Introduction: Upstream stimulating factor 2 (USF2) belongs to basic Helix-Loop-Helix-Leucine zipper transcription factor family, regulating expression of genes involved in immune response or energy metabolism network. Role of USF2 in neuropathic pain was evaluated. Methods: Mice were intraspinally injected with adenovirus for knockdown of USF2 (Ad-shUSF2) and then subjected to spinal nerve ligation (SNL) to induce neuropathic pain. Distribution and expression of USF2 were detected by western blot and immunofluorescence. Mechanical and thermal pain sensitivity were examined by paw withdrawal thresholds (PWT) and paw withdrawal latency (PWL). Chromatin immunoprecipitation (ChIP) and luciferase activity assays were performed to detect binding ability between USF2 and SNHG5. Results: The expression of USF2 was elevated and colocalized with astrocytes and microglia in L5 dorsal root ganglion (DRG) of SNL-induced mice. Injection of Ad-shUSF2 attenuated SNL-induced decrease of PWT and PWL in mice. Knockdown of USF2 increased the level of IL-10 but decreased TNF-α, IL-1β, and IL-6 in SNL-induced mice. Silence of USF2 enhanced protein expression of CD206 while reducing expression of CD16 and CD32 in SNL-induced mice. USF2 binds to promoter of SNHG5 and weakens SNL-induced up-regulation of SNHG5. SNHG5 binds to miR-181b-5p, and miR-181b-5p to interact with CXCL5. Conclusion: Silence of USF2 ameliorated neuropathic pain, suppressed activation of M1 microglia, and inhibited inflammation in SNL-induced mice through regulation of SNHG5/miR-181b-5p/CXCL5 axis. Therefore, USF2/SNHG5/miR-181b-5p/CXCL5 might be a promising target for neuropathic pain. However, the effect of USF2/SNHG5/miR-181b-5p/CXCL5 on neuropathic pain should also be investigated in further research.

Neuropathic pain is a debilitating and refractory disease in the nervous system and characterized by allodynia, hyperalgesia, and spontaneous pain [1]. Surgery or diseases can induce peripheral nerve injury and result in abnormal chronic pain and pain hypersensitivity reaction during development of neuropathic pain, thus adversely affecting the quality of life of patients [2]. Limited therapies are available for neuropathic pain due to incomplete understanding of pathogenesis and mechanisms [3]. Dysregulation of genes in the dorsal root ganglion (DRG) was regarded as molecular basis of development of neuropathic pain [4]. Therefore, identification of dysregulated genes might facilitate the diagnosis and treatment of neuropathic pain.

Upstream stimulating factors (USFs) include USF1 and USF2 and belong to member of basic Helix-Loop-Helix-Leucine zipper transcription factor family [5]. USFs interact with cognate E-box regulatory elements and regulate expression of genes involved in lipid and glucose metabolism, cell proliferation and cycle, stress, and immune responses [5]. USF2 was aberrantly expressed in refractory rheumatoid arthritis and promoted secretion of IL-17A, IFN-γ, IL-22 in Th17 cells [6]. USF2 regulated inflammatory responses in endometritis through binding with promoter of TREM1 [7]. Moreover, study has shown that USF2 bind to E-box control element in the promoter region of neuron-specific K-Cl cotransporter and might mediate physiological process in developing brain [8]. USF2 was also up-regulated in brain of aging mice [9]. USF2/CEACAM network was involved in development of the spinal cord through analysis of mouse single-cell sequencing dataset [10]. However, the role of USF2 in neuropathic pain remains unknown.

MiRNAs were dysregulated in neuropathic pain, modulating genes involved in neuroinflammation and pain and mediating development of neuropathic pain [11]. MiRNAs were considered to be biomarkers and potential therapeutic targets of neuropathic pain [12]. For example, miR-23a regulated TXNIP/NLRP3 inflammasome in spinal glial cells through targeting CXCR4 and ameliorated SNL-induced neuropathic pain [13]. USF2 binds to promoter of miR-34a, reducing expression of BMP3 to promote osteogenic differentiation of BMSCs [14]. Therefore, USF2 might mediate miRNAs expression during the development of neuropathic pain.

In this study, expression of USF2 in SNL-induced mice with neuropathic pain was first investigated by western blot and immunofluorescence. Role of USF2 on neuropathic pain was evaluated through the injection of Ad-shUSF2 and assessed via mechanical and thermal pain sensitivity, microglia activation, and neuroinflammation. The downstream target of USF2 was also assessed with ChIP and luciferase activity assays. The results might provide a novel therapeutic target for treating neuropathic pain.

Animal Model

Male C57BL/6 mice, aged 6–8 weeks and weighed 20–22 g (N = 150), were acquired from SLAC Laboratory (Shanghai, China). Experiments were approved by the Experimental Animal Ethics Committee of Guizhou Medical University (Approval No.: 2201438). Mice were divided into two groups: sham (N = 10) and SNL (N = 140). Mice in the SNL group were anesthetized and surgically exposed to the transverse lumbar process. Transverse lumbar process was removed, and L5 spinal nerve was ligated with silk 4–0 thread. The layers of muscle and skin were then closed. Mice in the sham group received the same operation without ligation of the L5 spinal nerve. Two days later, mechanical and thermal pain tests were performed, and L5 DRG was removed for functional assays.

Mice in the SNL group were divided into 8 groups: sham with Ad-GFP, sham with Ad-shUSF2, SNL with Ad-GFP, SNL with Ad-shUSF2, sham with NC agomir, sham with miR-181b-5p agomir, SNL with NC agomir, SNL with miR-181b-5p agomir, with 15 mice in each group. Ad-GFP, Ad-shUSF2, NC agomir, and miR-181b-5p agomir were purchased from Invitrogen (Carlsbad, CA, USA). For intraspinal injection of Ad-GFP and Ad-shUSF2, mice were anesthetized and received hemilaminectomy at L1-L2 vertebral segments. A glass micropipette was used to inject 20 μL Ad-GFP or Ad-shUSF2 (l × 1011 pfu/mL) into L5 DRG daily for 3 consecutive days. Mice were then subjected to SNL surgery. For intrathecal injection of NC agomir or miR-181b-5p agomir, mice were held firmly, and a 30-gauge needle attached to a microsyringe was inserted into region between the L5 and L6 vertebrae. A total of 10 μL NC agomir or miR-181b-5p agomir (20 μm) were also injected into mice daily for 3 consecutive days. The mice were also subjected to SNL surgery.

qRT-PCR

L5 DRG was lysed in Trizol (Invitrogen) or miRcute miRNA isolation kit (Tiangen, Beijing, China) to isolate total RNAs or miRNAs, respectively. RNAs were then reverse-transcribed into cDNAs and subjected to SYBR Green Master (Roche, Mannheim, Germany) to determine expression of USF2, SNHG5, miR-181b-5p, and CXCL5. GAPDH and U6 were used as endogenous controls. The primer sequences are showed in Table 1.

Table 1.

Primers for qRT-PCR

GenesForward (5′-3′)Reverse (5′-3′)
GAPDH ACC​ACA​GTC​CAT​GCC​ATC​AC TCC​ACC​ACC​CTG​TTG​CTG​TA 
USF2 CTG​TGA​TCC​AAA​ATC​CCT​TCA​GC GGT​CTG​TGG​TCT​GTA​CGG​AC 
SNHG5 GCC​ATT​GTT​CTT​CGC​GTC​TT TCT​CAC​TGG​TCA​GCA​TTC​ACC 
miR-181b-5p CAA​TCA​ACA​TTC​ATT​GCT​GTC​GG GGC​CAC​AGT​TGC​ATT​CAT​TGT​T 
CXCL5 GCA​TTT​CTG​CTG​CTG​TTC​ACA​CT GGT​TAA​GCA​AAC​ACA​GCG​TAG​CT 
U6 CTCGCTTCGGCAGCACA AAC​GCT​TCA​CGA​ATT​TGC​GT 
GenesForward (5′-3′)Reverse (5′-3′)
GAPDH ACC​ACA​GTC​CAT​GCC​ATC​AC TCC​ACC​ACC​CTG​TTG​CTG​TA 
USF2 CTG​TGA​TCC​AAA​ATC​CCT​TCA​GC GGT​CTG​TGG​TCT​GTA​CGG​AC 
SNHG5 GCC​ATT​GTT​CTT​CGC​GTC​TT TCT​CAC​TGG​TCA​GCA​TTC​ACC 
miR-181b-5p CAA​TCA​ACA​TTC​ATT​GCT​GTC​GG GGC​CAC​AGT​TGC​ATT​CAT​TGT​T 
CXCL5 GCA​TTT​CTG​CTG​CTG​TTC​ACA​CT GGT​TAA​GCA​AAC​ACA​GCG​TAG​CT 
U6 CTCGCTTCGGCAGCACA AAC​GCT​TCA​CGA​ATT​TGC​GT 

Western Blot

L5 DRG was lysed in RIPA buffer (Beyotime, Beijing, China) to isolate protein samples. Samples were separated by SDS-PAGE and then electro-transferred onto PVDF membrane. Membranes were blocked in 5% BSA and probed with primary antibodies: anti-USF2 and anti-β-actin (1:2,000), anti-CD206 and anti-CD16 (1:3,000), anti-CD32, and anti-CXCL5 (1:4,000). After incubation with horseradish peroxidase-labeled secondary antibody (1:5,000), membranes were subjected to enhanced chemiluminescence (KeyGen Biotech). Immunoreactivities were then detected and analyzed by ImageJ software. All the proteins were purchased from Abcam (Cambridge, MA, USA).

Immunofluorescence

L5 DRG was fixed with 10% formalin and embedded in paraffin. Tissues were cut into 4-µm sections, and the sections were treated with 0.5% Triton-100. Sections were immersed in Tris-EDTA buffer containing 0.05% Tween 20 and blocked with 5% BSA. Sections were incubated overnight with anti-USF2 (1:100; Abcam), anti-GFAP (1:80; Abcam), or anti-IBA1 (1:80; Abcam). Sections were then treated with secondary antibody, which were then stained with DAPI and examined under fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Mechanical and Thermal Pain Sensitivity

Mice were placed in plexiglass boxes, and von Frey hairs with increasing stiffness (1–30 g) attached to Electronic Von Frey (IITC Life Science Inc., Woodland Hill, CA, USA) were used to stimulate pressure on the plantar surface of the hind paw. PWT was calculated as maximum value (g) when the paws of mice were released. Mice received the test 5 times with intervals of 3 min per day for 15 consecutive days. Mean value of five repetitions was counted as PWT. For thermal pain sensitivity analysis, an infrared beam was used to stimulate heat on the plantar surface of the hind paw. A 20-s cutoff was used to eliminate tissue damages. PWL was calculated as time passed from initiation of stimuli to paw withdrawal. Mice also received the test five times with intervals of 5 min per day for 15 consecutive days. Mean value of five repetitions was counted as PWL.

ELISA

L5 DRG was lysed in RIPA buffer and then centrifuged at 12,000 g for 60 min. Supernatants were harvested, and levels of TNF-α, IL-1β, IL-6, and IL-10 were determined by ELISA kits (Cwbiotech Beijing, China).

ChIP

HEK293 was fixed in 0.75% paraformaldehyde for 15 min and then lysed in lysis buffer. Cell lysates were subjected to ultrasound to isolate DNA fragments from 200 to 1,000 bp. Anti-IgG and anti-USF2 were incubated with the lysates, and QIAquick Gel Extraction Kit (Qiagen) was used to precipitate and purify the chromatin fragments. The purified DNAs were performed with PCR using following primers: (primer 1: Forward: 5′-GCC​AGC​TTT​AAC​AAA​ACG​GT-3′ and Reverse: 5′-CCT​AGA​ATA​GGG​CCA​GGC​A-3′; primer 2: Forward: 5′-GCC​TAT​TGT​CCC​AAC​ATC​TGT​G-3′ and Reverse: 5′-AGT​GGC​ACA​AGA​TGC​CAA​GG-3′; primer 3: Forward: 5′-GCC​ATG​ATT​GCA​CCT​CTG​TC-3′ and Reverse: 5′-TTT​TAG​CCT​GGT​CTG​TTG​CC-3′).

Dual Luciferase Reporter Assay

Sequence of promoter region in SNHG5 was subcloned into pmirGLO vector (Promega, Madison, WI, USA). HEK293 was cotransfected with pmirGLO-SNHG5 and shUSF2, shNC, pcDNA-USF2 (USF2), or pcDNA vector (NC). Cells were subjected to Lucifer Reporter Assay System (Promega) to detect the luciferase activities 2 days later. Sequences of wildtype or mutant 3′-UTR of SNHG5 or CXCL5 were also subcloned into pmirGLO vector. HEK293 was cotransfected with pmirGLO vectors and miR-181b-5p mimic or NC mimic. The luciferase activities were also determined 2 days later.

RIP

Cell lysates of HEK293 were collected and treated overnight with protein G sepharose beads (GE Healthcare, Eindhoven, The Netherlands) coated with anti-IgG or anti-AGO2 antibodies. The precipitates were subjected to qRT-PCR to detect expression of SNHG5 and miR-181b-5p.

Statistical Analysis

All the data were expressed as mean ± SEM and analyzed via the Student’s t test or one-way analysis of variance in GraphPad Prism software. P < 0.05 was considered statistically significant.

USF2 Was Elevated in Spinal Cord of SNL-Induced Mice

To induce neuropathic pain, mice were subjected to SNL operation. Levels of PWT and PWL were reduced in SNL-induced mice (Fig. 1a). Expression of USF2 was up-regulated in L5 DRG of SNL-induced mice compared to the sham-operated mice (Fig. 1b, c). USF2 was colocalized with GFAP and IBA1 (Fig. 1d), suggesting that elevated USF2 in both astrocytes and microglia in the spinal cord might regulate the development of neuropathic pain.

Fig. 1.

USF2 was elevated in the spinal cord of SNL-induced mice. a Levels of PWT and PWL were reduced in SNL-induced mice. b mRNA expression of USF2 was up-regulated in L5 DRG of SNL-induced mice. c Protein expression of USF2 was up-regulated in L5 DRG of SNL-induced mice. The original figures of WB are listed in the supplementary file. d Immunofluorescence analysis showed that USF2 was colocalized with GFAP and IBA1 in L5 DRG of SNL-induced mice. **versus sham, p < 0.01. The detailed p value is listed in the supplementary p value file.

Fig. 1.

USF2 was elevated in the spinal cord of SNL-induced mice. a Levels of PWT and PWL were reduced in SNL-induced mice. b mRNA expression of USF2 was up-regulated in L5 DRG of SNL-induced mice. c Protein expression of USF2 was up-regulated in L5 DRG of SNL-induced mice. The original figures of WB are listed in the supplementary file. d Immunofluorescence analysis showed that USF2 was colocalized with GFAP and IBA1 in L5 DRG of SNL-induced mice. **versus sham, p < 0.01. The detailed p value is listed in the supplementary p value file.

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Knockdown of USF2 Ameliorated Neuropathic Pain in SNL-Induced Mice

To investigate the role of USF2 in neuropathic pain, intraspinal injection of Ad-shUSF2 was performed in mice. Injection of Ad-shUSF2 reduced expression of USF2 in the spinal cord (Fig. 2a). Silence of USF2 attenuated mechanical allodynia and heat hyperalgesia in SNL-induced mice through up-regulation of PWT and PWL (Fig. 2b).

Fig. 2.

Knockdown of USF2 ameliorated neuropathic pain in SNL-induced mice. a Intraspinal injection of Ad-shUSF2 reduced expression of USF2 in the spinal cord. The original figures of WB are listed in the supplementary file. b Intraspinal injection of Ad-shUSF2 attenuated SNL-induced decrease of PWT and PWL in mice. **versus sham + Ad-GFP, p < 0.01. #, ##versus SNL + Ad-GFP, p < 0.05, p < 0.01. &, &&versus sham + Ad-USF2, p < 0.05, p < 0.01. The detailed p value is listed in the supplementary p value file.

Fig. 2.

Knockdown of USF2 ameliorated neuropathic pain in SNL-induced mice. a Intraspinal injection of Ad-shUSF2 reduced expression of USF2 in the spinal cord. The original figures of WB are listed in the supplementary file. b Intraspinal injection of Ad-shUSF2 attenuated SNL-induced decrease of PWT and PWL in mice. **versus sham + Ad-GFP, p < 0.01. #, ##versus SNL + Ad-GFP, p < 0.05, p < 0.01. &, &&versus sham + Ad-USF2, p < 0.05, p < 0.01. The detailed p value is listed in the supplementary p value file.

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Knockdown of USF2 Suppressed Activation of Microglia and Neuroinflammation

Immunofluorescence analysis showed that the distribution of USF2 in astrocytes and microglia was inhibited by intraspinal injection of Ad-shUSF2 (Fig. 3a). Loss of USF2 increased protein expression of CD206 while decreasing CD16 and CD32 expression in the spinal cord of SNL-induced mice (Fig. 3b), thus driving microglia polarization to M2 phenotype. Level of IL-10 in SNL-induced mice was up-regulated, while TNF-α, IL-1β, and IL-6 were down-regulated by silence of USF2 (Fig. 3c), revealing anti-inflammatory effect of USF2 deficiency on neuropathic pain.

Fig. 3.

Knockdown of USF2 suppressed activation of microglia and neuroinflammation. a Intraspinal injection of Ad-shUSF2 reduced colocalization of USF2 with GFAP and IBA1 in L5 DRG of SNL-induced mice. b Intraspinal injection of Ad-shUSF2 increased protein expression of CD206, while decreased CD16 and CD32 expression in the spinal cord of SNL-induced mice. The original figures of WB are listed in the supplementary file. c Intraspinal injection of Ad-shUSF2 increased level of IL-10, while decreased TNF-α, IL-1β, and IL-6 in SNL-induced mice. **versus sham + Ad-GFP, p < 0.01. #, ##versus SNL + Ad-GFP, p < 0.05, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

Fig. 3.

Knockdown of USF2 suppressed activation of microglia and neuroinflammation. a Intraspinal injection of Ad-shUSF2 reduced colocalization of USF2 with GFAP and IBA1 in L5 DRG of SNL-induced mice. b Intraspinal injection of Ad-shUSF2 increased protein expression of CD206, while decreased CD16 and CD32 expression in the spinal cord of SNL-induced mice. The original figures of WB are listed in the supplementary file. c Intraspinal injection of Ad-shUSF2 increased level of IL-10, while decreased TNF-α, IL-1β, and IL-6 in SNL-induced mice. **versus sham + Ad-GFP, p < 0.01. #, ##versus SNL + Ad-GFP, p < 0.05, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

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USF2 Transcriptionally Activated SNHG5

Expression of SNHG5 was elevated in the spinal cord of SNL-induced mice (Fig. 4a). The binding site between USF2 and SNHG5 was predicted by JASPAR (https://jaspar.genereg.net/) (Fig. 4b). ChIP assay confirmed that USF2 binds to promoter region of SNHG5 (Fig. 4c). Over-expression of USF2 increased luciferase activity, and silence of USF2 reduced the luciferase activity of pmirGLO-SNHG5 (Fig. 4d). Intraspinal injection of Ad-shUSF2 attenuated SNL-induced increase of SNHG5 (Fig. 4e), demonstrating that USF2 transcriptionally up-regulated SNHG5 in neuropathic pain.

Fig. 4.

USF2 transcriptionally activated SNHG5. a Expression of SNHG5 was elevated in the spinal cord of SNL-induced mice. b Potential binding site between USF2 and SNHG5 was predicted by JASPAR (https://jaspar.genereg.net/). c ChIP assay confirmed that USF2 binds to promoter region of SNHG5. d Over-expression of USF2 increased luciferase activity and silence of USF2 reduced the luciferase activity of pmirGLO-SNHG5. e Intraspinal injection of Ad-shUSF2 attenuated SNL-induced increase of SNHG5 in L5 DRG of SNL-induced mice. **versus sham, IgG or NC, p < 0.01. ##versus shNC or SNL + Ad-GFP, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

Fig. 4.

USF2 transcriptionally activated SNHG5. a Expression of SNHG5 was elevated in the spinal cord of SNL-induced mice. b Potential binding site between USF2 and SNHG5 was predicted by JASPAR (https://jaspar.genereg.net/). c ChIP assay confirmed that USF2 binds to promoter region of SNHG5. d Over-expression of USF2 increased luciferase activity and silence of USF2 reduced the luciferase activity of pmirGLO-SNHG5. e Intraspinal injection of Ad-shUSF2 attenuated SNL-induced increase of SNHG5 in L5 DRG of SNL-induced mice. **versus sham, IgG or NC, p < 0.01. ##versus shNC or SNL + Ad-GFP, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

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SNHG5 Binds to miR-181b-5p

miR-181b-5p was down-regulated in spinal cord of SNL-induced mice (Fig. 5a). ENCORI (https://starbase.sysu.edu.cn/) predicted binding site between SNHG5 and miR-181b-5p (Fig. 5b). RIP assay confirmed binding ability between SNHG5 and miR-181b-5p (Fig. 5c). Moreover, luciferase activity of pmirGLO-SNHG5-WT was reduced by over-expression of miR-181b-5p (Fig. 5d). Knockdown of USF2 weakened SNL-induced increase of SNHG5 and decrease of miR-181b-5p in the spinal cord of mice (Fig. 5e).

Fig. 5.

SNHG5 bind to miR-181b-5p. a miR-181b-5p was down-regulated in the spinal cord of SNL-induced mice. b ENCORI (https://starbase.sysu.edu.cn/) predicted binding site between SNHG5 and miR-181b-5p. c RIP assay confirmed binding ability between SNHG5 and miR-181b-5p. d Over-expression of miR-181b-5p decreased luciferase activity of pmirGLO-SNHG5-WT. e Intraspinal injection of Ad-shUSF2 weakened SNL-induced increase of SNHG5 and decrease of miR-181b-5p in the spinal cord of mice. **versus sham, anti-IgG or NC mimic, p < 0.01. ##versus shNC or SNL + Ad-GFP, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

Fig. 5.

SNHG5 bind to miR-181b-5p. a miR-181b-5p was down-regulated in the spinal cord of SNL-induced mice. b ENCORI (https://starbase.sysu.edu.cn/) predicted binding site between SNHG5 and miR-181b-5p. c RIP assay confirmed binding ability between SNHG5 and miR-181b-5p. d Over-expression of miR-181b-5p decreased luciferase activity of pmirGLO-SNHG5-WT. e Intraspinal injection of Ad-shUSF2 weakened SNL-induced increase of SNHG5 and decrease of miR-181b-5p in the spinal cord of mice. **versus sham, anti-IgG or NC mimic, p < 0.01. ##versus shNC or SNL + Ad-GFP, p < 0.01. &&versus sham + Ad-USF2, p < 0.01. The detailed p value is listed in the supplymentary p value file.

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miR-181b-5p Interacted with CXCL5

CXCL5 was up-regulated in the spinal cord of SNL-induced mice (Fig. 6a, b). Binding site between miR-181b-5p and CXCL5 was predicted by ENCORI (Fig. 6c) and confirmed by luciferase activity assay (Fig. 6d). Intrathecal injection of miR-181b-5p agomir enhanced miR-181b-5p expression compared to injection of NC agomir (Fig. 6e). Over-expression of miR-181b-5p reduced expression of CXCL5 in the spinal cord of SNL-induced mice (Fig. 6e, f).

Fig. 6.

miR-181b-5p interacted with CXCL5. a CXCL5 mRNA was up-regulated in the spinal cord of SNL-induced mice. b CXCL5 protein was up-regulated in the spinal cord of SNL-induced mice. c Potential binding site between miR-181b-5p and CXCL5 was predicted by ENCORI. d Over-expression of miR-181b-5p decreased luciferase activity of pmirGLO-CXCL5-WT. e Intrathecal injection of miR-181b-5p agomir enhanced miR-181b-5p expression and reduced CXCL5 in the spinal cord of SNL-induced mice. f Intrathecal injection of miR-181b-5p agomir reduced CXCL5 protein in the spinal cord of SNL-induced mice. **versus sham, NC agomir or NC mimic, p < 0.01. ##versus sham + miR-181b-5p agomir, p < 0.01. The original figures of WB are listed in the supplementary file. The detailed p value is listed in the supplymentary p value file.

Fig. 6.

miR-181b-5p interacted with CXCL5. a CXCL5 mRNA was up-regulated in the spinal cord of SNL-induced mice. b CXCL5 protein was up-regulated in the spinal cord of SNL-induced mice. c Potential binding site between miR-181b-5p and CXCL5 was predicted by ENCORI. d Over-expression of miR-181b-5p decreased luciferase activity of pmirGLO-CXCL5-WT. e Intrathecal injection of miR-181b-5p agomir enhanced miR-181b-5p expression and reduced CXCL5 in the spinal cord of SNL-induced mice. f Intrathecal injection of miR-181b-5p agomir reduced CXCL5 protein in the spinal cord of SNL-induced mice. **versus sham, NC agomir or NC mimic, p < 0.01. ##versus sham + miR-181b-5p agomir, p < 0.01. The original figures of WB are listed in the supplementary file. The detailed p value is listed in the supplymentary p value file.

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Peripheral nerve injury induces alteration in expression of genes in immune cells, glial, and sensory neurons [15]. Therefore, transcription factors, with the ability to regulate “genetic reprogramming,” are implicated in distinct processes in the development of neuropathic pain [15]. For example, octamer transcription factor 1 was elevated in L4/5 DRG of chronic constriction injury-induced rats with neuropathic pain [16]. Block of octamer transcription factor 1 improved morphine analgesia, ameliorated cold allodynia, heat hyperalgesia, and mechanical allodynia in rats with neuropathic pain [16]. This study first found that USF2 functioned as a transcription factor to stimulate expression of SNHG5, and loss of USF2 attenuated mechanical allodynia and heat hyperalgesia in SNL-induced mice with neuropathic pain.

USF2 was first identified to be up-regulated in L5 DRG of SNL-induced mice. Moreover, USF2 was colocalized with GFAP and IBA1 in L5 DRG, suggesting that USF2 was elevated in both astrocytes and microglia in the spinal cord of mice with neuropathic pain. Silence of USF2 reduced expression of endogenous CGRP RNA through down-regulation of mitogen-activated protein kinase regulation of the promoter of CGRP in trigeminal ganglion neurons [17]. Multiple pathways, such as WNT-β catenin signaling [18] and ERK signaling [19], important for kinase proliferation, differentiation, and survival of the neural cells, are implicated in the pathogenesis of neurodegenerative disease. Therefore, USF2 might regulate neuropathic pain through regulating MAPK signaling.

Previous study has shown that glia cells, including astrocytes and microglia, were activated in response to peripheral nerve damage [20]. Activation of glia released glutamate and substance P and facilitated pain signaling [21]. Activation of microglia stimulated secretion of growth factors, chemokines, and proinflammatory cytokines, including TNF-α, IL-1β, and IL-6, to trigger neuroinflammation, activation of astrocytes, and central sensitization, thus potentiating the development of neuropathic pain [22]. Moreover, glial also interacted with neurons and mediated nociceptive transmission in neuropathic pain [23]. Therefore, glia, especially activated microglia, was regarded as a potential target for the treatment of neuropathic pain [20, 22]. Dickkopf3 provoked transformation of pro-inflammatory M1 microglia into anti-inflammatory M2 microglia and suppressed neuroinflammation to alleviate neuropathic pain [24]. Intraspinal injection of Ad-shUSF2 in this study reduced the colocalization between USF2 and GFAP/IBA1 in SNL-induced mice. Furthermore, knockdown of USF2 increased expression of CD206, decreased CD16 and CD32 expression, and potentiated microglia polarization from M1 to M2. USF2 deficiency reduced proinflammatory cytokines, IL-22, IFN-γ, and IL-17A, to inhibit inflammation in refractory rheumatoid arthritis [6]. Our results showed that silence of USF2 increased production of anti-inflammatory factor IL-10 but decreased TNF-α, IL-1β, and IL-6 levels, thus repressing neuroinflammation in SNL-induced mice. These results demonstrated that USF2 deficiency attenuated neuropathic pain through suppression of M1 microglia polarization and neuroinflammation. Astrocytes were also implicated in the pathogenesis of neuropathic pain [25]. The role of USF2 in activation of astrocytes during neuropathic pain should be investigated in further research.

Non-coding RNAs, including lncRNAs and miRNAs were dysregulated in the spinal cord dorsal horn, DRG and damaged nerve in response to peripheral nerve injury [12, 26]. LncRNAs could regulate pro-inflammatory factors, signaling, receptors, miRNAs, and RNA-associated proteins, and participate in pain transmission during development of neuropathic pain [27]. Therefore, lncRNAs were identified as potential targets for treating neuropathic pain [28]. Previous study has shown that SNHG5 was significantly up-regulated in mouse SNL model, and knockdown of SNHG5 increased expression of miR-154-5p, decreased CXCL13, and inhibited activation of astrocytes and microglia, thus ameliorating neuropathic pain [29]. Here, SNHG5 was also found to be up-regulated in L5 DRG of SNL-induced mice in this study. USF2 binds to promoter region of SNHG5, and knockdown of USF2 reduced SNHG5 expression. SNHG5 interacted with miR-181a-5p and decreased the miRNA expression [30]. Our study confirmed that miR-181b-5p was a binding target of SNHG5. Moreover, down-regulation of miR-181b reduced PWT and PWL, promoted inflammation, and aggravated neuropathic pain [31]. Our results indicated that knockdown of USF2 increased miR-181b-5p expression in L5 DRG of SNL-induced mice. Therefore, USF2 deficiency might attenuate neuropathic pain through down-regulation of SNHG5 and up-regulation of miR-181b-5p. However, the effect of USF2/SNHG5/miR-181b-5p on neuropathic pain should be investigated in further research.

Emerging evidence has shown that chemokines contribute to neuroinflammation, activation of astrocytes, and central sensitization during development of neuropathic pain [22, 32]. CXCL5 was elevated in sciatic nerves of chronic constriction injury-induced neuropathic pain in rats [33]. CXCL5 induced nociceptive hypersensitivity and aggravated neuropathic pain [33], and blockage of CXCL5 alleviated the neuropathic pain [33]. CXCL5 was also up-regulated in L5 DRG of SNL-induced mice and identified as target gene of miR-181b-5p in this study. Intrathecal injection of miR-181b-5p agomir increased miR-181b-5p and decreased CXCL5 in mice with neuropathic pain.

Collectively, USF2 deficiency attenuated mechanical allodynia and heat hyperalgesia and inhibited microglial activation and neuroinflammation in SNL-induced mice. USF2 deficiency decreased SNHG5 to increase miR-181b-5p, thus reducing CXCL5. However, the effect of USF2/SNHG5/miR-181b-5p/CXCL5 on neuropathic pain should also be investigated in further research.

Ethical approval was obtained from the Experimental Animal Ethics Committee of Guizhou Medical University (Approval No.: 2201438).

The authors state that there are no conflicts of interest to disclose.

This work was supported by the Science and Technology Fund Project of Guizhou Provincial Health Commission (Grant No.: gzwkj2023-394), the colleges and universities Youth Science and technology talent development project of Guizhou Province (Grant No.: KY-[2022]236), and Innovative training program for college students (Grant No.: S202210660081).

Conceptualization, methodology, and writing – original draft were performed by Mi Chen; formal analysis, resources, and investigation were performed by Yang Yang; formal analysis, visualization, and data curation were performed by Jiatian Cui; project administration, supervision, and validation were performed by Li Qiu; and validation, supervision, and writing – review and editing were performed by Xiaohua Zou and Xianggang Zeng. All authors read and approved the final manuscript.

All data generated or analyzed during this study are included in this published article. The data that support the findings of this study are not publicly available due to privacy reasons but are available from the corresponding author upon reasonable request.

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