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. 2018 Mar;59(2):191-200.
doi: 10.1080/03008207.2017.1330333. Epub 2017 Jun 8.

Link protein N-terminal peptide and fullerol promote matrix production and decrease degradation enzymes in rabbit annulus cells

Ching-Hua Yeh  1   2 , Dennis Chen  1 , Bayan Aghdasi  1 , Li Xiao  1 , Mengmeng Ding  1 , Li Jin  1 , Xudong Li  1
Affiliations

Link protein N-terminal peptide and fullerol promote matrix production and decrease degradation enzymes in rabbit annulus cells

Ching-Hua Yeh et al. Connect Tissue Res. 2018 Mar.

Abstract

Purpose: Intervertebral disc degeneration is a major cause of back pain. Novel therapies for prevention or reversal of disc degeneration are needed. It is desirable for potential therapies to target both inflammation and matrix degeneration.

Materials and methods: The combined regenerative potential of link protein N-terminal peptide (LN) and fullerol on annulus fibrosus (AF) cells was evaluated in a 3D culture model.

Results: Interleukin-1α (IL-1α)-induced AF cell degeneration was counteracted by fullerol, LN, and fullerol + LN, with the latter having the greatest effect on matrix production as evaluated by real-time polymerase chain reaction and glycosaminoglycan assay. IL-1α-induced increases in pro-inflammatory mediators (interleukin-6 and cyclooxygenase-2) and matrix metalloproteinases (MMP-1, -2, -9, and -13) were also counteracted by fullerol and LN.

Conclusion: Our data demonstrate that LN and fullerol individually, and in combination, promote matrix production and have anti-inflammatory and anti-catabolic effects on AF cells.

Keywords: Back pain; fullerol; inflammation; intervertebral disc degeneration; link N peptide.

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Conflict of interest statement

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article.

Figures

Figure 1
Figure 1
IL-1α-induced AF cell degeneration in a dose-dependent manner. Rabbit AF cells were cultured in a monolayer for 3 days. Cells were treated with or without IL-1α to induce AF cell degeneration. Cells were harvested for RNA isolation and the gene expression of collagen II and aggrecan was measured with real-time reverse transcription PCR. The target gene was normalized to 18s. *p < 0.05 versus control.
Figure 2
Figure 2
Link N and fullerol increased both collagen II and aggrecan expression in monolayer culture. Rabbit AF cells were cultured in a monolayer for 7 days. Cells were treated with or without IL-1α with addition of LN, fullerol, or LN + fullerol for 7 days in monolayer culture. The gene expression of collagen II and aggrecan was measured with real-time reverse transcription PCR. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group.
Figure 3
Figure 3
Link N and fullerol increased both collagen II and aggrecan expression in a 3D culture model. Cells were treated with or without IL-1α with addition of LN, fullerol, or LN + fullerol for 7 days. The cell pellets were harvested for RNA isolation and the gene expression of collagen II and aggrecan was measured with real-time reverse transcription PCR. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group.
Figure 4
Figure 4
Link N and fullerol increased collagen II and aggrecan expression in 3D culture for 14 days. Cells were treated with or without IL-1α with addition of LN, fullerol, or LN + fullerol. The cell pellets were harvested for RNA isolation and the gene expression of collagen II and aggrecan was measured with real-time reverse transcriptionPCR. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group.
Figure 5
Figure 5
Biochemistry assay shows Link N and fullerol combined increased GAG expression. Rabbit AF cells were cultured in pellet culture for 7 days. Cells were treated with or without IL-1α with addition of LN, fullerol, or LN + fullerol. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group.
Figure 6
Figure 6
Link N and fullerol decreased mRNA expression levels of IL-6 and COX-2. Cells were treated with or without IL-1α with addition of LN, fullerol, or LN + fullerol in a pellet culture for 7 days. The cell pellets were harvested for RNA isolation and the gene expression of IL-6 and COX-2 was measured with real-time reverse transcriptionPCR. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group.
Figure 7
Figure 7
Link N and fullerol increased anabolic metabolites through decreasing MMP expression and activity. A, the gene expression of MMP-1 and -3 were measured with real-time reverse transcriptionPCR. B, zymographic images. The culture media were subjected to gelatin and collagen zymography. Bar graphs show intensity of gel bands. *p < 0.05 versus control; #p < 0.05 versus IL-1α-treated group. TGF-β3 was used as a positive control.
Figure 8
Figure 8
Possible mechanisms under which fullerol and LN act on rabbit AF cells.
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