(A) Ghrelin is detected in the extracellular matrix of the cell clusters formed in NP tissue from degenerated discs, as measured by immunohistochemistry. Left panel is negative control. Samples from disc degeneration patients were collected and were stained with anti-ghrelin antibody (brown), and consequently counterstained with methyl green (green). The insert indicates the higher magnification view of cell clusters. Scale bar, 25μm. (B) Ghrelin is expressed in plasma of primary human NP cells, as detected by cell immunostaining approach. NP cells were isolated, and cell immunostaining was performed for ghrelin. (C) RNA level of ghrelin was reduced by IL-1β, as assessed by real time PCR. (D, E) Protein level of ghrelin is reduced post- IL-1β treatment, as assayed by Western blot. The values are the mean±SD. *p < 0.05 and ** p < 0.01 vs. Control group. Scale bar=25μm. Each experiment was run in triplicate.