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. 2024 Feb;12(2):e1132.
doi: 10.1002/iid3.1132.

Silencing of secreted phosphoprotein 1 attenuates sciatic nerve injury-induced neuropathic pain: Regulating extracellular signal-regulated kinase and neuroinflammatory signaling pathways

Haiyu Xie  1 , Feng Lu  1 , Xiaoling Li  1 , Enfu Wang  1 , Jiao Mo  1 , Weidong Liang  1
Affiliations

Silencing of secreted phosphoprotein 1 attenuates sciatic nerve injury-induced neuropathic pain: Regulating extracellular signal-regulated kinase and neuroinflammatory signaling pathways

Haiyu Xie et al. Immun Inflamm Dis. 2024 Feb.

Abstract

Background: Neuropathic pain (NP) is a chronic pathological pain that affects the quality of life and is a huge medical burden for affected patients. In this study, we aimed to explore the effects of secreted phosphoprotein 1 (SPP1) on NP.

Methods: We established a chronic constriction injury (CCI) rat model, knocked down SPP1 via an intrathecal injection, and/or activated the extracellular signal-regulated kinase (ERK) pathway with insulin-like growth factor 1 (IGF-1) treatment. Pain behaviors, including paw withdrawal threshold (PWT), paw withdrawal latency (PWL), lifting number, and frequency, were assessed. After sacrificing rats, the L4-L5 dorsal root ganglion was collected. Then, SPP1 levels were determined using quantitative polymerase chain reaction (qPCR) and western blot analysis. The levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-10, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)-β were determined using qPCR and enzyme-linked immunosorbent assay. The levels of ERK pathway factors were determined via western blot analysis.

Results: We found that CCI decreased PWT and PWL, increased the lifting number and frequency, and upregulated SPP1 levels. The loss of SPP1 reversed these CCI-induced effects. Additionally, CCI upregulated IL-1β, TNF-α, IL-6, EGF, and VEGF levels, downregulated TGF-β levels, and activated the ERK pathway, while silencing of SPP1 abrogated these CCI-induced effects. Moreover, IGF-1 treatment reversed the effects of SPP1 loss.

Conclusions: The data indicate that silencing SPP1 attenuates NP via inactivation of the ERK pathway, suggesting that SPP1 may be a promising target for NP treatment.

Keywords: ERK pathway; SPP1; neuropathic pain; sciatic nerve injury.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Secreted phosphoprotein 1 (SPP1) expression is upregulated in the chronic constriction injury (CCI) rat model. (A) Paw withdrawal threshold, (B) paw withdrawal latency, (C) paw lifting number, and (D) frequency were determined after CCI operation for 0, 1, 3, 5, 7, and 14 days. SPP1 levels were determined via (E) quantitative polymerase chain reaction and (F) western blot. *p < .05; **p < .01; ***p < .001.
Figure 2
Figure 2
Knockdown secreted phosphoprotein 1 (SPP1) relieves neuropathic pain in chronic constriction injury (CCI)‐induced rats. (A) Levels were determined via quantitative polymerase chain reaction following transfection. (B) Paw withdrawal threshold, (C) paw withdrawal latency, (D) paw lifting number, and (E) frequency were measured after SPP1 knockdown. **p < .01; ## p < .01.
Figure 3
Figure 3
Depletion of secreted phosphoprotein 1 (SPP1) alleviates the inflammation response. The levels of inflammatory factors, interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, IL‐6, and IL‐10, were determined via (A) enzyme‐linked immunosorbent assay and (B) quantitative polymerase chain reaction. **p < .01; ## p < .01.
Figure 4
Figure 4
Loss of secreted phosphoprotein 1 (SPP1) inhibits angiogenesis induced by chronic constriction injury (CCI). The levels of epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)‐β were determined via (A) enzyme‐linked immunosorbent assay and (B) quantitative polymerase chain reaction. **p < .01; ## p < .01.
Figure 5
Figure 5
Knockdown secreted phosphoprotein 1 (SPP1) inactivates the extracellular signal‐regulated kinase (ERK) pathway. (A) Serine/threonine kinase (AKT), mitogen‐activated protein kinase (MEK), ERK, p‐AKT, p‐MEK, and p‐ERK protein levels were determined via western blot. (B) The ratios of p‐ERK/ERK, p‐AKT/AKT, and p‐MEK/MEK were quantified. **p < .01; ## p < .01.
Figure 6
Figure 6
Silencing of secreted phosphoprotein 1 (SPP1) relieves neuropathic pain by regulating the extracellular signal‐regulated kinase (ERK) pathway. (A) Paw withdrawal threshold, (B) paw withdrawal latency, (C) paw lifting number, and (D) frequency were measured following SPP1 knockdown and insulin‐like growth factor 1 treatment. **p < .01; ## p < .01; # p < .05.
Figure 7
Figure 7
Silencing of secreted phosphoprotein 1 (SPP1) alleviates inflammation via the extracellular signal‐regulated kinase (ERK) pathway. (A) The protein levels of AKT, MEK, ERK, p‐AKT, p‐MEK, and p‐ERK was examined using western blot. The interleukin (IL)‐1β, tumor necrosis factor (TNF)‐α, and IL‐6 levels were tested by (B) enzyme‐linked immunosorbent assay and (C) quantitative polymerase chain reaction. **p < .01; ## p < .01.
Figure 8
Figure 8
Loss of secreted phosphoprotein 1 (SPP1) suppresses angiogenesis via the extracellular signal‐regulated kinase (ERK) pathway. (A) Enzyme‐linked immunosorbent assay and (B) quantitative polymerase chain reaction were conducted to determine the epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and transforming growth factor (TGF)‐β levels. **p < .01; ## p < .01; # p < .05.
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