Spang Christoph, Backman Ludvig J, Le Roux Sandrine, Chen Jialin, Danielson Patrik
Department of Integrative Medical Biology, Anatomy, Umeå University, SE-901 87, Umeå, Sweden.
Department of Clinical Sciences, Ophthalmology, Umeå University, Umeå, Sweden.
BMC Musculoskelet Disord. 2017 May 25;18(1):218. doi: 10.1186/s12891-017-1575-4.
A body of evidence demonstrating changes to the glutaminergic system in tendinopathy has recently emerged. This hypothesis was further tested by studying the effects of glutamate on the tenocyte phenotype, and the impact of loading and exposure to glucocorticoids on the glutamate signaling machinery.
Plantaris tendon tissue and cultured plantaris tendon derived cells were immunohisto-/cytochemically stained for glutamate, N-Methyl-D-Aspartate receptor 1 (NMDAR1) and vesicular glutamate transporter 2 (VGluT2). Primary cells were exposed to glutamate or receptor agonist NMDA. Cell death/viability was measured via LDH/MTS assays, and Western blot for cleaved caspase 3 (c-caspase 3) and cleaved poly (ADP-ribose) polymerase (c-PARP). Scleraxis mRNA (Scx)/protein(SCX) were analyzed by qPCR and Western blot, respectively. A FlexCell system was used to apply cyclic strain. The effect of glucocorticoids was studies by adding dexamethasone (Dex). The mRNA of the glutamate synthesizing enzymes Got1 and Gls, and NMDAR1 protein were measured. Levels of free glutamate were determined by a colorimetric assay.
Immunoreactions for glutamate, VGluT2, and NMDAR1 were found in tenocytes and peritendinous cells in tissue sections and in cultured cells. Cell death was induced by high concentrations of glutamate but not by NMDA. Scleraxis mRNA/protein was down-regulated in response to NMDA/glutamate stimulation. Cyclic strain increased, and Dex decreased, Gls and Got1 mRNA expression. Free glutamate levels were lower after Dex exposure.
In conclusion, NMDA receptor stimulation leads to a reduction of scleraxis expression that may be involved in a change of phenotype in tendon cells. Glutamate synthesis is increased in tendon cells in response to strain and decreased by glucocorticoid stimulation. This implies that locally produced glutamate could be involved in the tissue changes observed in tendinopathy.
最近有大量证据表明肌腱病中谷氨酰胺能系统发生了变化。通过研究谷氨酸对肌腱细胞表型的影响，以及加载和暴露于糖皮质激素对谷氨酸信号传导机制的影响，进一步验证了这一假说。
对跖肌腱组织和培养的跖肌腱衍生细胞进行免疫组织化学/细胞化学染色，检测谷氨酸、N-甲基-D-天冬氨酸受体1（NMDAR1）和囊泡谷氨酸转运体2（VGluT2）。将原代细胞暴露于谷氨酸或受体激动剂NMDA。通过乳酸脱氢酶/甲基噻唑基四唑（LDH/MTS）检测法测量细胞死亡/活力，并通过蛋白质免疫印迹法检测裂解的半胱天冬酶3（c-caspase 3）和裂解的聚（ADP-核糖）聚合酶（c-PARP）。分别通过实时定量聚合酶链反应（qPCR）和蛋白质免疫印迹法分析硬骨素mRNA（Scx）/蛋白质（SCX）。使用FlexCell系统施加循环应变。通过添加地塞米松（Dex）研究糖皮质激素的作用。检测谷氨酸合成酶谷草转氨酶1（Got1）和谷氨酰胺酶（Gls）的mRNA以及NMDAR1蛋白。通过比色法测定游离谷氨酸水平。
在组织切片和培养细胞的肌腱细胞和腱周细胞中发现了谷氨酸、VGluT2和NMDAR1的免疫反应。高浓度谷氨酸可诱导细胞死亡，但NMDA不会。响应于NMDA/谷氨酸刺激，硬骨素mRNA/蛋白质表达下调。循环应变增加，而Dex降低，Gls和Got1 mRNA表达。Dex暴露后游离谷氨酸水平降低。
总之，NMDA受体刺激导致硬骨素表达减少，这可能与肌腱细胞表型变化有关。肌腱细胞中谷氨酸合成响应于应变而增加，并受到糖皮质激素刺激的抑制。这意味着局部产生的谷氨酸可能参与了肌腱病中观察到的组织变化。
