Exploring the mechanism of Nav1.3 in the ION-CCI rat model based on the TLR4/TRAF6/NF-κB pathway

Background : Trigeminal neuralgia (TN) is a common and difficult-to-treat neuropathic pain disorder in clinical practice. Previous studies have shown that Toll-like receptor 4 (TLR4) modulates the activation of the NF-κB pathway to affect neuropathic pain in rats. Voltage-gated sodium channels (VGSCs) are known to play an important role in neuropathic pain electrical activity


Introduction
Neuropathic pain caused by a lesion or disease of the somatosensory nervous system is a common chronic pain condition with major impact on quality of life [8].Trigeminal neuralgia (TN) is a common but difficult-to-treat neuropathic pain disorder in the clinical setting [18].TN is mostly caused by microvascular compression of neurosensory roots, leading to demyelination of nerve fibers, which results in neuropathic pain [1].
Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain.After ION-CCI, rats exhibit changes in spontaneous behavior and in response to tactile stimulation that are indicative of persistent pain and mechanical allodynia [21].This behavior in ION-CCI rats bears strong resemblance to spontaneous pain observed in patients.
Electrical signaling in excitable cells depends on a variety of specialized transmembrane channel proteins.Increasing research suggests that voltage-gated sodium channels (VGSCs) play an essential role in TN electrical activity [12].VGSCs include ten subtypes, among which Nav1.3/SCN3A, whose expression is upregulated after nerve damage, is particularly associated with the development of pain [13,14].
Activation of Toll-like receptor 4 (TLR4), a member of the Toll-like receptor family, leads to an increase in inflammatory factors, causing pain [22].Spinal cord stimulation has also been shown to effectively attenuate neuropathic pain in CCI rats by inhibiting the activation of the TLR4/NF-κB signaling pathway [23].However, the mechanism of action of TLR4 in trigeminal nerve injury is not clear yet.
Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a member of the TRAF family, which are known downstream proteins of several receptor families with immunomodulatory functions, including TLRs [15].It is hypothesized that TRAF6 plays a role in TLR4-mediated trigeminal nerve injury.
Injury-induced transcriptional reprogramming requires ATF3, a transcription factor that is induced rapidly after injury and necessary for axonal regeneration and functional recovery [17].
In previous studies, ION-CCI provided good models for studying trigeminal nerve injury [7].Based on the above, this experiment aimed to investigate the effects of TLR4 on the expression of TRAF6, NF-κB p65, p-p65, and Nav1.3, and to explore the possible mechanism of TLR4 regulation of Nav1.3 in ION-CCI rats.

Animals
Male Sprague-Dawley (SD) rats (age 6-8 weeks, 200-250 g) were provided by the Animal Experiment Center of Anhui Medical University (LLSC20190705).The rats were randomly divided into cages, with no more than five rats per cage.This study was conducted under the approval of the Animal Ethics Committee of Anhui Medical University, following the premise of animal ethics.

ION-CCI model
After 1 week of adaptive training, SD rats were subjected to infraorbital nerve chronic constriction injury (ION-CCI) [2].In the sham surgery group, rats only received a 5 mm incision at the intersection of the left zygomatic arch and nasal bone, without direct ligation of the infraorbital nerve.

Experimental design
After 1 week of adaptive training, the rats were randomly divided into two groups: Sham group and CCI group, with six rats per group.
On the 14th day after modeling, the rats were randomly divided into the following six groups: Sham group, CCI group, CCI + LPS-rs (TLR4 antagonist) group, CCI + C25-140 (TRAF6 inhibitor) group, CCI + PDTC (NF-κB p65 inhibitor) group, and CCI + Normal Saline (NS) group, with six rats per group.To investigate the role of the TLR4/TRAF6/NF-κB pathway in inducing neuropathic pain in rats, on the 14th day after ION-CCI surgery, the TLR4 antagonist LPS-rs, the TRAF6 inhibitor C25-140, and the NF-κB p65 inhibitor PDTC were injected separately through the infraorbital foramen on the operated side.Mechanical pain thresholds were measured 1 h before injection and at 1, 2, and 3 h post-surgery.qRT-PCR and western blot were performed to investigate the expression of TLR4, TRAF6, p65, p-p65, and Nav1.3 in the TG after injection 2 h of each group of rats.As shown in Fig. 1 and Fig. 2.

Behavioral testing
Von-Frey filaments were used to test the mechanical pain threshold of the rats on the day before surgery, as well as on postoperative days 1, 3, 5, 7, 9, 11, 14, 21, and 42.
The mechanical pain threshold testing was conducted between 10:00 and 12:00, with the rats placed in a quiet environment 1 h before each test.Von-Frey filaments were used to vertically stimulate the operated side of the rat's vibrissal pad.The bending forces of the Von-Frey filaments were sequentially applied as follows: 0.008, 0.07, 0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, and 8 g.The testing started with the smallest force and gradually increased.Each filament was applied five times, with an interval of at least 30 s between each stimulation, until the rat exhibited a positive response (Fig. 3).
The following three responses will be considered positive [21]: a. Withdrawal response.b.Aggression or escape response.c.Asymmetric facial grooming behavior.
The criteria for a positive response include the occurrence of any one or more of the above three behaviors at least three times.The corresponding force value of the filament at that point will be considered the mechanical pain threshold of the operated side's vibrissal pad.

Intrathecal injection
On the 14th day after modeling, the corresponding inhibitors or antagonists were injected into each group of rats.After administering  anesthesia, a sterile insulated microinjection needle was inserted into the inner side of the zygomatic process palpation area (1-2 mm) at an angle of 10 • to the midline of the head.The needle tip was advanced approximately 22 mm along the infraorbital canal [16].LPS-rs (1 μg/1 μL) was injected; C25-140 (10 μg/μL) was injected; PDTC (20 μg/1 μL) was injected.

Tissue collection
After euthanizing the rats by excessive intraperitoneal injection of 10 % chloral hydrate, the heads were dissected to remove the TG from the operated side.The weight of the TGs was measured and recorded.

Real-time polymerase chain reaction (PCR)
Tissue samples were extracted using TRIzol reagent.Chloroform extraction was performed followed by isopropanol precipitation.The precipitate was then washed with 75 % ethanol, dried, and resuspended in DEPC-treated water for storage at -80 • C. cDNA was synthesized using the PrimeScript RT Kit (TaKaRa, Dalian, China).The expression levels of ATF3, TLR4, TRAF6, p65, p-p65, and SCN3A were measured using a fluorescence quantitative PCR instrument.Relative quantification study was conducted using the 2 -ΔΔCt method.The primer sequences used in the fluorescence quantitative PCR experiment are listed in Table 1.

Western blotting
For western blotting, 1 mg of tissue was added to 10 μL of RIPA lysis buffer (Beyotime, Shanghai, China) and ground using a low-temperature high-speed tissue grinder (Servicebio, Wuhan, China).Total protein concentration and the amount of protein were determined using a BCA protein analysis kit.The protein samples were loaded into the wells of the SDS-PAGE gel for electrophoresis.Subsequently, the proteins were transferred onto a PVDF membrane (Millipore, USA), which was subsequently placed in 5 % skim milk on a shaker for blocking.After blocking, the membrane was washed with TBST solution.The corresponding bands were incubated with the diluted primary antibodies (anti-ATF3, 1:500, bioss, bs-0519R; TLR4 antibody, 1:1000, bioss, bs-20594R; anti-TRAF6, 1:2000, bioss, bs-2830R; anti-SCN3A, 1:1000, Abcam, ab309473; anti-p65, 1:2000, Proteintech, 66535-1-Ig; and antip-p65, 1:2000, CST, 3033 T) overnight at 4 • C on a shaker.After washing the membrane, the secondary antibodies were added and incubated at room temperature for 2 h.Following another round of washing, the membrane was exposed to an imaging system (Peiqing, Shanghai, China) using an ECL Ultra-Sensitive Luminescence Kit (Thermo, Waltham, USA).After exposure, Image J software was used to perform statistical analysis on the grayscale values of the protein bands.

Statistical analysis
The experimental results were analyzed using GraphPad Prism 8.0 software (version 6.01).The mechanical pain threshold results were analyzed using a two-way analysis of variance (ANOVA).An independent samples t-test was performed for the western blotting and qRT-PCR results between the CCI and Sham groups.A one-way ANOVA was conducted for the western blotting and qRT-PCR results among the CCI, CCI + LPS-rs, CCI + C25-140, CCI + PDTC, and CCI + NS groups.A significance level of p < 0.05 was considered statistically significant for all analyses.

CCI group rats showed a decreased mechanical pain threshold
The results revealed no significant differences in the mechanical pain threshold between the two groups on the day before surgery, whereas both groups showed an increase in mechanical pain threshold 1 day after surgery.At 3 days after surgery, the mechanical pain threshold began to decrease, but there were no significant differences between the two groups.At 5 days after surgery, the two groups showed differences in mechanical pain threshold, with the maximum difference observed at day 14.After 14 days, the mechanical pain threshold in the CCI group gradually increased, and there were no significant differences between the two groups at 42 days.

Elevated ATF3 levels in the TG of CCI rats
In order to more objectively demonstrate that ION-CCI causes neuropathic pain, we examined the expression level of the pain marker ATF3 within the TG on the operated side of rats 14d after modelling.In Fig. 4, the western blot results demonstrated a significant increase in ATF3 protein expression in the TG of CCI rats compared with the Sham group (n = 3, **p < 0.01).In Fig. 5, the PCR results revealed that the CCI group exhibited higher ATF3 expression compared with the Sham group.
These findings indicate that ION-CCI surgery induced neuropathic pain in rats.

ION-CCI surgery induces activation of the TLR4/TRAF6/NF-κB pathway and upregulation of Nav1.3 in the TG
As shown in Fig. 6, the Western blot results revealed significant increases in the expression levels of TLR4, TRAF6, p-p65, and SCN3A proteins in the CCI group compared with the Sham group (n = 3, p < 0.001), while there was no significant difference in p65 expression between the two groups (n = 3, p > 0.05).Consistent trends were observed in qRT-PCR results, as depicted in Fig. 7.
Therefore, the TG was harvested 2 h after inhibitor injection, and changes in the expression of TLR4, TRAF6, p65, p-p65, and Nav1.3/SCN3A proteins were examined using western blot analysis, as illustrated in Fig. 6A and 6C.
In the CCI + LPS-rs group, the expression of TLR4, TRAF6, and p-p65 in the TG of rats was significantly inhibited, the expression of Nav1.3/SCN3A was decreased, and there was no significant difference in p65 expression compared with the control group.These findings indicate that the TLR4 inhibitor can suppress the expression of TRAF6 and phosphorylation of p65, as well as inhibit Nav1.3/SCN3A expression.
In the CCI + C25-140 group, the expression of TLR4, TRAF6, p-p65, and Nav1.3/SCN3A in the TG of rats was downregulated compared with that in the control group, with no significant change in p65 expression.When TRAF6 is inhibited, phosphorylation of p65 is also suppressed, suggesting that TRAF6 mediates the activation of the NF-κB pathway and voltage-gated sodium channels.
In the CCI + PDTC group, the expression of TLR4, TRAF6, p-p65, and Nav1.3/SCN3A in the TG of rats was downregulated compared with the control group, indicating that the NF-κB pathway is involved in regulating voltage-gated sodium channels.
Consistent trends were observed in mRNA expression using qRT-PCR analysis, as shown in Fig. 9.

Discussion
ION-CCI is a classical animal model of TN.In this study, we successfully established the ION-CCI rat model and observed that the mechanical pain threshold reached its lowest on the 14th day.Furthermore, we examined the expression of the neural pain marker ATF3 protein in the TG [20].The results showed significantly higher levels of ATF3 protein expression in the CCI group compared with the Sham group, confirming ION-CCI surgery caused neuropathic pain in rats.
It is important to note that both the Sham and CCI groups showed an increase in mechanical pain thresholds on the first day after ION-CCI surgery, which may be related to an incision wound in the territory of the injured nerve caused by the surgery.Some studies have found similar.During the first week after ION-CCI, rats no longer respond to mild or moderate tactile (i.e., von Frey) stimuli.This hyporesponsiveness is then abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior [6].In addition, 1 h after infraorbital nerve injection of the inhibitors, there were no significant differences in mechanical pain thresholds among the groups, which may be due to the time required for the inhibitors to enter the cells and exert their effects.However, after 2 h, the pain thresholds reached their peak and then decreased, with significant differences still present at the 3-h time point.Further refinement of the time gradient design is necessary for a more comprehensive analysis.
The role of voltage-gated sodium ion channels in neurological disorders has been reported in several previous studies, where they have been found to be associated with the activation of microglia after injury [11].The study of Nav1.3, also known as SCN3A, has been relatively in-     depth, and it is considered one of the markers induced after neural damage [5,10].This is consistent with the results of this experiment, where after using ION-CCI surgery to induce TN in rats, the expression of Nav1.3/SCN3A within the TG was higher, and the mechanical pain threshold was lower compared with those in the control group.
However, the mechanism by which SCN3A mediates neuropathic pain is complex owing to the complexity of the internal environment.Some researchers believe that TNF-α can increase sodium currents by activating the NF-κB and p38 MAPK signaling pathways in central nervous system neurons, thereby increasing the expression of VGSC [4].In this experiment, we found that inhibition of the NF-κB pathway inhibited the phosphorylation of p65, also led to the suppression of SCN3A expression and an improvement in mechanical pain threshold, validating the potential involvement of the NF-κB pathway in mediating SCN3A-induced neuropathic pain.
TLR4 has long been regarded as an upstream factor of NF-κB.In more recent years, the role of the TLR4/NF-κB pathway in neuroinflammation has gradually been discovered.Research has shown that targeting TLR4 to inhibit activation of the NF-κB pathway can improve neuroinflammation caused by sciatic nerve injury in rats, indicating that the TLR4/NF-κB pathway may play a role in the development of neuropathic pain [3].This finding is consistent with the results of our experiment, where inhibition of the NF-κB pathway by inhibiting phosphorylation of P65 through a TLR4 inhibitor led to a reduction in neuropathic pain.
Previous experiments conducted by our research team demonstrated that TRAF6 can induce the expression of SCN3A in TN.Therefore, we suspect that TRAF6 plays an important role in the TLR4/NF-κB pathway.However, the role of the TLR4/TRAF6/NF-κB signaling pathway in TN has not been confirmed.Here, for the first time, we validate that the TLR4/TRAF6/NF-κB pathway mediates the expression of SCN3A and induce ION-CCI rats neuropathic pain through voltage-gated sodium channels.
In this study, after modeling, the expression of TLR4, TRAF6, p-p65, and SCN3A was upregulated, and the pathway was activated, leading to a reduction in mechanical pain threshold in rats.However, after the injection of inhibitors, the expression of these factors was downregulated, the pathway was suppressed, and the mechanical pain threshold showed varying degrees of recovery.
This study provides supplementary information on the mechanism of neuropathic pain and proposes a novel target for its treatment.Based on our findings, further screening can be conducted to identify lead compounds with strong binding affinity to target sodium channel subtypes, good analgesic effects, low toxicity, and favorable pharmacokinetic properties, ultimately contributing to the development of better treatment methods for TN.

Limitations of the research
This study has certain limitations that warrant discussion.First, only the mechanical pain threshold was measured for the first 3 h after the inhibitor injection.Although the most significant differences from the control group were observed at the 2-h mark, there were still noticeable differences in the mechanical pain threshold of some experimental groups compared with the control group at 3 h.Therefore, a longer evaluation period should be considered.Second, as we only studied male SD rats in the ION-CCI model, there may be biases caused by sex differences.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 3 .
Fig. 3. Comparison of the changes in mechanical pain threshold at different time points between the Sham and CCI groups.Statistically significant differences were observed in the rats of the CCI group.Data represent the mean ± standard error of mean (SEM), n = 6 (**p < 0.01, ***p < 0.001).

Table 1
List of primer sets.