背景

复杂区域疼痛综合征（Complex regional pain syndrome, CRPS）是一种好发于四肢，以炎症反应为主要特征的慢性疼痛，严重影响患者的生活质量。大约7% 的骨折患者术后会进展成为CRPS，并伴有区域性自主神经功能紊乱及运动功能受损等，其中肢体的缺血再灌注（Ischemia-Reperfusion，I/R）损伤是重要机制之一。角质形成细胞作为非神经元细胞，是皮肤表皮的主要组成部分，可以“感知”局部氧化损伤信号参与炎症反应。研究证实Wnt信号通路的激活在神经病理性疼痛中起重要作用，但是其是否表达于皮肤角质形成细胞并参与CRPS尚未明确。本研究主要从CRPS外周敏化机制出发，通过建立体外氧糖剥夺/复氧细胞模型（Oxygen Glucose Deprivation/Reperfusion，OGD/R），模拟体外炎症反应特征，筛选Wnt信号通路参与CRPS局部氧化损伤的关键因子。通过构建大鼠肢体I/R损伤模型模拟CRPS患者临床病理特征，使用分子生物学手段和生物信息学手段探究皮肤角质形成细胞中Wnt5a参与CRPS外周敏化的潜在机制。

方法

    1)本研究首先通过大鼠皮肤提取原代角质形成细胞并构建OGD/R细胞模型，使用实时荧光定量PCR（Quantitative Real-time polymerase chain reaction，qPCR）方法筛选Wnt信号通路参与氧化损伤的关键启动因子。使用shRNA干扰序列对角质形成细胞中Wnt5a进行特异性敲减,采用免疫荧光（Immunofluorescence，IF）、蛋白免疫印迹实验（Western blot, WB）、透射电镜及线粒体膜电位试剂盒探究Wnt5a参与局部氧化损伤机制。

    2）基于人角质形成细胞转录组和高通量基因表达数据库（Gene Expression Omnibus, GEO）公共数据库中CRPS患者血液组织测序结果进行生物信息学分析。利用GEO2R工具筛选差异性基因（Differentially expressed genes, DEGs），通过富集分析方法鉴定参与CRPS发生机制的信号通路，使用蛋白互作网络分析寻找潜在关键蛋白。

3）构建大鼠慢性缺血后疼痛模型(Chronic post-ischemia pain，CPIP)模拟CRPS患者肢体I/R损伤病理过程, 通过行为学测试观察14天内大鼠机械痛和热痛阈值变化。采用IF和WB方法在皮肤和背根神经节水平进一步验证Wnt5a对下游炎症相关靶基因NMDA-NR2B亚基（N-methyl-D-aspartate 2B, NR2B）和基质金属蛋白酶9(Matrix Metalloprotein 9, MMP9)的调控，探究其在CRPS外周敏化中的作用。

结果

    1)本研究首先通过大鼠皮肤提取原代角质形成细胞并成功构建OGD/R体外细胞模型模拟体外氧化损伤。qPCR结果表明，Wnt5a经OGD/R处理后mRNA水平显著增加。IF及WB实验证实Wnt5a表达于角质形成细胞。与Control组相比，OGD/R组Wnt5a平均荧光强度及蛋白水平均显著上调，表明Wnt5a是参与CRPS氧化损伤的重要启动因子。

2)通过人角质形成细胞转录组和公共数据库中CRPS患者血液组织RNA-Seq分析，我们发现Wnt信号通路参与CRPS引起的炎症反应，MMP9是参与调控细胞氧化损伤的关键蛋白。

3）通过构建CPIP大鼠肢体I/R损伤疼痛模型，我们发现大鼠的机械痛及热痛阈值在造模后明显下降，并持续到术后14天, 表明大鼠肢体I/R损伤后发生外周痛觉敏化。皮肤及背根神经节活化的NR2B和MMP9能够促进外周敏化，并受到Wnt5a的调控。

结论

皮肤角质形成细胞中Wnt5a通过激活下游NR2B和MMP9参与了CRPS引起的外周敏化。Wnt5a抑制剂的应用可以为治疗CRPS提供新的治疗策略。

Background 

Complex regional pain syndrome (CRPS) is a chronic pain condition that commonly occurs in the extremities, characterized by inflammatory features, which seriously affects the life quality of patients. Approximately 7% of fracture patients will develop into CRPS after surgery, accompanied with regional autonomic dysfunction and impaired motor function, among which ischemia-reperfusion (I/R) injury is one of the important mechanisms. Keratinocytes, as non-neuronal cells, are the predominant cell type in the skin epidermis. They can sense local oxidative damage signaling and contribute to inflammatory response. Studies have confirmed that Wnt signaling pathway plays an important role in the pathogenesis of neuropathic pain. However, whether it is expressed in skin keratinocytes and involved in CRPS remain unclear. This study mainly focus on the peripheral mechanism of CRPS. The OGD/R (Oxygen Glucose Deprivation/Reperfusion) cell model in vitro was established to simulate the inflammatory response, aiming to identify the key factors of Wnt signaling pathway in CRPS-induced local oxidative damage. A rat CPIP model was set up in vivo to mimic the clinical pathophysiology of CRPS patients. The molecular biology and bioinformatics analysis were used to examine the underlying mechanism of Wnt5a in skin keratinocytes contributes to CRPS-induced peripheral sensitization.

Methods

1) In this study, primary keratinocytes were first extracted from rat skin, then we established an OGD/R cell model to simulate oxidative damage in vitro. qPCR (Quantitative Real-time polymerase chain reaction) was used to identify the key target in the Wnt signaling pathway associated with oxidative damage. The shRNAs (short hairpin RNA) were used to knock down Wnt5a in keratinocytes specifically, the immunofluorescence (IF), western blot (WB), transmission electron microscopy and mitochondrial membrane potential kit were used to investigate the role of Wnt5a in oxidative damage.

2) Bioinformatics analysis was conducted based on the transcriptome of human keratinocytes and blood tissue of CRPS patients in the Gene Expression Omnibus (GEO) public database. GEO2R tool was used to identify differentially expressed genes (DEGs), enrichment analysis was used to find the signaling pathways involved in CRPS, and protein-protein interaction networks was used to search for target proteins.

3) The chronic post-ischemia pain (CPIP) rat pain model was established to simulate limb ischemia reperfusion injury experienced by CRPS patients. Mechanical and heat pain thresholds in the hind paw were measured through behavioral testing within 14 days. IF and WB were used to further identify the regulation of Wnt5a on downstream NR2B and MMP9 in the skin and dorsal root ganglion, aiming to explore its role in peripheral sensitization of CRPS.

Results 

1) In this study, primary keratinocytes were extracted from the rat skin and the OGD/R cell was successfully established to simulate oxidative damage in vitro. qPCR results showed that mRNA levels of Wnt5a significantly increased after OGD/R treatment. From the IF and WB analysis, we found that Wnt5a is expressed in keratinocytes. Compared to the Control group, the mean fluorescence intensity and protein levels of Wnt5a in the OGD/R group were significantly upregulated, indicating that Wnt5a is an important initiator in CRPS-induced oxidative damage.

2) Through the RNA-Seq analysis of human keratinocytes and the blood tissue of CRPS patients, we found that Wnt signaling pathway is involved in the CRPS-induced inflammatory response, and MMP9 is a key protein in cellular oxidative damage.

3) From the limb I/R injury in CPIP rats, we found that the thresholds of mechanical pain and thermal pain decreased significantly and lasted until 14 days after modeling, indicating an increased pain sensitivity after I/R injury. The activation of NR2B and MMP9 regulated by Wnt5a in the skin and DRG may further exacerbate peripheral sensitization.

Conclusion 

Wnt5a in the skin keratinocytes contributes to CRPS-induced peripheral sensitization through the activation of NR2B and MMP9. The application of Wnt5a inhibitors is supposed to provide a new therapeutic strategy for CRPS.