Membrane protein Nav1.7 contributes to the persistent post-surgical pain regulated by p-p65 in dorsal root ganglion (DRG) of SMIR rats model

Abstract

Background: Persistent post-surgical pain is a difficult clinical problem. In this study, we intend to explore the mechanism underlying the persistent post-surgical pain in SMIR (skin/muscle incision and retraction) rats.

Methods: First of all, the expression of membrane protein Nav1.7 and p-p65 (Phosphorylation of p65) were detected in ipsilateral L4-6 DRGs of SMIR rats by western-blot and immunostaining. Then with ProTx-II (Nav1.7 blocker) or PDTC (p65 inhibitor) were intrathecally injected while the change of Nav1.7 expression and mechanical withdrawal threshold were detected. Finally chromatin immunoprecipitation assay method was used to detect whether could p-p65 bind in the Nav1.7 gene promoter region directly.

Results: The results shows that mechanical hyperalgesia occurs following SMIR model, from 5 day (d) and lasted more than 20d after surgery. Meanwhile, the expression of Nav1.7 was up-regulated at 10d, 15d and 20d after surgery compared with naïve group. The expression of p-p65 was up-regulated at 10d and 15d compared with incision group. The mechanical hyperalgesia induced by SMIR was reversed after blocking Nav1.7 or inhibiting p65. Furthermore, Nav1.7 expression was down-regulated when p-p65 was inhibited and p-p65 could combine with the Nav1.7 gene promoter region directly.

Conclusion: Membrane protein Nav1.7 could participate in the peripheral sensitization of persistent post-surgical pain, which may be regulated by p-p65.

Keywords: DRG; Mechanical hyperalgesia; Nav1.7; P-p65; Persistent post-surgical pain.

Fig. 1

a-d The PWT result showed that persistent postoperative pain can be evoked through prolonged tissue retraction, consistent with the previously reported. Incision group (incision), SMIR group (incision plus retraction). (**P < 0.01, ***P < 0.001, vs incision group, n = 8)

Fig. 2

a Western blotting of Nav1.7 using the total protein from ipsilateral L4-L6 DRGs of SMIR surgery rats, statistical summaries of western blot analysis: The expression of Nav1.7 protein was increased on days 10,15 and 20 after SMIR surgery (*P < 0.05,**P < 0.01, ***P < 0.001 vs naive group; n = 3). b-c Effect of pretreatment with i.t injection of ProTx-II (once daily from 1d before surgery until 7d after surgery) on the mechanical hyperalgesia induced by SMIR model rats. The result showed that selective Nav1.7 channel blocker can significantly reduced hyperalgesia induced by SMIR in a dose dependent way (*P < 0.05, ***P < 0.001 vs SMIR group; n = 6)

Fig. 3

a Double immunofluorescence staining (The ipsilateral DRGs after 10d of SMIR group rats were used). b The distribution of Nav1.7 positive cells diameter (DRG from 3 rats)

Fig. 4

a-b Effects of pretreatment with i.t injection of PDTC (once daily from 1d before surgery until 10d after surgery) on the mechanical hyperalgesia induced by SMIR model (**P < 0.01,***P < 0.001,n = 6). c-d Western blotting result of nucleoprotein p-p65 from ipsilateral L4-L6 DRGs of SMIR surgery rats. Statistical summaries of western blot analysis: The expression of p-p65 protein was increased at 10d (n = 4) and 15d (n = 5) after SMIR surgery compared with incision groups, whereas pretreatment with PDTC (once daily from 1d before surgery until 7d after surgery) can prevent the activation of NF-κB at 10d (n = 4) and 15d (n = 5) compared with SMIR group (*P < 0.05 vs incision group; #P < 0.05 vs SMIR group)

Fig. 5

a-b Western blotting result of Nav1.7 from ipsilateral L4–6 DRGs of SMIR rats. Statistical summary of the densitometric analysis: compared to SMIR group, pretreatment with PDTC can inhibit the up-regulation of Nav1.7 at 10d and 15d after surgery (*P < 0.05,**P < 0.01 vs incision group; ###P < 0.001 vs SMIR group, n = 3). c Double immunofluorescence staining of ipsilateral DRG (10d after SMIR surgery). d Products of PCR amplifications for P1 - P9 from the SCN9A proximal promoter region was separated by 1% agarose gel electrophoresis