Fig 2: Pituitary adenylate cyclase-activating polypeptide (PACAP) immunoreactivity in healthy and OA cartilage tissue. (A,B) PACAP immunohistochemistry in control cartilage (without ACLT) exhibited a very strong (ES = ++++; IS = 4) immunostaining in chondrocytes from rat femoral articular cartilage (superficial and middle zone). Magnification ×20; Scale bars: 100 µm; (C) PACAP immunohistochemistry in moderate/severe OA cartilage (with ACLT) exhibited a weak/absent (ES = +; IS = 1) immunostaining in chondrocytes from rat femoral articular cartilage (superficial and middle zone). Magnifications ×20; Scale bars: 100 µm; (D) No immunostaining was observed in the negative control (ES = 0; IS = 0) treated with PBS in place of the primary antibody. Magnifications ×20; Scale bars: 100 µm; (E) Immunohistochemistry: percentage of PACAP positive cells out of the total number of cells counted in control groups and in OA group. Results are presented as the mean ± SEM. One-way ANOVA followed by Tukey’s post-hoc test was applied to evaluate the statistical significance of the results. ** p < 0.01 vs. control groups.

Fig 3: PACAP and IL-1β concentration in the SF collected from the articular cavity of the knee joint from healthy and ACLT-induced OA rats. PACAP and IL-1β concentration in the SF was measured by ELISA method both in controls, sham-operated and OA rat groups. The concentration of PACAP and IL-1β in the box and whisker plots represent the lower and the upper quartile; the open circle in the middle of the box represents the median and the ends of the lines extend to the smallest and largest data point ≤1.5 IQR (interquartile range). (* p < 0.05, vs. control and sham).

Fig 4: Schematic illustration depicting PACAP regulation in the proposed model of OA and its beneficial effects in isolated chondrocytes after IL-1ß insult. Upon anterior cruciate ligament transection (ACLT), rats progressively develop clinical signs of OA, including cartilage deterioration and local inflammatory response [55,56]. In this scenario, PACAP immunoreactivity, which was detectable at high intensities in chondrocytes localized in the outer regions of the healthy cartilage is significantly reduced by experimental induction of OA. Such an event is accompanied by the reduction of PACAP concentration in the synovial fluid and a remarkable increase in intra-articular presence of the pro-inflammatory cytokine IL-1ß, suggestive of an active inflammatory process. To assess whether PACAP contributed to prevent chondrocyte death, these cells were isolated through enzymatic digestion and challenged with IL-1ß, to partly mimic the inflammatory milieu observed in vivo. Results show that PACAP treatment ameliorated most of the detrimental effects of the cytokine, acting both as a chondroprotective and anti-inflammatory molecule. PACAP conc. = PACAP concentration; PACAP-IR = PACAP immunoreactivity.

Fig 5: Effects of PACAP on IL-1ß induced iNOS and COX-2 expression levels. Chondrocytes were exposed to IL-1ß for 24 h at indicated concentrations and changes in iNOS and COX-2 protein levels were measured by Western blot analyses; Bands were quantified using ImageQuantTL software and normalized values were plotted in the histogram shown. Each value represents the mean band densities ± SEM from each group. (# p < 0.05 or ## p < 0.01 vs. vehicle). Experiments were repeated at least three times with similar results.