Ramanathan Rahul, Firdous Ayesha, Dong Qing, Wang Dong, Lee Joon, Vo Nam, Sowa Gwendolyn
Ferguson Spine Laboratory, Department of Orthopaedic Surgery University of Pittsburgh Pittsburgh Pennsylvania USA.
Department of Physical Medicine and Rehabilitation University of Pittsburgh Pittsburgh Pennsylvania USA.
JOR Spine. 2022 Mar 10;5(2):e1197. doi: 10.1002/jsp2.1197. eCollection 2022 Jun.
INTRODUCTION: Intervertebral disc degeneration (IDD) is closely related to heightened inflammation in the annulus fibrosis (AF) and nucleus pulposus (NP) cells in the intervertebral disc. An imbalanced matrix homeostasis has been shown to contribute to disc degeneration and associated discogenic low back pain. Metformin, a diabetes medication, has been noted to exhibit anti-inflammatory properties through upregulation of the AMPK pathway, leading to various anti-inflammatory-related responses in hepatocytes. However, it is still unclear how metformin influences disc cellular response to inflammatory stress and the corresponding mechanism. Hence, the objective of this study is to elucidate the effects of metformin on expression of key pro-inflammatory, catabolic, and anabolic factors within rat AF cells in response to inflammatory stimulation and mechanical tensile stress. METHODS: Five Fischer 344 rats were sacrificed and their spines isolated. AF cells were cultured and plated in flexible silicone membrane-based six-well plates. Wells were split into eight groups and subjected to metformin, IL-1β, mechanical stretch, and combined treatments. Relative gene expressions of MMP-13, COX-2, iNOS, AGC, and Col1 were assessed with quantitative real-time polymerase chain reaction (qRT-PCR), and downstream prostaglandin E (PGE2) production was quantified with enzyme-linked immunosorbent assay (ELISA). NF-kB nuclear translocation was also quantified. RESULTS: Metformin in the presence of the combined stress treatments (M + IL/S) significantly increased Col1, COX-2, and MMP-13 gene expression, decreased PGE2 production compared to IL/S conditions alone. Metformin treatment of cultured rat annulus fibrosus cells significantly reduced the nuclear translocation of NF-κB after 4 h of IL-1β treatment from 43.1% in case of IL-1β treatment down to 26.2% in the case of metformin + IL-1β treatment. DISCUSSION: The lack of metformin-mediated suppression of inflammatory response in the nonstretch groups indicates that metformin may be enacting its effects through a stretch-dependent manner. These results suggest a foundation for pursuing further research into metformin's potential role as an anti-inflammatory agent for curtailing intervertebral disc degeneration.
引言：椎间盘退变（IDD）与椎间盘纤维环（AF）和髓核（NP）细胞炎症加剧密切相关。基质稳态失衡已被证明会导致椎间盘退变及相关的盘源性下腰痛。二甲双胍是一种糖尿病药物，已被注意到可通过上调AMPK途径表现出抗炎特性，从而导致肝细胞中各种与抗炎相关的反应。然而，二甲双胍如何影响椎间盘细胞对炎症应激的反应及其相应机制仍不清楚。因此，本研究的目的是阐明二甲双胍对大鼠AF细胞中关键促炎、分解代谢和合成代谢因子表达的影响，以应对炎症刺激和机械拉伸应力。 方法：处死5只Fischer 344大鼠并分离其脊柱。将AF细胞培养并接种到基于柔性硅胶膜的六孔板中。孔被分成八组，并接受二甲双胍、白细胞介素-1β（IL-1β）、机械拉伸及联合处理。用定量实时聚合酶链反应（qRT-PCR）评估基质金属蛋白酶-13（MMP-13）、环氧化酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）、聚集蛋白聚糖（AGC）和I型胶原蛋白（Col1）的相对基因表达，并用酶联免疫吸附测定（ELISA）定量下游前列腺素E（PGE2）的产生。还对核因子κB（NF-κB）的核转位进行了定量。 结果：在联合应激处理（M + IL/S）存在的情况下，二甲双胍与单独的IL/S条件相比，显著增加了Col1、COX-2和MMP-13基因表达，降低了PGE2的产生。用二甲双胍处理培养的大鼠纤维环细胞，在IL-1β处理4小时后，NF-κB的核转位从IL-1β处理时的43.1%显著降低至二甲双胍 + IL-1β处理时的26.2%。 讨论：在非拉伸组中缺乏二甲双胍介导的炎症反应抑制表明，二甲双胍可能通过拉伸依赖的方式发挥其作用。这些结果为进一步研究二甲双胍作为一种抗炎剂在减少椎间盘退变方面的潜在作用奠定了基础。
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