Ulinastatin attenuates neuropathic pain induced by L5-VRT via the calcineurin/IL-10 pathway

Abstract

Previous studies have shown that ulinastatin, an effective inhibitor of the inflammatory response in clinical applications, can attenuate hyperalgesia in rodents. However, the underlying mechanism remains unclear. In the present study, we first examined the change in the calcineurin level, which plays an important role in regulating cytokine release in the nervous system, following lumbar 5 ventral root transection in the rat. Furthermore, we determined whether intraperitoneal (i.p.) injection of ulinastatin attenuated pain behavior via inhibition of the calcineurin-mediated inflammatory response induced by lumbar 5 ventral root transection. The results showed that the paw withdrawal threshold and paw withdrawal latency were significantly decreased following lumbar 5 ventral root transection compared to the sham group. Neuropathic pain induced by lumbar 5 ventral root transection significantly decreased the expression of calcineurin in the DRG, and calcineurin was mostly located with NF-200-positive cells, IB4-positive cells, and CGRP-positive cells and less with GFAP-positive satellite cells. Furthermore, intrathecal (i.t.) injection of exogenous calcineurin attenuated the pain behavior induced by lumbar 5 ventral root transection. Importantly, intraperitoneal injection of ulinastatin alleviated the pain behavior and calcineurin downregulation induced by lumbar 5 ventral root transection. Lastly, the cytokine IL-10 was significantly decreased following lumbar 5 ventral root transection, and application of calcineurin (intrathecal) or ulinastatin (intraperitoneal) inhibited the IL-10 downregulation induced by lumbar 5 ventral root transection. These results suggested that ulinastatin, by acting on the CN/IL-10 pathway, might be a novel and effective drug for the treatment of neuropathic pain.

Keywords: Calcineurin; DRG; IL-10; cytokine; neuropathic pain; ulinastatin.

Figure 1.

(a and b) L5-VRT induces significant decreases in the ipsilateral hindpaw withdrawal threshold and hindpaw withdrawal latency. **P < 0.01 versus the sham group. L5-VRT: lumbar 5 ventral root transection.

Figure 2.

(a and b) L5-VRT induces a significant decrease in CN in the DRG on days 7, 10, and 14 compared to the sham group (**P < 0.01 versus the sham group). (c–f) Double immunofluorescence staining shows CN (red) expression in the DRG neurons in sham rats. CN is mostly co-localized with NF-200-positive cells (c), CGRP-positive cells (e) and IB4-positive cells (f) and less with GFAP-positive satellite cells (d) Scale bars: (c–f) = 100 µm. L5-VRT: lumbar 5 ventral root transection; CN: calcineurin.

Figure 3.

(a and b) Intrathecal injection of exogenous CN (10 U) each day for seven days after L5-VRT increases the ipsilateral hindpaw withdrawal threshold and hindpaw withdrawal latency. *P < 0.05, **P < 0.01 versus the L5-VRT group. L5-VRT: lumbar 5 ventral root transection; CN: calcineurin.

Figure 4.

(a and b) Intraperitoneal injection of ulinastatin (10,000, 50,000, or 100,000 U/kg) for seven days before L5-VRT increases the ipsilateral hindpaw withdrawal threshold and hindpaw withdrawal latency; however, no significant difference was obtained between the ulinastatin 10,000U/kg group and the L5-VRT group or between the ulinastatin100,000 U/kg group and the ulinastatin 50,000 U/kg group. *P < 0.05, **P < 0.01 versus the L5-VRT group. (c and d) Intraperitoneal injection of ulinastatin (50,000 U/kg) for seven days prevents the downregulation of CN induced by L5-VRT in the ipsilateral DRG (**P < 0.01 versus the sham group, ^##P < 0.01 versus the L5-VRT group). L5-VRT: lumbar 5 ventral root transection; CN: calcineurin.

Figure 5.

L5-VRT induces a significant decrease in the IL-10 protein level in the L4-L5 DRG on days 7, 10, and 14. (a) IL-10 upregulation was induced in the ipsilateral DRG after intrathecal injection of exogenous CN (10 U/day) for seven days. (b) IL-10 was also upregulated after intraperitoneal injection of ulinastatin (50,000 U/kg) for seven days. **P < 0.01 versus the sham group. ^#P < 0.05, ^##P < 0.01 versus the L5-VRT group. The IL-10 levels were determined with an ELISA. L5-VRT: lumbar 5 ventral root transection; CN: calcineurin.