Role of receptor-interacting protein 1/receptor-interacting protein 3 in inflammation and necrosis following chronic constriction injury of the sciatic nerve

Abstract

Nerve damage often leads to nervous system dysfunction and neuropathic pain. The serine-threonine kinases receptor-interacting protein 1 (RIP1) and 3 (RIP3) are associated with inflammation and cell necrosis. This study aimed to explore the role of RIP1 and RIP3 in sciatic nerve chronic constriction injury (CCI) in mice. On a total of thirty mice, sciatic nerve CCI was performed. The paw withdrawal threshold was measured using Von Frey filaments. The mRNA expression and protein levels of inflammatory factors RIP1 and RIP3 in the dorsal root ganglion (DRG), spinal cord (SC) and hippocampus (HIP) were also determined. We found that paw withdrawal threshold was significantly reduced from the second day after the operation, and the levels of tumour necrosis factor-α and interferon-γ in DRG, SC and HIP were significantly increased on the eighth and 14th days in CCI mice. Furthermore, the downstream signalling molecules of RIP1 and RIP3, GTPase dynamin-related protein-1, NLR family pyrin domain containing-3 (NLRP3) and nuclear factor κB-p65 were upregulated. Increased protein levels of programmed cell death protein 1, which indicate cell death of peripheral and central nervous tissue, were induced by CCI of the sciatic nerve. Overall, this study showed that RIP1 and RIP3 were highly expressed in DRG, SC and HIP of the sciatic nerve in CCI mice and may be involved in chronic neuroinflammation and neuronecrosis.

Fig. 1

Chronic constriction injury (CCI) promoted the release of tumour necrosis factor (TNF)-α and interferon (IFN)-γ. A total of 30 mice were used to induce a CCI model through ligation of the sciatic nerve. (a) The paw withdrawal threshold (PWT) was measured using Von Frey filaments. (b, c) The mRNA expressions of TNF-α and IFN-γ in the dorsal root ganglion (DRG), spinal cord (SC) and hippocampus (HIP) were measured by RT-PCR. (d, e) Protein levels of TNF-α and IFN-γ in the DRG, SC and HIP were measured by enzyme-linked immunosorbent assay. n=10. *P<0.05, ***P<0.001, versus the Sham group.

Fig. 2

Sciatic nerve chronic constriction injury (CCI) upregulated mRNA expression of RIP1 and RIP3 and increased the protein level of receptor-interacting protein (RIP)1/RIP3 in dorsal root ganglion (DRG), spinal cord (SC) and hippocampus (HIP). The mRNA expression of RIP1 (a), RIP3 (b) and western blotting bands of RIP1/RIP3 in the dorsal root ganglion, spinal cord and hippocampus on the 14th day after the operation. n=3. *P<0.05, versus the Sham group.

Fig. 3

Sciatic nerve chronic constriction injury (CCI) activated inflammasome and nuclear factor (NF)-κB signalling. Fourteen days after ligation, the protein levels of dynamin-related protein (DRP)1, NLR family pyrin (NLRP3) and NF-κB p65 in the dorsal root ganglion (a), spinal cord (b) and hippocampus (c) were measured by western blotting.

Fig. 4

The sciatic nerve chronic constriction injury (CCI) increased the protein level of PD1. Fourteen days after ligation, the protein levels of PD1 in the dorsal root ganglion (a), spinal cord (b) and hippocampus (c) were measured by western blotting. DRG, dorsal root ganglion; SC, spinal cord; HIP, hippocampus.