Preclinicalex-vivoTesting of Anti-inflammatory Drugs in a Bovine Intervertebral Degenerative Disc Model
Preclinicalex-vivoTesting of Anti-inflammatory Drugs in a Bovine Intervertebral Degenerative Disc Model
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DOI：
10.3389/fbioe.2020.00583
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发表时间：
2020-06-10
影响因子：
5.7
通讯作者：
Lang, Gernot
中科院分区：
文献类型：
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作者：
Li, Zhen;Gehlen, Yannik;Lang, Gernot
椎间盘源性腰痛 (LBP) 是导致残疾的主要原因，而炎症被认为是症状性椎间盘退变 (IDD) 的主要驱动因素。目前正在研究抗炎药，因为它们被证明可以减轻 IDD 患者的症状。然而，它们潜在的抗炎和再生活性却鲜为人知。本研究旨在探讨依那西普和托法替尼在 IVD 生物反应器内的临床前椎间盘 (IVD) 器官培养模型中维持椎间盘稳态的潜力，从而实现动态负荷和营养物质交换。将牛尾部 IVD 在生物反应器系统中培养 4 天，以模拟生理或退行性条件：（1）生理生理负荷（0.02-0.2 MPa；0.2 Hz；2 小时/天）和高葡萄糖 DMEM 培养基（4.5 g/L）； (2)Deg+肿瘤坏死因子α(TNF-α)-变性负荷(0.32-0.5MPa；5Hz；2小时/天)和低葡萄糖DMEM培养基(2g/L)，注射TNF-α。椎间盘内注射依那西普，同时将托法替尼补充到培养基中。通过 RT-qPCR 测量 IVD 组织中的基因表达。分析了一氧化氮 (NO)、白细胞介素 8 (IL-8) 和糖胺聚糖 (GAG) 释放到 IVD 条件培养基中的情况。使用乳酸脱氢酶和乙锭同二聚体-1 染色评估 IVD 中的细胞活力。进行免疫组织化学评估 IVD 组织中 IL-1β、IL-6、IL-8 和 II 型胶原的蛋白表达。依那西普和托法替尼下调髓核（NP）组织中IL-1β、IL-6、IL-8、基质金属蛋白酶1（MMP1）和MMP3的表达以及纤维环（AF）组织中IL-1β、MMP3、环氧化酶-2（COX2）和神经生长因子（NGF）的表达。此外，依那西普显着减少了外部 AF 和 NP 区域的 IL-1 β 阳性染色细胞。托法替尼显着减少 AF 区域内 IL-1 β 和 IL-8 阳性染色的细胞。依那西普和托法替尼均将 GAG 损失降低至生理培养条件下的水平。依那西普和托法替尼能够中和 IDD 器官培养模型中的促炎和分解代谢环境。然而，可能需要联合抗炎和合成代谢治疗来限制 IDD 加速并缓解炎症引起的背痛。
Discogenic low back pain (LBP) is a main cause of disability and inflammation is presumed to be a major driver of symptomatic intervertebral disc degeneration (IDD). Anti-inflammatory agents are currently under investigation as they demonstrated to alleviate symptoms in patients having IDD. However, their underlying anti-inflammatory and regenerative activity is poorly explored. The present study sought to investigate the potential of Etanercept and Tofacitinib for maintaining disc homeostasis in a preclinical intervertebral disc (IVD) organ culture model within IVD bioreactors allowing for dynamic loading and nutrient exchange. Bovine caudal IVDs were cultured in a bioreactor system for 4 days to simulate physiological or degenerative conditions: (1) Phy-physiological loading (0.02-0.2 MPa; 0.2 Hz; 2 h/day) and high glucose DMEM medium (4.5 g/L); (2) Deg+Tumor necrosis factor alpha (TNF-alpha)-degenerative loading (0.32-0.5 MPa; 5 Hz; 2 h/day) and low glucose DMEM medium (2 g/L), with TNF-alpha injection. Etanercept was injected intradiscally while Tofacitinib was supplemented into the culture medium. Gene expression in the IVD tissue was measured by RT-qPCR. Release of nitric oxide (NO), interleukin 8 (IL-8) and glycosaminoglycan (GAG) into the IVD conditioned medium were analyzed. Cell viability in the IVD was assessed using lactate dehydrogenase and ethidium homodimer-1 staining. Immunohistochemistry was performed to assess protein expression of IL-1 beta, IL-6, IL-8, and collagen type II in the IVD tissue. Etanercept and Tofacitinib downregulated the expression of IL-1 beta, IL-6, IL-8, Matrix metalloproteinase 1 (MMP1), and MMP3 in the nucleus pulposus (NP) tissue and IL-1 beta, MMP3, Cyclooxygenase-2 (COX2), and Nerve growth factor (NGF) in the annulus fibrosus (AF) tissue. Furthermore, Etanercept significantly reduced the IL-1 beta positively stained cells in the outer AF and NP regions. Tofacitinib significantly reduced IL-1 beta and IL-8 positively stained cells in the inner AF region. Both, Etanercept and Tofacitinib reduced the GAG loss to the level under physiological culture condition. Etanercept and Tofacitinib are able to neutralize the proinflammatory and catabolic environment in the IDD organ culture model. However, combined anti-inflammatory and anabolic treatment may be required to constrain accelerated IDD and relieving inflammation-induced back pain.