Mechanism of Wnt5a on Keratinocyte Regulating MMP9 for CRPS-Ⅰ Peripheral Sensitization

  Objective  To explore the mechanism of Wnt5a on keratinocyte involved in the peripheral sensitization of complex regional pain syndrome type-Ⅰ (CRPS-Ⅰ) by regulating the expression of MMP9, and search for potential therapeutic strategies.

  Methods  Cultured HaCaT cells were treated with oxygen glucose deprivation/re-oxygenation (OGD/R). The early stage of mitochondrial damage and membrane potential changes after OGD/R and the effects of Box5 (Wnt5a inhibitor) at different concentrations (20 μmol/L, 40 μmol/L) on MMP9 were explored. Adult male Sprague-Dawley rats were divided into Control group(n=8), CPIP group (n=8), Box5 (20) group (n=8) and Box5 (40) group (n=8). The rat chronic post-ischemia pain (CPIP) model was established to mimic the pathophysiological process of CRPS-Ⅰ. Box5 (20) group and Box5 (40) group were treated with intraplantar injection of 20 μmol/L and 40 μmol/L Box5 100 μL, respectively. The changes of mechanical withdrawal threshold and thermal withdrawal latency were measured within two weeks, and the skin inflammatory infiltration and keratosis were observed by HE staining. The expression of MMP9 was observed by immunofluorescence, and the levels of IL-1β and TNF-α in dorsal root ganglion of different groups were detected by ELISA.

  Results  Vitro experiment: After OGD/R treatment, the mitochondrial atrophy was observed in OGD/R group under transmission electron microscope and the average fluorescence intensity of MMP9 was found to increase significantly (P<0.001). Compared with Control group, the mitochondrial membrane potential in OGD/R group decreased significantly by JC-1 detection (P=0.027). Compared with OGD/R group, only Box5 (40) group had a statistically significant increase in mitochondrial membrane potential (P=0.046). Animal experiment: Behavioral tests showed that the mechanical pain threshold and thermal pain threshold of CPIP group were significantly decreased at each time point (D1, D2, D4, D10, D14) (all P<0.05). HE staining indicated that there was a large-scale infiltration of inflammatory cell in the dermis and excessive keratosis in the epidermis, and the thickness of stratum granulosum and stratum spinosum increased significantly (P < 0.001). Immunofluorescence analysis showed that the expression of MMP9 in CPIP group was significantly increased (P<0.001). Compared with CPIP group, the fluorescence intensity of MMP9 in Box5 (20) group (P=0.002) and Box5 (40) group (P<0.001) were significantly decreased. ELISA results showed that the concentrations of IL-1β (P=0.048) and TNF-α (P=0.002) in CPIP group were significantly increased. Compared with CPIP group, the concentrations of IL-1β (P=0.047) and TNF-α (P=0.047) were significantly decreased in Box5 (40) group.

  Conclusions  Peripheral ischemia reperfusion injury leads to overexpression of MMP9 on keratinocytes, resulting in CRPS-Ⅰ peripheral sensitization. Targeted inhibition of Wnt5a/MMP9 pathway can reverse pain behavior in rat model of CPIP, thus providing a strategy for clinical treatment of chronic pain.