Abstract
This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation.
hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 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. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. 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 RT-PCR. 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 and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin).
Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001) mRNA levels. r-irisin pretreatment of hNPCs cultured in pro-inflammatory conditions resulted in a rescue of metabolic activity (p<0.001) and a decrease of IL-1β (p<0.05) levels. Similarly, incubation of hNPCs with IL-1β and subsequent exposure to r-irisin increased hNPC metabolic activity (p<0.001), COL2 gene expression (p<0.05) and decreased IL-1β (p<0.05) and ADAMTS-5 levels (p<0.01).
Irisin stimulates hNPC proliferation, metabolic activity, and anabolism by reducing IL-1β and catabolic enzyme expression while promoting matrix synthesis.
