含量显著降低(P<0.05),IL-6、TNF-α、IL-1β 含量升高(P<0.05),OARSI 评分显著升高(P<0.05);与 G 组比较,H 组 CTX含量升高(P<0.05),OC、IL-6、TNF-α、IL-1β 含量差异无统计学意义(P>0.05),OARSI 评分无明显变化(P>0.05),组织学观察显示胫骨软骨缺失程度相似。结论ICA 对透明软骨、钙化软骨及软骨下骨均有保护作用,并且能在一定程度上改善 OA 软骨下骨的骨重建,且 ICA 对 OA 早期干预作用更明显。

ObjectiveTo investigate the effects of icariin (ICA) on serum bone turnover markers expressions and histological changes of cartilage and subchondral bone in mouse osteoarthritis (OA) model.MethodsEighty 8-week-old male C57BL/6J mouse were randomly divided into 8 groups (n=10). The OA model was established by anterior cruciate ligament transaction (ACLT). Group A: sham operation/early-stage normal saline administration; group B: sham operation/early-stage ICA administration; group C: ACLT/early-stage normal saline administration; group D: ACLT/early-stage ICA administration; group E: sham operation/late-stage normal saline administration; group F: sham operation/late-stage ICA administration; group G: ACLT/late-stage normal saline administration; group H: ACLT/late-stage ICA administration. Each animal received either ACLT or simply opening joint capsule, respectively. For groups B and D, ICA was given by gavage [10 mg/(kg·day)] on the first day after ACLT. For groups F and H, ICA was given with the same volume at 4 weeks after operation. The blood serum of the mouse was collected and prepared at 8 weeks after operation. Serum bone turnover markers and cytokines, including C-telopeptide of type I collagen (CTX), osteocalcin (OC), interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-1β, were measured by ELISA. Tissue samples from the knee were stained by alcian blue/hematoxylin & orange G (AB/H&OG). Histological changes of cartilage and subchondral bone were observed and evaluated by Osteoarthritis Research Society International (OARSI) scoring system.ResultsComparison between each group with early-stage administration (groups A, B, C, and D): Compared with groups A and B, the levels of CTX and OC in group C were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score was significantly increased (P<0.05). Compared with group C, the levels of CTX and OC in group D were significantly increased (P<0.05); the level of IL-6 was significantly reduced (P<0.05); the levels of TNF-α and IL-1β were not changed (P>0.05), and OARSI score was significantly reduced (P<0.05). Histological observation showed that the tibial cartilage loss was significantly improved. Comparison between each group with late-stage administration (groups E, F, G, and H): Compared with groups E and F, the levels of CTX and OC in group G were significantly reduced (P<0.05); the levels of IL-6, TNF-α, and IL-1β and OARSI score were significantly increased (P<0.05). Compared with group G, the level of CTX in group H were increased (P<0.05); the levels of OC, IL-6, TNF-α, and IL-1β and OARSI score were not changed (P>0.05). Histological observation showed that the tibial cartilage loss had no changes after late-stage ICA administration.ConclusionICA plays protective effects on subchondral bone, hyaline, and calcified cartilage. Meanwhile, ICA can improve bone remodeling in subchondral bone of OA to some extent. The consistent changes of serum bone markers and pathological morphology suggest that early intervention of ICA on OA is more effective.

关键词: 淫羊藿苷; 骨关节炎; 软骨下骨; 骨重建; 小鼠

Key words: Icariin; osteoarthritis; subchondral bone; bone remodeling; mouse

引用本文: 高堪达, 王漱阳, 王秋根. 淫羊藿苷治疗小鼠骨关节炎后血清学及组织学改变的实验研究. 中国修复重建外科杂志, 2017, 31(8): 963-969. doi: 10.7507/1002-1892.201703044 复制

登录后 ,请手动点击刷新查看全文内容。 没有账号,
登录后 ,请手动点击刷新查看图表内容。 没有账号,
1. Hügle T, Geurts J. What drives osteoarthritis?-synovial versus subchondral bone pathology. Rheumatology (Oxford), 2016. [Epub ahead of print]
2. Henrotin Y, Pesesse L, Sanchez C. Subchondrol bone and osteosrthritis: biological and cellur aspects. Osteoporos Int, 2012, 23 Suppl 8: S847-851.
3. 于德刚, 汤亭亭, 朱振安. 骨关节炎软骨下骨改变及其作用研究进展. 国际骨科学杂志, 2015, 36(3): 172-178.
4. Daghestani HN, Kraus VB. Inflammatory biomarkers in osteoarthritis. Osteoarthritis Cartilage, 2015, 23(11): 1890-1896.
5. Imamura M, Ezquerro F, Marcon Alfieri F, et al. Serum levels of pro-inflammatory cytokines in painful knee osteoarthritis and sensitization. Int J Inflam, 2015, 2015: 329792.
6. Moskowitz RW. Osteoarthritis cartilage histopathology: grading and staging. Osteoarthritis Cartilage, 2006, 14(1): 1-2.
7. Fan JJ, Cao LG, Wu T, et al. The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells. Molecules, 2011, 16(12): 10123-10133.
8. Ma HP, Ming LG, Ge BF, et al. Icariin is more potent than genistein in promoting osteoblast differentiation and mineralization in vitro. J Cell Biochem, 2011, 112(3): 916-923.
9. Hsieh TP, Sheu SY, Sun JS, et al. Icariin inhibits osteoclast differentiation and bone resorption by suppression of MAPKs/NF-κB regulated HIF-1α and PGE (2) synthesis. Phytomedicine, 2011, 18(2-3): 176-185.
10. 翟吉良, 翁习生, 邱贵兴. 骨关节炎动物模型的建立及选择.中国矫形外科杂志, 2007, 15(11): 843-845.
11. Van Spil WE, Welsing PM, Bierma-Zeinstra SM, et al. The ability of systemic biochemical markers to reflect presence, incidence, and progression of early-stage radiographic knee and hip osteoarthritis: data from CHECK. Osteoarthritis Cartilage, 2015, 23(8): 1388-1397.
12. Ishijima M, Kaneko H, Kaneko K. The evolving role of biomarkers for osteoarthritis. Therapeutic Advances in Musculoskeletal Disease, 2014, 6(4): 144-153.
13. Zoch ML, Clements TL, Riddle RC. New insights into the biology of osteocalcin. Bone, 2016, 82: 42-49.
14. Papalia R, Vadalà G, Torre G, et al. The cytokinome in osteoarthritis, a new paradigm in diagnosis and prognosis of cartilage disease. J Biol Regul Homeost Agents, 2016, 30(4 Suppl 1): 77-83.
15. Latourte A, Cherifi C, Maillet J, et al. Systemic inhibition of IL-6/Stat3 signalling protects against experimental osteoarthritis. Ann Rheum Dis, 2016, 76(4): 748-755.
16. Mabey T, Honsawek S. Cytokines as biochemical markers for knee osteoarthritis. World J Orthop, 2015, 6(1): 95-105.
17. 石婷, 高戈, 闫祖炜, 等. 兔实验性骨关节炎六种血清学生物标志物的变化. 细胞与分子免疫学杂志, 2015, 31(12): 1620-1623, 1628.
18. 查振刚, 黄良任, 姚平, 等. 膝骨关节炎患者血清 TNF-α 与 IL-6 水平及其临床意义. 广东医学, 2005, 26(2): 191-193.
19. Charlier E, Relic B, Deroyer C, et al. Insights on Molecular Mechanisms of Chondrocytes Death in Osteoarthritis. Int J Mol Sci, 2016, 17(12): 2146.
20. Hosseinzadeh A, Kamrava SK, Joghataei MT, et al. Apoptosis signaling pathways in osteoarthritis and possible protective role of melatonin. J Pineal Res, 2016, 61(4): 411-425.
21. Zeng L, Rong XF, Li RH, Wu XY. Icariin inhibits MMP1, MMP3 and MMP13 expression through MAPK pathways in IL1β stimulated SW1353 chondrosarcoma cells. Mol Med Rep, 2017, 15(5): 2853-2858.
22. Zhang L, Zhang X, Li KF, et al. Icariin promotes extracellular matrix synthesis and gene expression of chondrocytes in vitro. Phytother Res, 2012, 26(9): 1385-1392.
23. Fu S, Yang L, Hong H, et al. Wnt/β-catenin signaling is involved in the Icariin induced proliferation of bone marrow mesenchymal stem cells. J Tradit Chin Med, 2016, 36(3): 360-368.
24. Qin S, Zhou W, Liu S, et al. Icariin stimulates the proliferation of rat bone mesenchymal stem cells via ERK and p38 MAPK signaling. Int J Clin Exp Med, 2015, 8(5): 7125-7133.
25. Wu Y, Xia L, Zhou Y, et al. Icariin induces osteogenic differentiation of bone mesenchymal stem cells in a MAPK-dependent manner. Cell Prolif, 2015, 48(3): 375-384.