Tajima Takuya, Sekimoto Tomohisa, Yamaguchi Nami, Taniguchi Noboru, Kurogi Syuji, Maruyama Masugi, Chosa Etsuo
Division of Orthopaedic Surgery, Department of Medicine of Sensory and Motor Organs, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, 889-1692, Japan.
Department of Medical Sciences Applied Physiology, Faculty of Medicine, Graduate School of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki, Miyazaki, 889-1692, Japan.
BMC Musculoskelet Disord. 2017 Nov 14;18(1):449. doi: 10.1186/s12891-017-1815-7.
ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteins play an important pathological role in matrix degeneration. Aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family members, we examined the influence of Hb by synovial cells in an in vitro experimental system.
Synovial tissues were obtained from five young patients with meniscal injury under arthroscopic surgery. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 μg/ml). The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with and without 100 μg/ml Hb, and culture media were taken at 6, 12, and 24 h. To identify the proteins responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, -8, -9, and -10; enzyme-linked immunosorbent assay (ELISA); and gene expression for ADAMTS-5 and -9 were examined. Statistical comparisons between each group were performed using paired t-tests.
Western blot analysis revealed that Hb-treatment resulted in the expression of ADAMTS-5 and -9. Neither control group nor Hb-treated medium showed immunoreactivity against ADAMTS-8 or -10. In a dose-dependency study, the Hb-treated group showed significantly higher levels of ADAMTS-5 and -9 compared with the control (p < 0.05). There was no significant difference between 25, 50, and 100 μg/ml Hb-treated groups. In a time-course study, the ADAMTS-5 and -9 levels in the conditioned medium had significantly increased expression at 6, 12, and 24 h in the Hb-treated group (p < 0.05). Hb evoked significant expression of ADAMTS-9 mRNA at 12 and 24 h (p < 0.05).
These findings indicate that Hb induces the expression of ADAMTS-5 and -9 by synovial cells at low doses, even at an acute phase, and suggests a possible role for Hb in cartilage damage after intra-articular hemorrhage. The results also suggest a new potential therapeutic target by inhibiting the activities of ADAMTS-5 and -9 to prevent cartilage damage after intra-articular hemorrhage.
含血小板反应蛋白基序的解聚素和金属蛋白酶（ADAMTS）蛋白在基质退变中发挥重要的病理作用。蛋白聚糖降解是早期骨关节炎中的一个重要且关键的事件。为了确定血红蛋白（Hb）对滑膜组织产生ADAMTS家族成员能力的影响，我们在体外实验系统中检测了Hb对滑膜细胞的影响。
从5名接受关节镜手术的半月板损伤年轻患者获取滑膜组织。用人膝关节滑膜细胞原代培养物分别用不同剂量的人Hb（0、25、50、100μg/ml）处理。Hb处理24小时后收集培养基。在时间进程研究中，细胞分别用100μg/ml Hb处理和不处理，在6、12和24小时收集培养基。为鉴定负责蛋白聚糖酶活性的蛋白，使用抗人ADAMTS - 5、- 8、- 9和- 10的抗体进行蛋白质印迹分析；酶联免疫吸附测定（ELISA）；并检测ADAMTS - 5和- 9的基因表达。每组之间的统计比较采用配对t检验。
蛋白质印迹分析显示，Hb处理导致ADAMTS - 5和- 9的表达。对照组和Hb处理组培养基均未显示出针对ADAMTS - 8或- 10的免疫反应性。在剂量依赖性研究中，与对照组相比，Hb处理组的ADAMTS - 5和- 9水平显著更高（p < 0.05）。25、50和100μg/ml Hb处理组之间无显著差异。在时间进程研究中，Hb处理组条件培养基中的ADAMTS - 5和- 9水平在6、12和24小时时表达显著增加（p < 0.05）。Hb在12和24小时时引起ADAMTS - 9 mRNA的显著表达（p < 0.05）。
这些发现表明，即使在急性期，低剂量的Hb也能诱导滑膜细胞表达ADAMTS - 5和-9，并提示Hb在关节内出血后软骨损伤中可能发挥作用。结果还提示通过抑制ADAMTS - 5和- 9的活性来预防关节内出血后软骨损伤的一个新的潜在治疗靶点。
