中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2019, Vol. 14 ›› Issue (6): 1013-1024.doi: 10.4103/1673-5374.250620

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

大鼠蛛网膜下腔出血后早期脑损伤中wnt/Frizzled信号通路介导的神经保护

  

  • 出版日期:2019-06-15 发布日期:2019-06-15
  • 基金资助:

    安徽省自然科学基金(1508085QH184)

Neuroprotection mediated by the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage

Yang Wang 1 , De-Jun Bao 1 , Bin Xu 2 , Chuan-Dong Cheng 1 , Yong-Fei Dong 1 , Xiang-pin Wei 1 , Chao-Shi Niu 1, 3   

  1. 1 Department of Neurosurgery, First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui Province, China
    2 Anhui Medical University Auhui Province Medical Genetic Center, Hefei, Anhui Province, China
    3 Anhui Province Key Laboratory of Brain Function and Brain Disease, Hefei, Anhui Province, China
  • Online:2019-06-15 Published:2019-06-15
  • Contact: Chao-Shi Niu, MD, ah_neurosurgery@163.com.
  • Supported by:

    This study was supported by the Natural Science Foundation of Anhui Province of China, No. 1508085QH184 (to YW).

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摘要：

wnt/Frizzled 信号通路参与多种炎症相关性疾病，然而，wnt/Frizzled信号通路介导的炎症反应在实验性蛛网膜下腔出血(subarachnoid hemorrhage，SAH)中的作用尚未见深入研究。因此，实验评估了大鼠SAH后早期脑损伤中wnt/Frizzled信号通路的潜在作用，同时探究相关的神经保护机制。实验通过视交叉前池注入自体动脉血构建活体SAH模型大鼠。(1)实验1：将42只建模成功的成年大鼠按SAH损伤不同时间点分为SAH (6 h, 12 h, 24 h, 48 h, 72 h，1 周)组，同时设假手术组注射相同体积生理盐水作对照，观察wnt/Frizzled信号通路在SAH后早期脑损伤中的表达；(2)实验2：于SAH 48 h后，将大鼠分别采用重组人wnt1(recombinant human wnt1，rhwnt1)，小干扰wnt1(small interfering wnt1，siwnt1)RNA 和Frizzled1 单克隆抗体 (anti-Frizzled1)分别进行干预，以观察 wnt/Frizzled信号通路在SAH后的神经保护机制。(3)评估方法：采用Western Blot和免疫荧光染色评估各组wnt1，Frizzled1，β-连环蛋白，过氧物酶体-γ，CD36和核转录因子的表达，应用免疫荧光及ELISA法评估各组小胶质细胞分型及炎症因子释放情况。(4)结果发现：与假手术组相比，wnt1，Frizzled1和β-catenin的表达在SAH后明显下调，在SAH后48 h达到最低，并在SAH后1周内回到基线水平，Rhwnt1干预可以明显增加wnt1水平并能减轻SAH后早期(72 h之内)脑损伤 (包括皮质细胞凋亡，脑水肿及神经行为缺陷)，同时也增加了β-连环蛋白，CD36 和过氧物酶体-γ的水平及减低核转录因子κB水平；(5)值得注意的是，Rhwnt1干预可以促进M2 型小胶质细胞转化和抑制炎症因子(白细胞介素1β，6和肿瘤坏死因子α)释放，而siwnt1 RNA和anti-Frizzled1处理后起到了相反的效果；(6)上述数据证实：wnt/Frizzled信号通路可能通过抑制炎症反应参与SAH后的早期脑损伤，作用为调整M2型小胶质细胞的生成和减少炎症因子释放。

orcid: 0000-0001-8508-567X (Chao-Shi Niu)

关键词: 蛛网膜下腔出血, wnt/Frizzled 信号通路, 早期脑损伤, NF-&kappa, B, M2型小胶质细胞, 过氧化物酶体增殖物激活受体&gamma, 炎症因子, 神经再生

Abstract:

The Wnt/Frizzled signaling pathway participates in many inflammation-linked diseases. However, the inflammatory response mediated by the Wnt/Frizzled signaling pathway in experimental subarachnoid hemorrhage has not been thoroughly investigated. Consequently, in this study, we examined the potential role of the Wnt/Frizzled signaling pathway in early brain injury in rat models of subarachnoid hemorrhage. Simultaneously, possible neuroprotective mechanisms were also investigated. Experimental subarachnoid hemorrhage rat models were in¬duced by injecting autologous blood into the prechiasmatic cistern. Experiment 1 was designed to examine expression of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. In total, 42 adult rats were divided into sham (injection of equivalent volume of saline), 6-, 12-, 24-, 48-, 72-hour, and 1-week subarachnoid hemorrhage groups. Experiment 2 was designed to exam¬ine neuroprotective mechanisms of the Wnt/Frizzled signaling pathway in early brain injury induced by subarachnoid hemorrhage. Rats were treated with recombinant human Wnt1 (rhwnt1), small interfering Wnt1 (siwnt1) RNA, and monoclonal antibody of Frizzled1 (an¬ti-Frizzled1) at 48 hours after subarachnoid hemorrhage. Expression levels of Wnt1, Frizzled1, β-catenin, peroxisome proliferator-activated receptor-γ, CD36, and active nuclear factor-κB were examined by western blot assay and immunofluorescence staining. Microglia type con¬version and inflammatory cytokine levels in brain tissue were examined by immunofluorescence staining and enzyme-linked immunosorbent assay. Our results show that compared with the sham group, expression levels of Wnt1, Frizzled1, and β-catenin were low and reduced to a minimum at 48 hours, gradually returning to baseline at 1 week after subarachnoid hemorrhage. rhwnt1 treatment markedly increased Wnt1 expression and alleviated subarachnoid hemorrhage-induced early brain injury (within 72 hours), including cortical cell apoptosis, brain edema, and neurobehavioral deficits, accompanied by increasing protein levels of β-catenin, CD36, and peroxisome proliferator-activated re¬ceptor-γ and decreasing protein levels of nuclear factor-κB. Of note, rhwnt1 promoted M2-type microglia conversion and inhibited release of inflammatory cytokines (interleukin-1β, interleukin-6, and tumor necrosis factor-α). In contrast, siwnt1 RNA and anti-Frizzled1 treatment both resulted in an opposite effect. In conclusion, the Wnt/Frizzled1 signaling pathway may participate in subarachnoid hemorrhage-induced early brain injury via inhibiting the inflammatory response, including regulating microglia type conversion and decreasing inflammatory cytokine release. The study was approved by Animal Ethics Committee of Anhui Medical University and First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China (approval No. LLSC-20180202) in May 2017.

Key words: nerve regeneration, subarachnoid hemorrhage, Wnt/Frizzled signaling pathway, early brain injury, nuclear factor-κB, M2 type microglia, peroxisome proliferator-activated receptor-γ, inflammatory cytokines, neural regeneration