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角质形成细胞Wnt5a调控MMP9参与CRPS-Ⅰ型外周敏化机制研究

朱贺 闻蓓 许力 黄宇光

朱贺, 闻蓓, 许力, 黄宇光. 角质形成细胞Wnt5a调控MMP9参与CRPS-Ⅰ型外周敏化机制研究[J]. 协和医学杂志, 2024, 15(2): 335-343. doi: 10.12290/xhyxzz.2023-0551
引用本文: 朱贺, 闻蓓, 许力, 黄宇光. 角质形成细胞Wnt5a调控MMP9参与CRPS-Ⅰ型外周敏化机制研究[J]. 协和医学杂志, 2024, 15(2): 335-343. doi: 10.12290/xhyxzz.2023-0551
ZHU He, WEN Bei, XU Li, HUANG Yuguang. Mechanism of Wnt5a on Keratinocyte Regulating MMP9 for CRPS-Ⅰ Peripheral Sensitization[J]. Medical Journal of Peking Union Medical College Hospital, 2024, 15(2): 335-343. doi: 10.12290/xhyxzz.2023-0551
Citation: ZHU He, WEN Bei, XU Li, HUANG Yuguang. Mechanism of Wnt5a on Keratinocyte Regulating MMP9 for CRPS-Ⅰ Peripheral Sensitization[J]. Medical Journal of Peking Union Medical College Hospital, 2024, 15(2): 335-343. doi: 10.12290/xhyxzz.2023-0551

角质形成细胞Wnt5a调控MMP9参与CRPS-Ⅰ型外周敏化机制研究

doi: 10.12290/xhyxzz.2023-0551
基金项目: 

国家自然科学基金 82271262

详细信息
    通讯作者:

    许力,E-mail:pumchxuli@163.com

    黄宇光,E-mail:garypumch@163.com

  • 中图分类号: R614; R364

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

Funds: 

National Natural Science Foundation of China 82271262

More Information
  • 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。
    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

    图  5  大鼠CPIP后DRG组织IL-1β、TNF-α水平改变

    CPIP:同图 3

    Figure  5.  Level of IL-1β and TNF-α for DRG in rats after CPIP

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出版历程
  • 收稿日期:  2023-11-20
  • 录用日期:  2023-12-07
  • 网络出版日期:  2023-12-19
  • 刊出日期:  2024-03-30

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