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. 2019 Apr 18:12:1287-1296.
doi: 10.2147/JPR.S197168. eCollection 2019.

Intrathecal TRPM8 blocking attenuates cold hyperalgesia via PKC and NF-κB signaling in the dorsal root ganglion of rats with neuropathic pain

Song Cao  1   2 , Qingmei Li  3 , Jingfeng Hou  1 , Zhourui Li  1 , Xinya Cao  1 , Xiaohong Liu  4 , Bangyong Qin  1
Affiliations

Intrathecal TRPM8 blocking attenuates cold hyperalgesia via PKC and NF-κB signaling in the dorsal root ganglion of rats with neuropathic pain

Song Cao et al. J Pain Res. .

Abstract

Background: TRPM8 channel plays central roles in the sensitization of nociceptive transduction and is thought as one of the potential targets for the treatment of neuropathic pain. However, the specific molecular mechanisms are still less clear. Methods: Sciatic chronic constriction injury (CCI) rats were intrathecally administered with AMTB (TRPM8-selective antagonist) or PDTC (nuclear factor-kappa B (NF-κB) inhibitor). Cold-, thermal- and mechanical-pain thresholds were examined in CCI and sham-operated rats before and after intrathecal administration of AMTB or PDTC. Protein expression levels of TRPM8 and NF-κB p65, p-PKC/PKC value and p-PKA/PKA value in the CCI ipsilateral L4-6 dorsal root ganglions (DRGs) were analyzed. In addition, the co-expression of TRPM8 and NF-κB was evaluated in DRG. Results: Intrathecal injection of AMTB decreased the cold hypersensitivity and aggravated the thermal-hyperalgesia in the next 2 weeks after CCI surgery. The protein expression of TRPM8 and NF-κB p65 in the ipsilateral DRGs significantly increased after CCI surgery, which can be reversed by intrathecal administration of AMTB. The PKC, PKA, p-PKC/PKC and p-PKA/PKA values showed significantly increase after CCI surgery, while intrathecal AMTB administration offset the expression increase of PKC, p-PKC and p-PKC/PKC but PKA or p-PKA/PKA in the DRG. NF-κB inhibitor not only efficiently increased the cold-, thermal-pain threshold of CCI rats, but also enhanced AMTB's anti-cold pain effect although exerted no anti-thermal hyperalgesia effect compared with TRPM8 blockade group. Immunofluorescence results showed co-expression of TRPM8 and NF-κB in DRG neurons. Conclusion: TRPM8 channels in DRGs participate in the pathogenesis of cold and thermal hyperalgesia (not mechanical allodynia) in rats with neuropathic pain, which could be regulated by PKC (not PKA) and NF-κB signaling. TRPM8 channel, PKC and NF-κB are potential targets for cold hyperalgesia treatment in neuropathic pain patients.

Keywords: NF-κB; PKC; TRPM8; dorsal root ganglia; neuropathic pain.

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Conflict of interest statement

The authors report no conflicts of interest in this work.

Figures

Figure 1
Figure 1
TRPM8 blocker reduces cold pain, aggravates thermal pain but has no effect on mechanical pain in chronic constriction injury  (CCI) rats. (A) Cold pain sensitivity assessed by cold plate tests, which significantly decreased in AMTB group compared with CCI group on the 3rd, 7th, 10th and 14th day after CCI surgery. (B) The thermal withdrawal latencies assessed by a beam-thermal system were decreased in AMTB rats compared with CCI rats on the 3rd, 7th, 10th and 14th day after CCI surgery. (C) Mechanical withdrawal thresholds (MWTs) were assessed with the electronic von Frey plantar aesthesiometer on ipsilateral hind paws of CCI, CCI+TRPM8 blockade and sham-operated rats, which showed no obvious change between CCI and AMTB group. Data are expressed as mean±SD, n=12 for the three experiments. Statistical analyses consisted of repeated measures two-way ANOVA tests. *P<0.05 vs Sham group, #P<0.05 vs CCI group.
Figure 2
Figure 2
TRPM8 blockade reduces chronic constriction injury (CCI)-induced TRPM8 overexpression in DRGs. The TRPM8 protein expression was evaluated with Western blotting. Compared with the Sham group, the expression of TRPM8 in the L4-6 dorsal root ganglions (DRGs) was significantly increased on the 7th and 14th day after CCI surgery. Compared with the CCI group, the expression level of TRPM8 protein in AMTB group was significantly decreased on the 7th and 14th day after CCI surgery. Data are expressed as mean±SD and analyzed with one-way ANOVA. *P<0.05 vs Sham group, #P<0.05 vs CCI group.
Figure 3
Figure 3
Intrathecal injection of AMTB decreased chronic constriction injury  (CCI)-induced overexpression of PKC and elevated p-PKC/PKC value. (A) Compared with the Sham group, CCI increased the expression of PKC, p-PKC as well as the value of p-PKC/PKC. (B) In addition, CCI increased the expression of PKA and p-PKA although no significant difference was found for p-PKA/PKA between Sham and CCI rats. Compared with CCI group, intrathecal injection of AMTB decreased CCI-induced elevation of PKC and p-PKC/PKC value (A), but did not affect PKA, p-PKA and p-PKA/PKA (B). Data are expressed as mean±SD, n=3. *,#P<0.05 vs Sham group at the same time point, ▲, ●P<0.05 vs CCI group.
Figure 4
Figure 4
NF-κB participates in the neuropathic pain regulation of TRPM8 in DRGs of chronic constriction injury (CCI) rats. To exam whether NF-κB involved in the pain regulation of TRPM8, NF-κB p65 expression (A and B) and pain thresholds (C and D) were detected in Sham, CCI rats and CCI rats with TRPM8 blockade (with AMTB) or TRPM8+NF-κB blockade (with PDTC). (A and B) Western blotting images and statistics of NF-κB expression levels. Data are expressed as mean±SD, n=3. *P<0.05 vs Sham group, #P<0.05 vs CCI group (C and D). Pain behavior tests showed that compared with the CCI group, NF-κB blockade with PDTC significantly decreased the paw lift times and reduced thermal hyperalgesia. When the TRPM8 and NF-κB were both blocked, the paw lift times on cold plate decreased more on the 3rd, 7th, 10th and 14th day in CCI rats compared with that of NF-κB blockade CCI rats, but no difference of thermal hyperalgesia was found between NF-κB inhibition group and NF-κB + AMTB blockade group at the time points. Data are expressed as mean±SD, n=12. *P<0.05 vs NF-κB blockade group. (E) The co-localized expression of TRPM8 and NF-κB was assessed by immunofluorescence detected by confocal microscopy. Both TRPM8 and NF-κB were localized in the cytoplasm in Sham and CCI rat dorsal root ganglion. NF-κB p65 (shown in red) and TRPM8 (shown in red) localized in the same cells. Cellular nuclei were stained with DAPI (shown in blue).
Figure 5
Figure 5
Schematic presentation of the mechanisms of TRPM8 blockade on cold hyperalgesia in the dorsal root ganglions of chronic constriction injury (CCI) rats. CCI increases expression of TRPM8, PKC, p-PKC and NF-κB p65 in the ipsilateral DRGs of CCI rats. Intrathecal injection of AMTB blocks TRPM8, and offset these effects induced by CCI. NF-κB inhibitor PDTC enhances AMTB’s anti-cold pain effect. These suggest that TRPM8 channels in DRGs participate in the pathogenesis of cold hyperalgesia in rats with neuropathic pain, which could be regulated by PKC and NF-κB signaling. Arrowheads=activate, short bars=inhibition.
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References

    1. Baron R. Mechanisms of disease: neuropathic pain–a clinical perspective. Nat Clin Pract Neurol. 2006;2(2):95–106. doi:10.1038/ncpneuro0113 - DOI - PubMed
    1. Truini A, Garcia-Larrea L, Cruccu G. Reappraising neuropathic pain in humans–how symptoms help disclose mechanisms. Nat Rev Neurol. 2013;9(10):572–582. doi:10.1038/nrneurol.2013.180 - DOI - PubMed
    1. Colloca L, Ludman T, Bouhassira D, et al. Neuropathic pain. Nat Rev Dis Primers. 2017;3:17002. doi:10.1038/nrdp.2017.2 - DOI - PMC - PubMed
    1. Meacham K, Shepherd A. Neuropathic pain: central vs. peripheral mechanisms. Curr Pain Headache Rep. 2017;21(6):28. - PubMed
    1. Yin K, Zimmermann K, Vetter I, Lewis RJ. Therapeutic opportunities for targeting cold pain pathways. Biochem Pharmacol. 2015;93(2):125–140. doi:10.1016/j.bcp.2014.09.024 - DOI - PubMed