Antagonism of the prokineticin system prevents and reverses allodynia and inflammation in a mouse model of diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease

PC1 does not affect body weight, blood glucose and insulin levels.

<p>Effect of PC1 administration from day 21 to day 35 on body weight (panel A), blood glucose levels (panel B) and insulin levels (panel C). Plasma insulin levels were measured at the end of the PC1 therapeutic treatment, i.e. on day 35 after MLD-STZ. Effect of preventive PC1 administration (from day 0 to 14) on body weight (panel D), blood glucose levels (panel E) and insulin levels (panel F). Plasma insulin levels were measured at the end of the PC1 therapeutic treatment, i.e. on day 14 after MLD-STZ. ***p<0.001 vs vehicle; * p<0.05 vs vehicle</p

Effects of therapeutic PC1 administrations on cytokine production by macrophage and splenocytes.

<p>(A) Effect of therapeutic PC1 administrations (s.c. 150 μg/ kg, twice-daily for 14 days from day 21 to 35 after MLD-STZ) on IL-1β (a) and IL-10 (b) production by peritoneal macrophages. Data represent mean± SEM of 6–8 mice per group. (B) Effect of therapeutic PC1 administrations (s.c. 150 μg/ kg, twice-daily for 14 days from day 21 to 35 after MLD-STZ) on IFN-γ (a), IL-2 (b), IL-4 (c) and IL-10 (d) production by splenocytes. (C) Splenocyte Th1/Th2 profile was expressed as IFN-γ/IL-4 ratio and calculated by setting IFN-γ/IL-4 ratio of the control as 100%. Cytokine content, evaluated by ELISA, was reported as protein concentrations in culture media. Data represent mean ±SEM of 6 mice per group. One way ANOVA was used for statistical evaluation, followed by Tukey’s test for multiple comparisons. *p<0.05 vs vehicle/ CTR; °p<0.05 vs STZ.</p

Anti-allodynic effect of PKR antagonist (PC1) administration.

<p>Anti-allodynic effect of PC1 administration as a single bolus (A) or as repeated administrations (B, C). A: acute PC1(s.c. 150 μg/ kg) was administered 21 days after MLD-STZ. B: therapeutic PC1 protocol- PC1 was administered (s.c. 150 μg/ kg, twice-daily) for 14 days, from day 21 to 35 after MLD-STZ. C: preventive PC1 treatment-PC1 was administered (s.c. 150 μg /kg, twice-daily) for 14 days, starting from day 0, time point corresponding to the first STZ administration. Data represent mean± SEM of 6 mice / group. Two way ANOVA was used for statistical evaluation, followed by Bonferroni’s test. ***p<0.001 vs vehicle/CTR and CTR + PC1; °°p<0.01, °°°p<0.001 vs STZ.</p

PC1 administration prevents GluN2B overexpression in spinal cord.

<p>Effect of preventive PC1 administrations (14 days) on GluN1 (A), GluN2A (B), GluN2B (C), GluA1 (D) and GluA2/3 (E) subunit content and representative Western blot bands in spinal cord 14 days after MLD-STZ. Levels of glutamate receptor subunits were normalized on tubulin. Na/K ATPase was used as controls for the integrity of plasma membrane proteins (F). The values are the mean ± SEM of 3 replications for each antibody, performed on 3 separate pools. *p<0.05,**p<0.01 ***p<0.001 vs vehicle.</p