Upregulated TNF-α and lactate following ERK-SGK1 activation in the spinal dorsal horn underlies chronic postsurgical pain Li Y, Shi W, Dai J, Jia Q, Guo G, Zhang Y, Zhang W,

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Upregulated TNF-α and lactate following ERK-SGK1 activation in the spinal dorsal horn underlies chronic postsurgical pain

Yuying Li¹, Wenjuan Shi², Juanli Dai³, Qi Jia¹, Gang Guo¹, Yanling Zhang⁴, Weihong Zhang¹
¹ School of Medical Technology and Nursing, Shenzhen Polytechnic, Shenzhen, Guangdong, China
² Affiliated Shenzhen Maternity and Child Healthcare Hospital, Southern Medical University, Shenzhen, Guangdong, China
³ Department of Neurology, Xiehe Shenzhen Hospital, Huazhong University of Science and Technology, Shenzhen, Guangdong, China
⁴ Shenzhen Eye Hospital, Shenzhen, Guangdong, China

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Date of Submission	25-Sep-2022
Date of Decision	24-Nov-2022
Date of Acceptance	16-Jan-2023
Date of Web Publication	25-May-2023

Abstract

Skin/muscle incision and retraction (SMIR) during surgeries can lead to chronic postsurgical pain (CPSP). The underlying mechanisms are still unclear. In the present study, we showed that SMIR of the thigh induced phosphorylation of extracellular signal-regulated kinase (ERK), followed by serum- and glucocorticoid-inducible kinase-1 (SGK1) activation in the spinal dorsal horn. Intrathecal injection of PD98059, an ERK inhibitor, or GSK650394, a SGK1 inhibitor, significantly attenuated mechanical pain hypersensitivity in SMIR rats. The level of tumor necrosis factor α and lactate in spinal cord was significantly decreased by PD98059 or GSK650394 injection. Furthermore, PD98059 decreased the activation of SGK1 in the spinal dorsal horn. These results indicate that ERK-SGK1 activation followed by proinflammatory mediator release in the spinal dorsal horn underlies CPSP.

Keywords: Chronic postsurgical pain, ERK, lactate, SGK1, spinal dorsal horn, tumor necrosis factor alpha

How to cite this URL:
Li Y, Shi W, Dai J, Jia Q, Guo G, Zhang Y, Zhang W. Upregulated TNF-α and lactate following ERK-SGK1 activation in the spinal dorsal horn underlies chronic postsurgical pain. Chin J Physiol [Epub ahead of print] [cited 2023 May 29]. Available from: https://www.cjphysiology.org/preprintarticle.asp?id=377576

Introduction

Chronic postsurgical pain (CPSP) is prevalent. Approximately 10%–50% of patients who have undergone surgeries such as breast reconstruction, thoracic surgery, and leg amputation suffer from CPSP.^[1] Despite extensive studies, an effective treatment for chronic postoperative pain is still an unmet need. It is clear that not only peripheral sensitization but also central sensitization contributes to CPSP.^[2],[3]

Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), which transmits diverse extracellular-to-intracellular signals,^[4] is involved in the regulation of a variety of growth and differentiation pathways through phosphorylation cascades. ERK plays a critical role in neuronal plasticity.^[5],[6] ERK1/2 is activated in the spinal dorsal horn after peripheral nerve injury, high-frequency stimulation of the sciatic nerve or peripheral inflammation and plays an important role in chronic pain development.^[7],[8],[9]

Serum- and glucocorticoid-inducible kinase (SGK) 1, one member of the SGK family, is a downstream target of the MAPK/ERK signaling cascade. It has been reported that spinal SGK1 phosphorylation is increased after peripheral nerve injury, inflammation-associated hyperalgesia, or morphine-induced analgesic tolerance.^[10],[11] Moreover, inhibition of SGK1 antagonizes the mechanical allodynia induced by nerve injury or complete Freund's adjuvant.^[11] These results indicate that spinal SGK1 activation is involved in both inflammatory and neuropathic pain. To date, almost all studies demonstrating the role of SGK1 in chronic pain are based on animal models with afferent nerve injury or peripheral inflammation. In contrast, the role of SGK1 in CPSP, which involves no obvious afferent nerve injury or peripheral inflammation, has not been investigated.^[12],[13] In response to nervous tissue damage, proinflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), IL-6, and lactate, are increased.^{[14],[15],[16],[17]} These cytokines can change the expression or activity of receptors and ion channels and regulate the excitability of neurons, and thus contribute to neuropathic pain by regulating the excitability of neurons.^{[18],[19],[20]} Studies have shown that activation of ERK1/2 is involved in pathologic pain by releasing inflammatory factors.^[21],[22] It has been reported that the increased expression of TNF-α in the spinal dorsal horn critically contributes to the development of CPSP.^[13]

In the present study, we established chronic postoperative pain state of rats with skin/muscle incision and retraction (SMIR) surgery, which simulates the process of common surgical procedures and can induce persistent mechanical hypersensitivity for a long time with higher incidence than other currently available models.^[23] We assessed the activation of ERK1/2 and SGK1 in the spinal dorsal horn by immunofluorescence staining. The level of TNF-α and lactate in the spinal cord after SMIR was also determined by an ELISA and a lactate assay kit, respectively. Furthermore, direct intrathecal injection of specific inhibitors of ERK1/2 and SGK1 was performed to investigate the role of the ERK1/2-SGK1 pathway in CPSP.

Materials and Methods

Animals

Male Sprague-Dawley rats weighing 200–250 g, obtained from Guangdong Medical Laboratory Animal Center (China), were housed under a 12-h light/dark cycle. Rats were housed with free access to sterile water and standard laboratory food in a temperature-controlled room maintained at 24°C ± 1°C and 50%–60% humidity. All experimental procedures were approved by the Animal Care Committee of Shenzhen Polytechnic (No. 2022010) and followed the guidelines of the Regulations for the Administration of Affairs Concerning Experimental Animals (China) and the ethical guidelines for the investigation of experimental pain in conscious animals. All of the experiments were conducted during the light phase in a double-blinded fashion.

Skin/muscle incision and retraction surgery

SMIR surgery was performed following the previous procedure.^[23] Briefly, under anesthesia with isoflurane (1.5%–2.5%) and a mixture of 30% N₂O and 70% O₂, the muscle of the thigh was exposed by making a 1.5–2 cm incision in the skin of the medial thigh, approximately 4 mm medial to the saphenous vein. An incision (7–10 mm long) was then made in the superficial muscle layer of the thigh, followed by insertion of a microdissecting retractor (Biomedical Research Instruments Inc., USA). The skin and superficial muscle of the thigh was retracted by 2 cm for 1 h. The sham group of rats received skin incision but no retraction.

Assessment of mechanical sensitivity

Mechanical sensitivity of rats was assessed using up-down method, with a series of filaments of 0.6–15 g (0.6, 1, 1.4, 2, 4, 6, 8, 15 g, Stoelting Co, USA). After habituation on a Plexiglass box with a wire grid floor, filaments were applied in either ascending or descending strengths to determine the filament strength closest to the hind paw withdrawal threshold. Each filament was applied for a maximum of 6 s in each trial. Quick withdrawal or licking on the paw in response to the stimulus was considered a positive response. The 50% paw withdrawal thresholds were then calculated.

Immunofluorescence staining

Rats were anesthetized with pentobarbital (50 mg/kg) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB). The L3 and L4 spinal cords were dissected, postfixed for 3 h, and kept overnight in 30% sucrose in 0.1 M PB. The spinal cord sections (25 μm sections) were made on a cryostat (Leica CM 1900, Heidelberg, Germany). For immunofluorescence staining, sections were blocked with 1% donkey serum in 0.3% Triton for 1 h at room temperature and then incubated overnight at 4°C with a primary antibody for phosphorylated ERK (p-ERK) (1:300, Cell Signaling), p-SGK1 (1:200, Abcam), glial fibrillary acidic protein (GFAP, 1:500, Cell Signaling Technology, Danvers, MA, USA), neuronal-specific nuclear protein (NeuN) (1:500, mouse monoclonal; Chemicon, Temecula, CA, USA) or goat polyclonal anti-ionized calcium-binding adaptor molecule 1 (Iba1, 1:500, Abcam). The sections were then incubated with a corresponding secondary antibody (1:300; Invitrogen) for 1 h at the room temperature.

Double staining was performed using the same procedure as described above except that two antibodies were added together.

The optical density of positive signals per section was measured by ImageJ Version: K 1.45. Every fifth spinal section was picked, 4–6 sections for each animal were selected randomly, and the average was calculated.

ELISA

The dorsal quadrants of L3/L4 spinal dorsal horn ipsilateral to the SMIR surgery were harvested and cut into pieces in precooled PBS. Then, the samples were then handled with an Ultrasonic Cell Disruptor (Sonics & Materials Inc., USA). The extracts were centrifuged at 10,000 g and 4°C for 15 min to obtain the supernatants. The concentrations of TNF-α were assayed with ELISA (BD Biosciences, USA) following the manufacturer's protocols.

Lactate detection

The dorsal L3/L4 spinal cord was dissected under sodium pentobarbital anesthesia, after which approximately 10–25 mg of the tissue was homogenized in 1 ml of a dry-ice chilled solution containing 80% methanol in H₂O. The homogenates were then centrifuged at 12,000 rpm for 15 min to remove the insoluble fraction. The supernatants that contain soluble metabolites were then lyophilized and resuspended in 50 μl of sterile H₂O. The amount of lactate was detected by the Lactate Assay Kit (MAK065, Sigma), following the manufacturer's guidelines. The results were then normalized by the tissue weight.

Direct transcutaneous intrathecal injection

Direct transcutaneous intrathecal injection was performed as follows. Briefly, a 25-Ga 3 10 needle connected to a Hamilton syringe was inserted into the tissues between the dorsal aspects of L5 and L6 of rats, perpendicular to the vertebral column. The injection was considered successful if a tail-flick was observed immediately. 10 μl PD98059 or GSK650394 dissolved in 10% DMSO (purchased from MedChemExpress, NJ, USA) was administrated.

Statistics analysis

Data are expressed as means ± standard error of the mean and were analyzed with GraphPad Prism 8.0 software (San Diego, CA, USA). For the behavioral tests, data between testing days were analyzed with Friedman ANOVA for repeated measurements, followed by the Wilcoxon matched-pairs test when appropriate. The data between groups on a given testing day were analyzed with Mann–Whitney U test. Immunohistochemistry and ELISA data were analyzed by one-way ANOVA followed by Tukey's post hoc test or two tailed unpaired Student's t-test. P < 0.05 was considered statistically significant. No statistical methods were used to predetermine the sample size but based on our previous experience.

Results

Skin/muscle incision and retraction-induced mechanical allodynia and activated the ERK pathway in the L3/L4 spinal dorsal horn

ERK1/2 is activated in the spinal dorsal horn after peripheral nerve injury.^[7] To test whether SMIR surgery, which has been reported to induce long-lasting mechanical sensitivity without nerve injury, also activated the ERK pathway in the spinal cord, immunofluorescence staining was performed on these rats that were confirmed to have developed mechanical sensitivity using the up-down method. The results showed that the 50% paw withdrawal threshold in the ipsilateral hind paw decreased at Day 4, after which the decrease reached a maximum at Day 7 and then gradually returned to the baseline level by Day 32 [Figure 1]. These results were consistent with previous studies and showed that CPSP developed. We further tested whether SMIR activated the MAPK/ERK pathway in the spinal dorsal horn at day 7 and day 14 following SMIR by immunostaining for the expression of p-ERK 1/2, an important ERK/MAPK signaling component. The results showed that compared to the sham group, the expression of p-ERK 1/2 (Thy202/Tyr204) was significantly increased at day 7 and day 14 after SMIR [Figure 2]a and [Figure 2]b. Double immunofluorescence staining showed that at day 7 after sham or SMIR operation, p-ERK was located mainly in GFAP positive astrocytes [Figure 2]e and NeuN-stained neurons [Figure 2]c but not in Iba1 positive microglia [Figure 2]g. Compared to the sham group, the percentage of neurons [Figure 2]d and astrocytes [Figure 2]f, but not microglia [Figure 2]h expressing p-ERK was increased at Day 7 after SMIR.

Figure 1: The time course of changes in the 50% paw withdrawal threshold following SMIR. The 50% paw withdrawal threshold in the ipsilateral hind paw decreased at day 4, after which the decrease reached a maximum at day 7 and then gradually returned to the baseline level by day 32. *P < 0.05, ***P < 0.001 compared with the sham group. SMIR: Skin/muscle incision and retraction.

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Figure 2: p-ERK was upregulated in spinal dorsal horn neurons and astrocytes after SMIR. (a) Cmpared to the sham group, the expression of p-ERK 1/2 was significantly increased at Day 7 and Day 14 after SMIR. (b) Quantification of p-ERK density in laminae I-V 7 days after sham and SMIR operations. (c) p-ERK located in NeuN-marked neurons was upregulated in the ipsilateral L3/L4 spinal dorsal horn at day 7 after SMIR. (e) Compared to the sham group, p-ERK located in GFAP-marked astrocytes was significantly upregulated 7 days after SMIR. (g) p-ERK was not located in Iba1-labeled microglia in either sham or SMIR rats at Day 7 after SMIR. (d, f and h) The percentages of neurons, astrocytes and microglia that expressed p-ERK in sham and SMIR rats at Day 7 are shown. ***P < 0.001 compared with the sham group. Scale bar = 200 μm. p-ERK: Phosphorylated extracellular regulated protein kinase, GFAP: Glial fibrillary acidic protein, SMIR: Skin/muscle incision and retraction.

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Skin/muscle incision and retraction upregulated SGK1 phosphorylation mainly in neurons in the spinal dorsal horn

It has been reported previously that SGK1 can be activated following MAPK/ERK activation.^[24] We therefore further investigated whether SMIR activated SGK1 in the spinal dorsal horn by immunofluorescence staining. The results showed that compared to the sham group, the density of phosphorylated SGK1 was significantly increased in laminae I-V at day 7 and day 14 after SMIR [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d. Moreover, double immunofluorescence staining showed that p-SGK1 was mainly located in neurons [Figure 3]e, [Figure 3]f, [Figure 3]g but not in astrocytes or microglia [Figure 3]i, [Figure 3]j, [Figure 3]k and [Figure 3]m, [Figure 3]n, [Figure 3]o. The percentage of neurons [Figure 3]h, but not astrocytes [Figure 3]l or microglia [Figure 3]p that expressed p-SGK1 was increased at day 7 after SMIR.

Figure 3: p-SGK1 was upregulated in spinal dorsal horn neurons after SMIR. (a-d) Compared to the sham group, p-SGK1 optical density in the spinal dorsal horn was significantly increased at day 7 and day 14 after SMIR. (e-p) p-SGK1 was located in NeuN-marked neurons (e-h) and to a much lesser extent in astrocytes (i-l) and microglia (m-p) in the ipsilateral L3/L4 spinal dorsal horn 7 days after SMIR. ***P < 0.001, ****P < 0.0001 compared with the sham group. a-c: Scale bar = 500 μm. e-g, i-k, m-o: Scale bar = 200 μm. p-SGK1: Phosphorylated serum- and glucocorticoid-inducible kinase-1, SMIR: Skin/muscle incision and retraction.

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Intrathecal injection of PD98059 reduced skin/muscle incision and retraction-induced mechanical allodynia and decreased the levels of TNF-α, lactate and p-SGK1 in the spinal dorsal horn following SMIR

Having demonstrated that spinal p-ERK and p-SGK1 were increased following SMIR, to determine the relationship between ERK and SGK1 signals in CPSP, we next investigated whether inhibition of the ERK pathway could alleviate the established mechanical allodynia by inhibiting SGK-1 phosphorylation. To determine the role of p-ERK in the L3/L4 spinal cord in SMIR-induced allodynia, a selective inhibitor of MEK (ERK kinase), PD98059 (10 μl of a 37.41 μM solution) was intrathecally injected at day 7 after SMIR surgery. Behavioral data showed that PD98059 treatment on day 7 reduced SMIR-induced mechanical allodynia [Figure 4]a. At 0.5 h after PD98059 treatment, the 50% paw withdrawal threshold in the ipsilateral hind paw remained at a low level, which was comparable to the predrug treatment, whereas 3 h after PD98059 treatment, the 50% paw withdrawal threshold was significantly enhanced. The increasing effect of PD98059 disappeared at 24 h after PD98059 injection. Intrathecal injection of the vehicle yielded no obvious effect on the 50% paw withdrawal threshold at any time point that was assessed. Immunofluorescence staining revealed that 3 h after PD98059 treatment, the phosphorylation of SGK-1 was decreased [Figure 4]b, [Figure 4]c, [Figure 4]d. A previous study showed that the release of proinflammatory mediators, including TNF-α and lactate, occurred during pain development.^[17] Consistently, the present study showed by ELISA that compared to the sham group, the level of TNF-α and lactate was significantly increased at day 7 following SMIR, whereas treatment with PD98059 substantially decreased the level of TNF-α and lactate in the spinal dorsal horn [Figure 4]e and [Figure 4]f.

Figure 4: Intrathecal injection of PD98059 reduced mechanical allodynia and decreased the density of p-SGK1 and the levels of TNF-α and lactate in the spinal dorsal horn in SMIR rats. (a) At Day 7 after SMIR, when the 50% paw withdrawal threshold was decreased, intrathecal injection of PD98059 (10 μl of a 37.41 μM solution) reduced SMIR-induced mechanical allodynia. The changes in the 50% paw withdrawal threshold at 0.5, 3 and 24 h after PD98059 treatment are shown. (b-d) The density of p-SGK1 in the spinal dorsal horn was decreased 3 h after PD98059 treatment. (e and f) The levels of TNF-α and lactate in the sham, vehicle-treated SMIR and PD98059 treated SMIR groups are shown. *P < 0.05, ***P < 0.001 compared with the vehicle group. Scale bar = 100 μm. TNF-α: Tumor necrosis factor α, p-SGK1: Phosphorylated serum- and glucocorticoid-inducible kinase-1, SMIR: Skin/muscle incision and retraction.

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Inhibition of SGK1 inhibited mechanical allodynia and decreased the levels of TNF-α and lactate in the spinal dorsal horn following skin/muscle incision and retraction

To determine the role of SGK1 phosphorylation in the L3/L4 spinal cord in SMIR-induced allodynia, a selective inhibitor of SGK1 GSK650394 (10 μl of a 30 μM solution) was intrathecally injected at day 7 after SMIR surgery. Behavioral data showed that GSK650394 treatment on day 7 reduced SMIR-induced mechanical allodynia [Figure 5]a. Compared to the predrug level, the 50% paw withdrawal threshold in the ipsilateral hind paw was significantly enhanced at 3 h but not at 0.5 h after GSK650394 treatment and had returned to the predrug level at 24 h. Intrathecal injection of vehicle had no obvious effect on the 50% paw withdrawal threshold at any time points that was assessed. The ELISA results showed that the levels of TNF-α and lactate in the spinal dorsal horn were significantly decreased by GSK650394 [Figure 5]b and [Figure 5]c.

Figure 5: Intrathecal injection of GSK650394 reduced mechanical allodynia and decreased the density of p-SGK1 and the levels of TNF-α and lactate in the spinal dorsal horn following SMIR. (a) At Day 7 after SMIR, when the 50% paw withdrawal threshold had been decreased, intrathecal injection of GSK650394 (10 μl of a 30 μM solution) reduced SMIR-induced mechanical allodynia. The changes in the 50% paw withdrawal threshold at 0.5, 3, and 24 h after GSK650394 treatment are shown. (b and c) The levels of TNF-α and lactate in the sham, vehicle-treated SMIR and SGK650394-treated SMIR groups are shown. *P < 0.05 compared with the vehicle group. p-SGK1: Phosphorylated serum- and glucocorticoid-inducible kinase-1, SMIR: Skin/muscle incision and retraction, TNF-α: Tumor necrosis factor α.

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Discussion

In the present study, we found that both p-ERK1/2 and p-SGK1 were upregulated in the spinal dorsal horn during the development of CPSP after SMIR of the thigh. Moreover, intrathecal injection of either the ERK inhibitor PD98059 or the SGK1 inhibitor GSK650394 alleviated CPSP and decreased the level of TNF-α and lactate in the spinal dorsal horn at day 7 after SMIR. Furthermore, PD98059 decreased the activation of SGK1. These results indicated that TNF-α and lactate release following ERK1/2-SGK1 activation in the spinal dorsal horn contributes to CPSP.

Different from the chronic pain induced by nerve injury or inflammation, SMIR, akin to a clinical procedure, probably cause pain state by prolonged tissue retraction, which induces a large amount of ATP release into the spinal cord to activate the P2X7R and TNF-α upregulation.^[13] CPSP is thought to be associated with peripheral and central inflammatory states.^[25],[26] Our study found that SMIR increased the level of two proinflammatory factors, TNF-α and lactate, in the spinal dorsal horn of rats. In the neurons of the spinal cord or cortex, TNF-α can act on the presynaptic or postsynaptic receptors of sensory neurons, upregulating the release of presynaptic transmitters and increasing the frequency or amplitude of excitatory postsynaptic currents (EPSCs).^[27],[28] Similarly, in addition to being an energy source for neurons, lactate can also act as a neural function modulator, which can increase the firing frequency and EPSCs of central neurons.^[20] These studies suggest the possibility of central sensitization mechanisms of proinflammatory factors during CPSP. Lactate can further activate glial cells to release pro-inflammatory cytokines under pathological conditions, including TNF-α, IL-1, and IL-6.^[29] It has also been reported that TNF-α, as a leading cytokine, can induce the expression of IL-1, IL-6.^[30] It should be noted that many other proinflammatory factors may also participate in the development of CPSP. For example, IL-1 levels have been found to be elevated in the spinal cord or hippocampus during CPSP.^[31],[32] Further studies are needed to elucidate their roles on CPSP.

In the present study, inhibition of ERK1/2 phosphorylation decreased the level of TNF-α and lactate and attenuated mechanical hyperalgesia, which is consistent with previous findings.^[33],[34]

Ubiquitously expressed SGK1 supports cellular glucose uptake and glycolysis, angiogenesis, cell survival, cell migration, and wound healing. The activation of SGK1 is involved in various signaling cascades, including 3-phosphoinositide-dependent kinase1, phosphatidylinositide-3-kinase, and mammalian target of rapamycin.^[35] Previously, it has been reported that the mRNA and protein levels of SGK1 are upregulated by several types of cell stress, including ischemia, radiation, hyperosmotic shock, and various pathological pain models.^[12] Inhibition of SGK1 has been demonstrated to alleviate morphine-induced analgesia tolerance and the development of pathological pain.^[12],[36] Consistently, in this study, we showed that SMIR of the thigh-induced SGK1 activation in the spinal dorsal horn. Once activated, SGK1 can regulate several channels and transporters, including calcium, chlorine and potassium channels, Na⁺/K⁺-ATPase, the Na⁺-K⁺-2Cl^- cotransporter, the NaCl cotransporter, Na⁺/H⁺ exchangers and diverse amino acid transporters, all of which are well known to play important roles in action potential generation. Thus, SGK1 activation presumably facilitates more action potential production by providing more energy to the pain signal projecting cells in the dorsal horn.^[37]

Furthermore, SGK1 influences transcription factors such as nuclear factor kappa-B (NF-κB), p53 tumor-suppressor protein, cAMP-responsive element-binding protein (CREB), activator protein-1 (AP-1), and forkhead box O3 protein (FOXO3a). Based on previous studies, our findings that SGK1 was upregulated in spinal cord neurons and that inhibition of SGK1 attenuated the established postsurgical pain were rational.

In the present study, we found that inhibition of SGK1 activation decreased the level of TNF-α and lactate and PD98059 inhibited the activation of SGK1. Moreover, p-ERK is colocalized with neurons and astrocytes, and pSGK is mainly colocalized with neurons. It is high likely that SGK is directly activated by p-ERK pathway in neurons. Lactate has been shown to be released by neurons during neuronal activity. Both neurons and astrocytes may increase glycolysis after stimulation, and neurons do not rely on import of astrocytic-produced lactate. Instead, they increase their own glycolytic rate and become net exporters of lactate.^[38] Double immunofluorescence staining showed that TNF protein is localized in spinal neurons after ventral root injury and TNF mRNA is upregulated in spinal cord neurons during coronary artery occlusion.^[39] Thus, it is highly possible that neurons can release lactate and TNF under pathological states, and combined with the results of this study, their release may be regulated by the ERK-SGK1 pathway. In addition, ERK activation in astrocytes can lead to transcription of inflammatory factors, such as excitatory amino acids, cytokines, and ATP.^[40],[41] These mediators from astrocytes can subsequently lead to SGK1 activation in the surrounded neurons. Further studies are needed to clarify whether these hypothesis are correct.

In the present study, SGK1 was activated in the spinal dorsal horn, which is remote from the thigh that underwent incision and retraction. The mechanisms for the activation, however, might be multiple. Consistent with its name, SGK1 is sensitive to glucocorticoids and is regulated by a wide variety of stimuli, including cytokines and growth factors, all of which have been repeatedly shown to play critical roles in neuropathic pain.^[42],[43] For example, IL-6, an important proinflammatory cytokine that is essential to neuropathic pain, can activate SGK1 via the p38 MAPK signaling pathway.^[44],[45] MAPK is also activated following cytokine release. Many kinds of proinflammatory cytokines, such as TNF-α, IL-1, IL-6, and IL-33, can promote the ERK pathway in spinal cord glial cells, thus causing the release of more inflammatory factors and maintaining pathological pain.^{[46],[47],[48]} Thus, there might be a positive loop between proinflammatory cytokines and ERK1/2-SGK1 phosphorylation; that is, increased proinflammatory mediators could activate ERK1/2-SGK1, and conversely, ERK1/2 and SGK1 activation could lead to increased production of proinflammatory mediators. Interrupting this positive loop in time could alleviate pain and therefore would do benefit those who have received surgeries and are prone to develop chronic pain.

Conclusion

In conclusion, the activation of the ERK1/2-SGK1 pathway in the spinal dorsal horn after SMIR of the thigh contributed to the maintenance of CPSP via TNF-α and lactate.

Financial support and sponsorship

This work was supported by grants from the Characteristic Innovation Program (Natural Science) of Guangdong Education Department (2018GKTSCX077) and Shenzhen Polytechnic Research Fund (6023310001K).

Conflicts of interest

There are no conflicts of interest.

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