Acacetin Alleviates Inflammation and Matrix Degradation in Nucleus Pulposus Cells and Ameliorates Intervertebral Disc Degeneration in vivo

Abstract

Purpose

Intervertebral disc degeneration (IDD) is one of the most prevalent musculoskeletal disorders. The nucleus pulposus is the major component of the intervertebral disc, and nucleus pulposus cells (NPCs) play a significant role in the normal functioning of the intervertebral disc. Reactive oxygen species (ROS) generation, inflammation and extracellular matrix degradation in NPCs contribute to the degeneration of intervertebral discs. Acacetin is a drug that exerts antioxidant and anti-inflammatory effects on many types of cells. However, whether acacetin can relieve the degeneration of NPCs remains unknown.

Methods

NPCs were extracted from rat intervertebral discs. The NPCs were treated with tert-butyl peroxide (TBHP) to simulate a high-ROS environment, and acacetin was subsequently added. The contents of ROS, inflammatory mediators (COX-2, iNOS) and extracellular matrix components (aggrecan, collagen II, MMP13, MMP9, MMP3) were measured. Components of related signaling pathways (Nrf2, MAPK) were also evaluated. To determine the effect of acacetin in vivo, we simulated disc degeneration via needle puncture. Acacetin was then applied intraperitoneally, and the degenerative status was evaluated using MRI and histopathological analysis.

Results

In vitro, acacetin alleviated TBHP-induced ROS generation and upregulated the expression of antioxidant proteins, including HO-1, NQO1, and SOD. In addition, acacetin relieved the TBHP-induced generation of inflammatory mediators (COX-2, iNOS) and degradation of the extracellular matrix (aggrecan, collagen II, MMP13, MMP9, and MMP3). Acacetin exerted its effect by activating the Nrf2 pathway and inhibiting p38, JNK and ERK1/2 phosphorylation. In vivo, acacetin ameliorated puncture-induced disc degeneration in a rat tail model, which was evaluated using MRI and histopathological analysis.

Conclusion

Acacetin alleviated IDD in vitro and in vivo and may have the potential to be developed as an effective treatment for IDD.

Keywords:

• acacetin
• intervertebral disc degeneration
• IDD
• reactive oxygen species
• ROS
• inflammation

Acknowledgments

We are grateful for the support from the central laboratory of Zhongshan Hospital.

Abbreviations

IDD, intervertebral disc degeneration; NPC, nucleus pulposus cell; ROS, reactive oxygen species; TBHP, tert-butyl peroxide; ECM, extracellular matrix; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; NO, nitric oxide; PGE2, prostaglandin E2; NF-κB, nuclear factor kappa-B; MAPK, mitogen-activated protein kinase; PVDF, polyvinylidene difluoride; CCK-8, Cell Counting Kit-8; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SOD, superoxide dismutase; HO-1, heme oxygenase-1; NQO1, NAD(P)H dehydrogenase, quinone 1; MMP, matrix metalloproteinase; JNK, c-JUN N-terminal kinase; ERK, extracellular signal-regulated kinase; Keap1, Kelch-like ECH-associated protein 1; ARE, antioxidant response element; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase 2.

Disclosure

The authors report no conflicts of interest in this work.