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, 9 (1), 2769

Silencing of Spinal Trpv1 Attenuates Neuropathic Pain in Rats by Inhibiting CAMKII Expression and ERK2 Phosphorylation

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Silencing of Spinal Trpv1 Attenuates Neuropathic Pain in Rats by Inhibiting CAMKII Expression and ERK2 Phosphorylation

Shao-Hui Guo et al. Sci Rep.

Abstract

Accumulating evidence suggests a potential role of transient receptor potential vanilloid 1 (TRPV1) channels in inflammatory and cancer-related pain. However, the role of TRPV1 in the maintenance of neuropathic pain remains elusive. The current study investigated the effects of transient Trpv1 gene silencing using a small interference RNA (siRNA) on neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. Seven days after CCI, the TRPV1 siRNA was intrathecally administered (5 µg/15 µl, once daily for 2 days). TRPV1 and Ca2+/calmodulin-dependent protein kinase II (CAMKII) expression and extracellular signal-regulated kinase (ERK) phosphorylation in the spinal cord were detected using western blotting. The thresholds to mechanical and thermal stimuli were determined before and after intrathecal TRPV1 siRNA administration. TRPV1 and CAMKII expression and ERK2 phosphorylation in the spinal cord were upregulated after CCI. Intrathecal administration of the TRPV1 siRNA not only attenuated behavioural hyperalgesia but also reduced the expression of TRPV1 and CAMKII, as well as ERK2 phosphorylation. Based on these results, silencing of the TRPV1 gene in the spinal cord attenuates the maintenance of neuropathic pain by inhibiting CAMKII/ERK2 activation and suggests that TRPV1 represents a potential target in pain therapy.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Behavioural assessment of neuropathic pain induced by loose ligation of the sciatic nerve in rats. (A) Chronic constriction injury induced significant mechanical hyperalgesia, as denoted by a decreased paw withdrawal threshold to mechanical stimuli on the ipsilateral side. (B) Chronic constriction injury induced significant thermal hyperalgesia, as evidenced by the decreased paw withdrawal latency on the ipsilateral side. Data are presented as means ± SEM, and error bars represent the SEM. ***P < 0.001 compared with the sham group or the contralateral side (t-test, n = 10 rats per group). Contra: contralateral; Ipsi: ipsilateral; CCI: chronic constriction injury.
Figure 2
Figure 2
Increased expression levels of TRPV1, CAMKII, and phosphorylated ERK in the ipsilateral dorsal horn induced by chronic constriction injury. (A) The loose ligation of the sciatic nerve significantly increased TRPV1 and CAMKII expression in the ipsilateral spinal dorsal horn 7 days after CCI. (B) Bar chart showing the protein content relative to the sham group. The levels of TRPV1 and CAMKII in the ipsilateral spinal dorsal horn of the CCI group were significantly higher than those in the sham group (P = 0.031 for TRPV1; P = 0.018 for CAMKII). (C) Loose ligation of the sciatic nerve significantly increased ERK phosphorylation in the ipsilateral spinal dorsal horn. (D,E) Bar charts depicting the protein content relative to that in the sham group. A significantly higher level of phosphorylated ERK2 was observed in the ipsilateral spinal dorsal horn of the CCI group than in the ipsilateral spinal dorsal horn of the sham group (P = 0.023). GAPDH or tubulin served as a loading control and was run on the same blot. *P < 0.05 compared with the sham group (ANOVA, n = 4–5 rats per group). Error bars represent the SEM. CCI: chronic constriction injury; Contra: contralateral to CCI.
Figure 3
Figure 3
The intrathecal injection of the TRPV1 siRNA in vivo attenuated the mechanical and thermal hyperalgesia induced by chronic constriction injury. (A) The administration of the TRPV1 siRNA (5 μg/15 μl) once daily for two days significantly increased the paw withdrawal threshold to mechanical stimuli on days 1 to 4 post-transfection compared to the CCI + PEI control group or to the pre-injection baseline. (B) The TRPV1 siRNA significantly increased the paw withdrawal latency in response to thermal stimuli on days 1 to 4 post-transfection compared to results in the CCI + PEI control group or the pre-injection baseline. **P < 0.01 and *P < 0.05 compared with the PEI group results; ##P < 0.01 and #P < 0.05 compared with the pre-injection baseline results (ANOVA, n = 5–6 rats per group). Error bars represent the SEM. PEI: polyethylenimine; CCI: chronic constriction injury.
Figure 4
Figure 4
Reduced TRPV1 and CAMKII expression, as well as decreased ERK phosphorylation, in the spinal dorsal horn following TRPV1 siRNA administration. Western blots showing the reduced expression of the TRPV1 protein in the spinal dorsal horn 24 hours after the intrathecal injection of the TRPV1 siRNA (5 μg/15 μl) once daily for 2 days, in both naive (A) and CCI (C) rats. (B,D) Bar chart depicting the level of TRPV1 protein relative to that in the group administered PEI alone; the level of TRPV1 in the spinal dorsal horn was significantly reduced. (E) Western blots showing the reduced expression of CAMKII following the intrathecal injection of the TRPV1 siRNA compared to the results in the CCI + PEI group. (F) Bar chart presenting the protein content relative to the CCI + PEI group; the CAMKII level was significantly reduced. (G) Western blots showing the reduced level of phosphorylated ERK2 following the intrathecal injection of the TRPV1 siRNA in comparison to the results in the CCI + PEI group. (H) Bar chart depicting the protein content relative to the CCI + PEI group; the level of phosphorylated ERK2 was significantly reduced in the CCI + siRNA group. Tubulin or GAPDH served as the loading control and was run on the same blot. TRPV1 levels were significantly reduced in the siRNA group compared with those in the PEI group in naive rats. **P = 0.001 compared with those in the PEI group (ANOVA, n = 5 rats per group). TRPV1 levels were significantly reduced in the CCI + siRNA group compared with those in the CCI + PEI group in CCI rats. **P = 0.003 compared with those in the CCI + PEI group (t-test, n = 4 rats per group). The level of CAMKII in the ipsilateral spinal dorsal horn of the CCI + siRNA group was significantly lower than that in the CCI + PEI group. *P = 0.026 compared with that in the CCI + PEI group (t-test, n = 3 rats per group). The level of phosphorylated ERK2 in the ipsilateral spinal dorsal horn of the CCI + RNAi group was significantly lower than that of the CCI + PEI group. **P = 0.002 compared with that in the CCI + PEI group (Mann-Whitney U test, n = 6 rats per group). Error bars represent the SEM. PEI: polyethylenimine; CCI: chronic constriction injury.

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