Irisin stimulates anabolism and matrix synthesis in human nucleopulpocytes in vitro: new insights into a cross-talk between the muscle and the intervertebral disc. (#06)
INTRODUCTION
Physical activity favors weight loss and ameliorates pain and function in patients suffering from discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleopulpocytes (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.
METHODS
hNPCs were isolated from IVD samples obtained during spine surgery and cultured in alginate beads. hNPCs were then exposed to either phosphate-buffered saline (PBS) or human recombinant irisin (r-irisin) for 7 days at a concentration of 5, 10 and 25 ng/mL (n = 4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay at 4, 10 and 14 days of culture. Glycosaminoglycan (GAG) content was measured using the dimethylmethylene blue (DMMB) assay and normalized to the DNA ratio at 7 days of culture. Metabolic activity was assessed with the MTT assay and the Griess Reagent System at 4 days of culture. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and -3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by real time-polymerase chain reaction (RT-PCR) at 7 days of culture. In addition, MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β for 7 days and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin for 7 days).
RESULTS
Irisin increased hNPCs proliferation (Fig. 1, p<0.001), metabolic activity (Fig. 2, p<0.05), and GAG content (Fig. 3, p<0.01), as well as COL2 (p<0.01), ACAN (p<0.05), TIMP-1 and -3 (p<0.01) gene expression, while decreasing mRNA levels of MMP-13 (p<0.05) and IL-1β (Fig. 4, p<0.001). r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (Fig. 5, p<0.001), as well as a decrease of IL-1b (Fig.6, p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin led to an increment of hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and a reduction of IL-1β (p<0.05) and ADAMTS-5 levels (Fig. 6, p<0.01).
DISCUSSION
The present study suggested that irisin may stimulate hNPC proliferation, metabolic activity, and anabolism by reducing the expression of IL-1β and catabolic enzymes while promoting the synthesis of extracellular matrix components. Furthermore, such myokine was able to blunt the catabolic effect of in vitro inflammation. Our results indicate that irisin may be one of the mediators by which physical exercise and muscle tissues modulate IVD metabolism, thus suggesting the existence of a biological cross-talk mechanism between the muscle and the IVD.
- Belavý DL, Quittner MJ, Ridgers N, Ling Y, Connell D, Rantalainen T. Running exercise strengthens the intervertebral disc. Sci Rep. 2017 Apr 19;7:45975. doi: 10.1038/srep45975.
- Vadalà G, Di Giacomo G, Ambrosio L, Cannata F, Cicione C, Papalia R, Denaro V. Irisin Recovers Osteoarthritic Chondrocytes In Vitro. Cells. 2020 Jun 17;9(6):1478. doi: 10.3390/cells9061478.