角质形成细胞Wnt5a调控MMP9参与CRPS-Ⅰ型外周敏化机制研究OA北大核心CSTPCD

Objective To explore the mechanism of Wnt5a on keratinocyte involved in the peripheral sensiti-zation 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 es-tablished 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 me-chanical withdrawal threshold and thermal withdrawal latency were measured within two weeks,and the skin in-flammatory infiltration and keratosis were observed by HE staining.The expression of MMP9 was observed by im-munofluorescence,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 in-crease 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 experi-ment: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 keratino-cytes,resulting in CRPS-Ⅰperipheral 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.