Department of Orthopedic Surgery, the Affiliated Hospital of Qingdao University, Qingdao, 266000, Shandong, China.
Department of Orthopedic Surgery, Qingdao Municipal Hospital (Group), Qingdao, 266011, Shandong, China.
BMC Musculoskelet Disord. 2021 Jan 13;22(1):77. doi: 10.1186/s12891-020-03934-7.
Intervertebral disc degeneration (IVDD) is a primary cause of degenerative disc diseases; however, the mechanisms underlying the degeneration remain unclear. The immunoinflammatory response plays an important role in IVDD progression. The inflammatory cytokine lymphotoxin-α (LTα), formerly known as TNFβ, is associated with various pathological conditions, while its role in the pathogenesis of IVDD remains elusive.
Real-time quantitative polymerase chain reaction (RT-qPCR), Western blotting (WB), and enzyme-linked immunosorbent assays were used to assess the levels of LTα in human nucleus pulposus (NP) tissues between degeneration and control groups. The plasma concentrations of LTα and C-reactive protein (CRP) were compared between healthy and IVDD patients. Rat primary NP cells were cultured and identified via immunofluorescence. Methyl-thiazolyl-tetrazolium assays and flow cytometry were used to evaluate the effects of LTα on rat NP cell viability. After NP cells were treated with LTα, degeneration-related molecules (Caspase-3, Caspase-1, matrix metalloproteinase (MMP) -3, aggrecan and type II collagen) were measured via RT-qPCR and WB.
The levels of both the mRNA and protein of LTα in human degenerated NP tissue significantly increased. Plasma LTα and CRP did not differ between healthy controls and IVDD patients. Rat primary NP cells were cultured, and the purity of primary NP cells was > 90%. Cell experiments showed inversely proportional relationships among the LTα dose, treatment time, and cell viability. The optimal conditions (dose and time) for LTα treatment to induce rat NP cell degeneration were 5 μg/ml and 48 ~ 72 h. The apoptosis rate and the levels of Caspase-3, Caspase-1, and MMP-3 significantly increased after LTα treatment, while the levels of type II collagen and aggrecan were decreased, and the protein expression levels were consistent with their mRNA expression levels.
This study demonstrated that elevated LTα is closely associated with IVDD and that LTα may induce NP cell apoptosis and reduce important extracellular matrix (ECM) proteins, which cause adverse effects on IVDD progress. Moreover, the optimal conditions for LTα treatment to induce NP cell degeneration were determined.
椎间盘退行性变（IVDD）是退行性椎间盘疾病的主要原因；然而，其退化的机制尚不清楚。免疫炎症反应在 IVDD 进展中起着重要作用。炎症细胞因子淋巴毒素-α（LTα），以前称为 TNFβ，与各种病理状况有关，但其在 IVDD 发病机制中的作用仍不清楚。
实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹（WB）和酶联免疫吸附试验用于评估退变组和对照组之间人髓核（NP）组织中 LTα 的水平。比较健康组和 IVDD 组患者之间 LTα 和 C-反应蛋白（CRP）的血浆浓度。培养大鼠原代 NP 细胞，并通过免疫荧光进行鉴定。噻唑蓝比色法和流式细胞术用于评估 LTα 对大鼠 NP 细胞活力的影响。用 LTα 处理 NP 细胞后，通过 RT-qPCR 和 WB 测量退变相关分子（Caspase-3、Caspase-1、基质金属蛋白酶（MMP）-3、聚集蛋白聚糖和 II 型胶原）。
人退变 NP 组织中 LTα 的 mRNA 和蛋白水平均显著升高。健康对照组和 IVDD 患者的血浆 LTα 和 CRP 无差异。培养大鼠原代 NP 细胞，原代 NP 细胞的纯度>90%。细胞实验表明 LTα 剂量、处理时间和细胞活力之间呈反比关系。LTα 诱导大鼠 NP 细胞退变的最佳条件（剂量和时间）为 5μg/ml 和 48~72h。LTα 处理后，细胞凋亡率和 Caspase-3、Caspase-1 和 MMP-3 的水平显著升高，而 II 型胶原和聚集蛋白聚糖的水平降低，蛋白表达水平与其 mRNA 表达水平一致。
本研究表明，升高的 LTα 与 IVDD 密切相关，LTα 可能诱导 NP 细胞凋亡并减少重要的细胞外基质（ECM）蛋白，从而对 IVDD 进展产生不利影响。此外，确定了 LTα 治疗诱导 NP 细胞退变的最佳条件。
