The role of TNF-alpha/NF-kappa B pathway on the up-regulation of voltage-gated sodium channel Nav1.7 in DRG neurons of rats with diabetic neuropathy
Introduction
Diabetic neuropathy (DN) is a major complication of diabetes mellitus (Gooch and Podwall, 2004, Toth et al., 2010, Xu et al., 2011). Clinical symptoms encountered in DN include abnormal sensations such as hyperalgesia, allodynia and spontaneous pain (Calcutt et al., 2004; Serra, 2012). However, the exact cause and mechanism of DN are still unclear.
In rodents, symptoms of DN can be studied after induction of diabetes with streptozotocin (STZ) (Calcutt, 2004; Tsuda et al., 2008). In diabetic rats with hyperalgesia, DRG neurons display increased frequency of action potential generation in response to sustained suprathreshold mechanical stimulation (Fox et al., 1999, Malcangio and Tomlinson, 1998, Ahlgren and Levine, 1993) and increased spontaneous activity (Said, 1996). Both effects are thought to contribute to the sensation of pain. Voltage-gated sodium channels generate and propagate action potentials in excitable cells. The tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel Nav1.7 expressed in nociceptive neurons of DRG plays a critical role in chronic pain. Expression of Nav1.7 is increased in rats with inflammatory pain and neuropathic pain (Chattopadhyay et al., 2008, Hong et al., 2004). Reduction of Nav1.7 in DRG neurons by vector mediated miRNA or by activation of delta opioid receptor attenuates pain-related behaviors evoked by STZ-induced diabetes (Chattopadhyay et al., 2012, Chattopadhyay et al., 2008).
Recently it has become clear that proinflammatory cytokines play an important role in the pathogenesis and maintenance of neuropathic pain. TNF-α has been shown to be directly involved in the production of pain in several models of nerve injury (Uçeyler et al., 2007, Xu et al., 2006, Bishnoi et al., 2011). Clinical study showed that plasma TNF-α level is higher in DN patients than in “common” diabetes patients without suffering from chronic pain, what’s more, the higher the plasma TNF-α levels, the more severe the pain (Purwata, 2011). TNF-α may activate NF-κB by binding to TNF receptor1 (TNFR1) (Baud and Karin, 2001) and initiate the transcription of a wide variety of target genes (Pahl, 1999). After nerve injury, TNF-α may induce neuropathic pain by increasing the expression and function of Nav1.3 and Nav1.8 in DRG neurons (He et al., 2010, Chen et al., 2011). Whether the up-regulation of Nav1.7 in DRG neurons of rats with DN shares the same mechanism as that in nerve injury still remains unknown.
This study was undertaken to investigate the role of TNF-α and its downstream signal molecule NF-κB on pain behaviors and Nav1.7 expression in DRG following diabetes. We found that Nav1.7 was up-regulated in DRG of rats with DN and the increased Nav1.7 was co-localized with large and small sized neurons. Inhibiting the synthesis of TNF-α which prevented DN, strongly blocked the up-regulation of TNF-α, Nav1.7 and p-NF-κB (p65). Furthermore, intrathecal administration of NF-κB inhibitor PDTC obstructed pain behaviors and over-expression of Nav1.7 in DRG neurons. These data suggest that increased TNF-α may be responsible for up-regulation of Nav1.7 in DRG neurons of rats with DN, and NF-κB signal pathway is possibly involved in this process.
Section snippets
Experimental animals
Male Sprafue-Dawley rats weighting 220–280 g were purchased from Guangdong laboratory animal center. All animal experimental procedures were carried out in accordance with the guideline of National Institutes of Health on animal care and the ethical guidelines.
Drug administration
Freshly prepared STZ (60 mg/kg, Sigma) in saline (pH 4.5 with 0.1 N citrate buffer) was injected intraperitoneally (i.p.) as described previously (Bishnoi et al., 2011). Sham group received citrate buffered saline alone. Thalidomide (Sigma)
STZ induces bilateral mechanical allodynia, thermal hyperalgesia and over-expression of Nav1.7 in DRG neurons
A single administration of STZ at a dose of 60 mg/kg caused a marked and prolonged hyperglycemia (397.34 ± 12.32 mg/dl) compared with sham group (103.72 ± 8.26 mg/dl), (data not shown). The 50% withdrawal threshold significantly reduced to 7.87 ± 1.22 g on week 4 after STZ administration compared with the value (15.12 ± 0.32 g, ∗p < 0.05) on week 0 or with the value of sham group (13.91 ± 0.64 g, ∗p < 0.05, Fig. 1A). Paw withdrawal latency significantly reduced to 12.5 ± 1.42 s on week 4 after STZ administration
Discussion
In the present study, we reported that STZ induced mechanical allodynia, thermal hyperalgesia, accompanied by up-regulation of Nav1.7, TNF-α and p-NF-κB in DRG. Inhibiting the synthesis of TNF-α which prevented DN, strongly blocked the up-regulation of TNF-α, Nav1.7 and p-NF-κB. Furthermore pain behaviors and increased expression of Nav1.7 in DRG neurons were obstructed by intrathecal administration of NF-κB inhibitor PDTC. These data suggest that increased TNF-α may be responsible for
Conclusions
In conclusion, our present data provided evidence that pain behaviors evoked by STZ might be associated with the up-regulation of Nav1.7 in DRG. Importantly, increased TNF-α in DRG may be responsible for up-regulation of Nav1.7 in rats with DN, and NF-κB signal pathway may be involved in this process. The findings might provide potential target for preventing diabetic neuropathy.
Acknowledgements
This work was supported by grants from Natural Science Foundation of Guangdong (No. S2012040006843; S2013010011889), National Natural Science Foundation of China (No. 30900436).
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