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Mechanism of Wnt5a on Keratinocyte Regulating MMP9 for CRPS-Ⅰ Peripheral Sensitization
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摘要:
目的 探究皮肤角质形成细胞Wnt5a通过靶向调控基质金属蛋白酶9(matrix metalloproteinase-9，MMP9)的表达参与复杂区域疼痛综合征(complex regional pain syndrome, CRPS)-Ⅰ型外周敏化的机制，寻找该慢性疼痛的潜在治疗策略。 方法 本研究分为两部分，第一部分为体外实验。体外培养人永生化角质形成细胞HaCaT进行氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation，OGD/R)处理，初步观察OGD/R早期(24 h内)线粒体损伤及膜电位变化, 并探究给予不同浓度Wnt5a抑制剂Box5对MMP9的影响。第二部分为动物实验。将大鼠随机分为慢性缺血后疼痛(chronic postischemia pain, CPIP)组、Box5(20)组、Box5(40)组和对照组，每组8只，CPIP组、Box5(20)组、Box5(40)组先建立大鼠患肢缺血再灌注CPIP模型，模拟CRPS-Ⅰ型病理生理过程，Box5(20)组和Box5(40)组在此基础上分别足底注射20 μmol/L和40 μmol/L Box5溶液100 μL，对照组和CPIP组则分别注射生理盐水100 μL。通过疼痛行为学测定观察4组大鼠2周内不同时间点(D1，D2，D4，D10，D14)机械痛和热痛阈值变化情况。HE染色观察大鼠皮肤炎症浸润及角化情况，免疫荧光染色观察4组MMP9的表达情况，ELISA检测4组背根神经节(dorsal root ganglion, DRG)的IL-1β及肿瘤坏死因子α(tumor necrosis factor-α，TNF-α)水平。 结果 体外实验：HaCaT细胞进行OGD/R处理后, MMP9平均荧光强度显著增加(P＜0.001)；透射电镜下观察到OGD/R组出现线粒体明显萎缩，线粒体膜电位检测显示，与对照组相比，OGD/R组提示线粒体膜电位下降明显(P=0.027)。动物实验：与OGD/R组相比，仅Box5(40)组线粒体膜电位上升具有统计学差异(P=0.046)。行为学检测发现CPIP组大鼠术后各时间点(D1，D2，D4，D10，D14)机械痛阈值和热痛阈值均显著降低(P均＜0.05)。HE染色提示CPIP组大鼠患足真皮层出现大量炎症细胞浸润，表皮出现过度角化，颗粒层及棘层厚度显著增加(P＜0.001)。免疫荧光试验显示，CPIP组角质形成细胞MMP9荧光强度显著增加(P＜0.001)；与CPIP组相比，Box5(20)组(P=0.002)和Box5(40)组(P＜0.001)MMP9荧光强度均显著下降。ELISA检测结果显示，CPIP组IL-1β(P=0.048)和TNF-α浓度(P=0.002)显著升高; 与CPIP组相比，Box5(40)组IL-1β(P=0.047)和TNF-α浓度(P=0.047)显著下降。 结论 外周局部缺血再灌注损伤可导致角质形成细胞MMP9过度表达，引起CRPS-Ⅰ型外周敏化。靶向抑制Wnt5a/MMP9可逆转CPIP大鼠疼痛行为，为临床治疗慢性痛提供了参考依据。 Abstract: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. -
Key words:
- complex regional pain syndrome /
- keratinocyte /
- Wnt5a /
- MMP9 /
- peripheral sensitization
作者贡献：朱贺负责实验设计、实验实施和论文撰写；闻蓓负责图表制作及数据分析；许力、黄宇光负责研究设计、论文修订与最终审核。利益冲突：所有作者均声明不存在利益冲突 -
图 1 HaCaT细胞OGD/R后不同处理组角质形成细胞MMP9表达变化(标尺=20 μm, ×20)
OGD/R (oxygen-glucose deprivation/reoxygenation)：氧糖剥夺/复氧
Figure 1. MMP9 expression in different treatment groups after OGD/R in HaCaT cells (scale=20 μm, ×20)
图 2 Wnt5a抑制剂Box5对HaCaT细胞线粒体影响
A. 透射电镜下OGD/R组线粒体萎缩情况(标尺=500 nm)；B. JC-1染色检测各组线粒体膜电位变化(标尺=20 μm, ×20)
Figure 2. The effect of Wnt5a inhibitor Box5 on the mitochondria of HaCaT cells
A. Mitochondrial atrophy in OGD/R group under transmission electron microscopy (scale=500 nm); B. Mitochondrial membrane potential in each group by JC-1 detection (scale=20 μm, ×20)
OGD/R: 同图 1
图 3 大鼠建模给药流程图(A)及造模后疼痛行为学改变(B)
CPIP(chronic postischemia pain)：慢性缺血后疼痛；与对照组比较，*P＜0.05; 与CPIP组比较，#P＜0.05
Figure 3. Drug administration process diagram for rat modeling (A) and pain behavioral changes after modeling (B)
图 4 CPIP后患足表皮HE染色及免疫荧光染色结果(×20)
A. 大鼠患足表皮HE染色; B. 大鼠患足表皮免疫荧光染色(标尺=20 μm)
Figure 4. HE staining and immunofluorescence staining results of the affected foot epidermis after CPIP (×20)
A. HE staining of the affected foot epidermis in rats; B. Immunofluorescence staining of rat foot epidermis (scale=20 μm)
CPIP: 同图 3 -
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