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INTRODUCTION
Neuropathic pain (NP) results as a direct consequence of a lesion or disease, affecting the somatosensory nervous system [1], particularly an overall prevalence estimated at 7%–10% globally. Therefore, conquering NP plays a major role in accelerating rehabilitation. Many scholars combat NP by altering of ion channels, activating immune cells, utilizing glial-derived mediators, and epigenetic regulation [2]. However, the precise mechanisms are not fully understood. Calmodulin-dependent protein kinase type II (CaMKII) is crucial to neuroinflammation-expressed activity and plasticity in mediating the transcription of Ca2+ channels. Ca2+-dependent phosphorylated level of CaMK II (p-CaMKII) can regulate the receptor damage and sensitization by Ca2+ signaling in the development of neuropathic allodynia [3]. Given Ca2+ channel activating NF-kappa B (NF-κB) function, it modulates the expression of proinflammatory genes by translocating to the nucleus, which further sensitizes nociception [4]. As a inflammatory neuropeptide, calcitonin gene-related peptide (CGRP) plays a vital role in promoting central sensitization of inflammation and neuroinflammation involving the CGRP nociceptive pathway [5,6]. Oxidative stress is another important signaling mechanism closely linked to NP associated with inflammation and playing an important role in central sensitization development [7,8]. NF-E2-Related Factor 2 (Nrf2) is a critical factor in regulating cellular oxidation, in cell defense, and as an anti-inflammatory, preventing central sensitization, which also activates production of many antioxidant enzymes such as heme oxygenase 1 (HO-1), catalase glutathione and superoxide dismutase [9]. HO-1 is an antioxidant enzyme which acts by regulating the levels of reactive oxygen species, as well as possessing an anti-inflammatory capacity [10]. However, whether Nrf2 can prevent pain signal conduction, alleviating NP and its mechanism, is still unknown. It may bring a novel outlook to therapy for NP.
Nowadays, pulsed radio frequency (PRF) is an advancement in therapeutic modality for NP characterized by a better analgesic efficacy and fewer adverse side effects which are responsible for accelerating rehabilitation. PRF may exacerbate neuropathy that regulates the activity of ion channels inducing anti-inflammatory and anti-oxidative stress mechanisms [11–13]. However, the role of Nrf2 in the analgesic effect of PRF in modulating the pain mechanism is still unknown. To provide effective safe and personalized treatment, a better understanding of the ideal PRF parameters for optimizing pain relief is still needed.
Hence, the current study aimed to identify optimal parameters for PRF treatment and to elucidate the analgesic effect of the PRF mechanism, especially how Nrf2 activation may block pain signal transmission and provide a protective effect.
MATERIALS AND METHODS
1. Animals
A total of 110 male Sprague-Dawley rats weighing 180 g to 220 g acquired from Liaoning Changsheng Biological Center Shenyang, China were maintained at temperature of 23˚C –25˚C, on a 12 hour/12 hour light/dark cycle with free access to food and water. The Institutional Animal Care and Use Committee (IACUC) of China Medical University approved the experimental procedures (IACUC no. 2019091), which followed the guidelines of the International Association for the Study of Pain. All nociceptive tests were performed and every effort made to minimize animal suffering. The investigator performing functional and behavioral testing was blinded to the animals’ group assignments, which were coded in accordance with the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines to minimize bias [14].
2. Chemicals and reagents
Nrf2 antibody (ab137550), anti-HO-1 (ab189491), anti-NF-κB p65 antibody (ab16502), anti-p-CaMKII (ab5683), and anti-CaMKII (ab50202) were purchased from Abcam. The CGRP enzyme-linked immunosorbent assay (ELISA) kit (cat. no. CSBE08211r; Servicebio Biotechnology). CGRP8-37 (HY-P0209), KN-93 (HY-15465) and bardoxolone methyl (HY-13324) were purchased from China Med Chem Express.
3. Induction of SNI and PRF treatment
Peripheral neuropathy in rats was induced by spared nerve injury (SNI) of the sciatic nerve as initially described by Decosterd and Woolf [15]. Concisely, after inhaling 3% isoflurane anesthesia, the sciatic nerve and its branches in the rats’ right hind limb were exposed, then the phrenic nerve and the common peroneal nerve were ligated and disconnected preserving the slender sural nerve. After 24 hours, a significant pain response typically manifested in the hind paw and the lateral aspect of the foot. For the sham surgery group animals, the sciatic nerve was exposed without ligation. Fifty rats were randomly assigned to five groups, each containing ten animals, which included sham control (Group A), SNI control (Group B), and three PRF treatment groups. Fourteen days after surgery, the distinct schemes of treatment were performed including PRF therapy. Once the rats’ right sciatic nerve was exposed, the trocar electrode needle (PMK-21-50; Baylis Medical Company, Inc.), connected to a PRF generator (PMG-230; Baylis Medical Company, Inc.), was vertically placed at sciatic nerve transection. Emission pulse frequency was set at 2 Hz with the temperature lower than 42˚C guaranteeing prevention of local tissue due to thermal injury. Three voltage parameters were chosen: 45 V for 5 minutes (Group C), 45 V for 15 minutes (Group D), and 90 V for 15 minutes (Group E). Rats in the sham-PRF treatment and SNI groups were exposed to the trocar and electrode needle, but no PRF current was applied. During the study, no mortality was observed in any group. The baseline (day 0) data was obtained before PRF treatment, the sciatic nerve transection was performed on the 14th day, and the sham surgery was also performed on the 14th day. After performing the PRF treatment, the rats were given right hindlimb behavioral tests over 9 days which were blinded to group assignments. Following the final behavioral testing, the rat was euthanized with 5 mL/kg of liquid sevoflurane liquid and put into the transparent sealed box. The ipsilateral lumbar L4-6 spinal cord (SC) was collected and stored at –80˚C to conduct on further experiments.
4. Intrathecal drugs
The remaining 60 14-day-SNI rats were treated with an intrathecal Nrf2 activator (bardoxolone methyl; 6 μM [16]; 10 μL), CGRP antagonist (CGRP8-37; 40 nM [17]; 10 μL), CaMKII antagonist (KN-93; 30 nM [18]; 10 μL), dimethyl sulfoxide (DMSO; 10 μL), or saline control (saline; 10 μL) after establishing the SNI model for a further 7 days, with each group containing 10 rats. To deliver the drug into the lumbar subarachnoid space, catheterization was inserted into the space as described previously [6,19]. While the rat was anesthetized by inhalation of 3% isoflurane, an incision was made at the midline on the dorsal side, and a tungsten wire (20G) was inserted into the subarachnoid space between L5 and L6 of the spine, confirming the correct localization by a tail-flicking action. A PE 10 catheter was inserted into a tungsten wire (20G), which was placed in the L4 spinal subarachnoid space. Intrathecal 2% lidocaine (2.5 μL) was then administered, leading to paralysis of the awake rat’s hind limbs. After the drug injection, 50% paw withdrawal threshold (PWT) was daily assessed as described below. The rats’ SCs were collected after the last round of injection.
5. Behavioral testing
As described, mechanical allodynia was quantitatively assessed using Von Frey testing [20]. Briefly, the rat was placed in an acrylic cage with a wire mesh floor. Von Frey filaments were used to vertically stimulate the hind paw. To estimate the 50% PWT, the animal’s paw was first stimulated using the “up and down” method to determine the mechanical force necessary to produce paw withdrawal. If the paw was sharply withdrawn, flinched immediately, or if the animal started licking or shaking the paw, it was regarded as a positive response. Otherwise, the rat was tested with the next stronger Von Frey filament. A total of 6 responses were obtained, and the 50% threshold was calculated using the following formula: 50% contraction threshold = (10 [Xf+Kδ]) / 10,000, where Xf was the value (in log units) of the strength of the final Von Frey filament and k was the tabular value for the diverse stimulation responses, δ = 0.224.
6. Enzyme-linked immunosorbent assay
Based on the manufacturer’s instructions for the ELISA kit (cat. no. CSBE08211r), the L4-L6 SC homogenized samples were centrifuged, and the supernatants were used to quantify the CGRP content at 450 nm absorbance on the microplate. The CGRP concentrations were determined using a standard curve.
7. Western blotting
The collected L4-6 SC samples were homogenized using radioimmune precipitation assay (RIPA) lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) in 400 µL of ice-cold buffer and incubated at 0˚C for 30 minutes. They were then shaken at 12,000 x g at 4˚C for 30 minutes. After centrifugation, the supernatants were collected and boiled for 5 minutes, making the protein denatured. Using the bicinchoninic acid protein assay to quantify total protein concentration, 20 µg total protein per line was loaded, separated onto 8% SDS-PAGE gels, and then electrophoretically transferred onto a polyvinylidene fluoride membrane. In order to detect the protein, the membrane was sealed in buffered 5% fat-free milk for an hour [6], then incubated with the following primary antibodies: rabbit anti-Nrf2 (1:1,000), rabbit anti-HO-1 (1:2,000), rabbit anti-NF-κB p65 (1:1,000), rabbit anti-p-CaMKII (1:1000), rabbit anti-CaMKII (1:5000), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2,000) at 4˚C overnight. After the blot was hatched with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000; cat. no. SA00001-2; Proteintech) for 1 hour at room temperature, the protein was detected by enhanced chemiluminescent reagent (Gel DocTMXR; 170-8170) using Image J software to analyze the band. Statistical analysis was performed by normalizing the optical density ratios to GAPDH (total protein-/GAPDH).
8. Statistical analysis
All the data was presented as the mean ± standard deviation and analyzed with version 20 of the Statistical Package for the Social Sciences software (SPSS 20.0; IBM). To compare data from behavioral tests, two-way repeated measures ANOVAs were used followed by Bonferroni post-hoc tests for inter-group comparisons. ELISA results were analyzed using two-factor ANOVAs, followed by Bonferroni post-hoc tests for inter-group comparisons. ELISA results were analyzed using two-factor ANOVAs, followed by Bonferroni post-hoc tests for inter-group comparisons. P values < 0.05 were deemed statistically significant.
RESULTS
1. PRF treatment reversed mechanical allodynia in SNI rats
Based on the nociceptive index, 50% PWTs of the rats revealed a significant decrease in Group B compared to Group A (P < 0.01) indicating SNI-induced mechanical hyperalgesia. On day 4, PRF treatment significantly attenuated SNI rats’ mechanical allodynia in Group E (P < 0.01) compared to Group B. On day 6, PRF treatment with long duration and high voltage significantly increased 50% PWTs in Group C (P < 0.01), Group D (P < 0.001), and Group E (P < 0.01) compared to Group B. On day 9, PRF treatment with high voltage and long duration significantly increased 50% PWTs in Group C (P < 0.01), Group D (P < 0.01), and Group E (P < 0.001) compared to Group B (Fig. 1).
2. Effect of PRF on the inflammation pathway in SNI rats
Analysis of ELISA revealed a significant reduction in spinal CGRP content in Group A compared to Group B. The CGRP content induced by PRF decreased in Group C, Group D, and Group E compared to Group B (Fig. 2A). Western blot result assessed a long-duration dependently decrease in spinal NF-κB p65 expression induced by PRF in Group C, Group D, and Group E compared to Group B (Fig. 2B).
3. Effect of PRF on CaMKII pathway in SNI rats
Western blot results verified a long-duration and high-voltage dependently decrease in spinal CaMKII expression induced by PRF in Group C, Group D, and Group E compared to Group B (Fig. 2C). In Fig. 2D, the p-CaMKII expression was significantly reduced in the spines treated by PRF in Group C, Group D, and Group E compared to Group B.
4. Effect of PRF on the antioxidant signal pathway in SNI rats
Western blot results showed that PRF intervention revealed a high-voltage and long-duration dependently significantly Nrf2 expression increase in spine induced by PRF in Group C (P < 0.01), Group D (P < 0.001), and Group E (P < 0.001) compared to Group B (Fig. 3A). The level of HO-1 expression was significantly elevated in spine treated by PRF in Group C (P < 0.001), Group D (P < 0.01), and Group E (P < 0.001) compared to Group B (Fig. 3B).
5. Effect of intrathecal treatment CaMKII antagonist
In SNI rats receiving intrathecal CaMKII antagonist KN-93 throughout 7 days, the 50% PWTs were significantly higher than the DMSO vehicle treated SNI rats (Fig. 4A). After intrathecal KN-93 treatment, CaMKII and p-CaMKII expression were decreased in the SC of SNI rats compared to the DMSO control (P < 0.001) (Fig. 4B, C). CGRP content was reduced in the SC with KN-93 compared to the DMSO control (P < 0.01) (Fig. 4D).
6. Effect of intrathecal treatment Nrf2 activator or CGRP antagonist
In SNI rats intrathecally injected with Nrf2 activator bardoxolone methyl throughout 7 days, 50% PWTs were significantly higher than those receiving the DMSO control (Fig. 5C). After receiving intrathecal bardoxolone methyl, the level of Nrf2 expression in the SC of SNI rats was significantly increased (P < 0.05) (Fig. 5D), while CaMKII (P < 0.01) (Fig. 5E), p-CaMKII (P < 0.05) (Fig. 5F), and NF-κB p65 (P < 0.01) (Fig. 5G) expression as well as CGRP content (P < 0.05) (Fig. 5H) were decreased compared to the DMSO control. As shown in Fig. 5A, in SNI rats given intrathecal CGRP8-37 continuously 7 days, the 50% PWT was significantly higher than in those given the saline vehicle. The CGRP content was reduced in the SC of SNI rats given intrathecal CGRP8-37 treatment compared to those given the saline vehicle (P < 0.05) (Fig. 5B).
DISCUSSION
To analyze the role of Nrf2 in NP signal transmission and mediating the inflammatory pathway, Nrf2 activator bardoxolone methyl was applied to SNI rats. This study confirmed that intrathecal treatment with Nrf2 activator evidently ameliorated the SNI rats’ mechanical hypersensitivity, highlighting the neuroprotective effect of Nrf2 on antioxidative stress. Furthermore, intrathecal injection of bardoxolone methyl led to overexpression of Nrf2, which significantly downregulated CaMKII, p-CaMKII, and NF-κB p65 expression, and inhibited CGRP content in the spine of SNI rats. The present study also discovered that with intrathecally injected KN–93, CaMKII expression was decreased, regulating p-CaMKII expression downregulation and inhibiting CGRP expression in the SNI rats’ spine, while the SNI rats’ 50% PWTs was increased. With intrathecally injected CGRP8-37, CGRP content in the SC was reduced leading to significantly increasing the 50% PWTs of the SNI rats. Altogether, the results indicated that activating Nrf2 regulated the CaMKII/NF-κB signaling pathway, suppressed oxidative stress and inflammation, eased central sensitization, and generated analgesic effects. The present study also found that, on the 9th day, PRF treatment resulted in a significant downregulation of p-CaMKII and CaMKII expression in the spine of SNI rats, which was dependent on high voltage. NF-κB p65 expression in the spine of SNI rats was downregulated in a manner dependent on the duration of PRF treatment. CGRP content was reduced in the SCs of SNI rats, particularly in those that had undergone high-voltage, long-duration PRF treatment. In addition, the authors discovered that high-voltage, long-duration PRF treatment increased spinal Nrf2 and HO-1 expression in a manner dependent on the treatment parameters, which, in turn, reduced oxidative stress injury. With high-voltage, long-duration PRF treatment, the 50% PWTs of SNI rats were mostly attenuated, exhibiting the most obvious relief from allodynia and hyperpathia. Above the intrathecal results, we deduced dependently high-voltage (the highest is 90 V) and long duration (up to 15 minutes) of PRF, applied to the site of an injured peripheral nerve, could significantly alleviate hyperalgesia and allodynia associated with NP rats by activating the Nrf2 expression-modulated CaMKII-/NF-κB signaling pathway, easing inflammation-mitigating central sensitization.
The SNI model has been widely used to imitate clinical symptoms of human NP involved in central sensitization and the nociceptive pathway mechanism. The authors’ previous study [13] confirmed that SNI led to long-lasting mechanical hyperalgesia in the ipsilateral hind paw. This was accompanied by the upregulation of p-CaMKII together with CaMKII, which were activated by Ca2+ mediating inflammation in vincristine-induced NP, revealing that Ca2+ modulated the CaMKII pathway promoting inflammation facilitated to NP [21]. Studies showed that CaMKII expression and activity were increased in the superficial layer of the spinal dorsal horn and dorsal root ganglia amplify pain signal output to the brain, generating central sensitization leading to NP [22,23]. Normally, NF-κB combines with its inhibitory factor IκBα, which retains the balance of inflammatory pathways. Loss of IκBα prevents the inhibition of NF-κB, leading to the release of the free NF-κB subunit p65, which translocates to the nucleus and regulates proinflammatory effects, resulting in hyperalgesia [24]. Immunofluorescence results suggested that the nucleus translocation of NF-κB p65 was increased when human NP cells on intervertebral disc degeneration were treated with CGRP [25]. The authors observed that CGRP content in the spine significantly increased after modeling SNI [26] revealed inflammation participating in NP. Apart from the inflammation, Shim et al. [7] found that oxidation stress was also a crucial aspect of central sensitization attributed to NP. We priored to find that Nrf2 in the SNI model was also significantly reduced after a slight increase for 1 day [13]. Therefore, inflammation and oxidative stress responses were responsible for NP development accelerating central sensitization. PRF treatment attenuated mechanical allodynia in the SNI rat model [11,13]. Many scholars believed that PRF generated its analgesic effect by interfering with nerve-signaling nociceptive pathways [27]. A recent study showed that TRPV1 activated Ca2+ significantly increased nuclear Nrf2 in osteoarthritis (OA) rats, ameliorating pain. KN-93 competitive inhibited p-CaMKII significantly reduced the nuclear density of Nrf2 under the TRPV1 activation in OA rats [28]. It unveiled that CaMKII mediated the effect of Ca2+, enhancing Nrf2 nuclear translocation. The authors speculated that PRF might upregulate Nrf2 expression by activating Ca2+. This study found that activating Nrf2-inhibited CaMKII alleviated mechanical hypersensitivity, which might reveal blocking Ca2+ pain signal transmission. CaMKII and NF-κB signaling is a classic signal pathway. The report showed that CaMKII phosphorylation of the Rpt6 subunit of the proteasome stimulated IκBα turnover. This resulted in the release of the p65-p50 complex, which activated NF-κB signaling via a pro-inflammatory pathway [29]. It indicated that inhibiting CaMKII silenced NF-κB signaling downregulation of the pro-infammatory pathway ameliorating hyperalgesia and allodynia. The authors also deduced that PRF might, by activating Ca2+, elevate Nrf2 expression, which was modulated CaMKII/NF-kB signaling pathway. This modulation might speculate that it blocked Ca2+ pain signal transmission, inhibited inflammation, and relieved central sensitization. Additionally, more and more clinical reports have suggested [30,31] that high-voltage and long-term PRF therapy could effectively alleviate the symptom of NP patients to accelerate rehabilitation. In a recent clinical study, Wen et al. [32] indicated that high-voltage, long-duration PRF therapy, repeated twice, generated satisfactory efficacy in patients with subacute postherpetic neuralgia and had no significant adverse reactions. We highlighted that targeting Nrf2 in the central nervous system might be a novel clinical therapy perspective on prevention of Ca2+ pain signal conduction to treat peripheral nervous injury-induced NP. Collectively, this demonstrated the hypothesis that optimal parameters of PRF treatment might function by activating Nrf2 expression regulating the CaMK/NF-κB signaling pathway blocking Ca2+ pain signal transmission to alleviate neuropathy, easing central sensitization, generating therapeutic benefits. Further study is needed to identify the specific underlying ion channel activity and molecular biology mechanisms that PRF uses to alleviate NP. The ideal voltage, duration, and other parameters of PRF to treat NP also need further study.
In conclusion, high-voltage, long-duration PRF markedly ameliorated NP and relieved central sensitization by activating the Nrf2 expression-regulated CaMKII/NF-κB signaling pathway blocking Ca2+-mediated pain signal transmission.
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