LINK N REGULATES IL-1Β AND MARKERS OF PAIN IN THE INTERVERTEBRAL DISC

Intervertebral disc degeneration (IVDD) is a leading cause of chronic back pain due to abnormal sensory input (nociception) by dorsal root ganglia neurons that innervate the disc, which is caused by IL-1β a key proinflammatory cytokine involved in the progression of degeneration and pain thus increasing sensory innervation throughout the annulus fibrosus (AF) and into the nucleus pulposus (NP). Currently, there is a lack of a gold standard to relieve discogenic pain. We have demonstrated that LN, a 16-residue peptide derived from link protein, acts as an anabolic agent in the intervertebral disc (IVD) both in vitro and in vivo. We have also demonstrated that LN can regulate markers of pain in human disc cells. However, it remains unclear as to the mechanism(s) of LN on pain modulation. We hypothesize that LN will provide sustained therapeutic benefit to the disc by regulating IL-1β activity in IVDs and DRG neurons. Histology Twenty New Zealand White rabbits, 4–6-month-old skeletally mature were used. Degeneration was induced in all rabbits at the L2/3 and L4/5 levels, by AF puncture in the ventral aspect into the NP using an 18G needle to a maximum depth of 5 mm. After two weeks punctured discs were injected intradiscally into the NP area with either saline (10 μL/disc) or (25 μg) LN, then biweekly for 12 weeks. After12 weeks of treatment, rabbits in each group were euthanatized and their discs were excised for histological evaluation. IVD sections were incubated with antibodies targeting either CGRP, protein gene product 9.5 (PGP9.5), and NGF. Ca2+-mobilization-DRG neurons were isolated from lumbar regions L2-5 in 15-week-old C57BL/6 mice and cultured in glass chamber slides for 7 days with IL-1β with or without LN [1 μg/mL]. Cells were loaded with Fura-4, and AM and imaged for changes in intracellular Ca2+ either at resting state or following stimulation with capsaicin [100 nM] using a Zeiss LSM800 confocal microscope. Western blotting: Isolated human NP cells were cultured as pellets and treated in Prime Growth Disc Cell serum-free media and incubated with IL-1β [1ng/mL] with and without LN [1 μg/mL] for up to 24 hours. Pellets and media were collected individually and processed for Western blotting to identify changes in NFκB activation. Immunoprecipitation (IP): Biotinylated LN or biotinylated scrambled LN (SC) was attached to avidin-labelled agarose beads and then incubated with IL-1β. Competitive IP was performed to determine the ability of LN to inhibit IL-1β-IL1R1 interaction using His-tagged IL-1β and recombinant IL1R1. Rabbit IVDs demonstrated significant decreases in markers of pain when compared to saline treated controls (NGF, CGRP, PGP9.5; p In this study we have determined the effects of LN on pain factors, in addition to direct effects on DRG neuronal activity. We demonstrate that LN can regulate markers of pain in a rabbit model of disc degeneration possibly through direct regulation of IL-1β. These results may support the use of LN in the treatment of discogenic pain.