中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2018, Vol. 13 ›› Issue (11): 1927-1936.doi: 10.4103/1673-5374.239439

• 原著:退行性病与再生 • 上一篇    下一篇

人参环氧炔醇促进PC12细胞轴突生长的分子机制

  

  • 收稿日期:2018-07-22 出版日期:2018-11-15 发布日期:2018-11-15
  • 基金资助:

    国家自然科学基金（30873057，81171245）；上海市科委重点基础工程赠款（08JC1413600，11JC1406600）

Molecular mechanism of panaxydol on promoting axonal growth in PC12 cells

Wei-Peng Li1, Ke Ma2, Xiao-Yan Jiang3, Rui Yang2, Pei-Hua Lu2, Bao-Ming Nie2, Yang Lu2   

  1. 1 Department of Nuclear Medicine, the First Affiliated Hospital of Bengbu Medical College, Bengbu, Anhui Province, China
    2 Department of Pharmacy, Shanghai Jiao Tong University School of Medicine, Shanghai, China
    3 Key Laboratory of Arrhythmias of Ministry of Education of China, Tongji University School of Medicine, Shanghai, China
  • Received:2018-07-22 Online:2018-11-15 Published:2018-11-15
  • Contact: Yang Lu or Bao-Ming Nie,PhD, huaxue@shsmu.edu.cn or baomingnie@gmail.com.
  • Supported by:

    The study was supported partly by the National Natural Science Foundation of China, No. 30873057, 81171245; a grant from the Key Basic Project of Shanghai Municipal Science and Technology Commission of China, No. 08JC1413600, 11JC1406600

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摘要：

神经生长因子主要通过调节RTK-RAS-MEK-ERK通路促进PC12细胞轴突生长。有研究报道传统中药三七中的成分之一炔醇类化合物有与神经生长因子类似的功能，可诱导PC12细胞的轴突生长，但分子机制尚不明确。课题组以往研究发现，炔醇类化合物人参环氧炔醇可升高胞内cAMP水平，并且ERK抑制剂U0126会抑制炔醇诱导的轴突生长，但cAMP下游通路如何激活ERK尚未进行深入研究。为此，实验设计以采用特异性信号通路抑制剂干预关键信号分子，观察人参环氧炔醇、神经生长因子和forskolin诱导PC12细胞轴突生长的分子机制。发现RTK抑制剂SU5416可明显抑制神经生长因子促轴突生长的作用，但不能抑制人参环氧炔醇促PC12细胞轴突生长；腺苷酸环化酶(AC)抑制剂SQ22536和cAMP依赖的蛋白激酶抑制剂RpcAMPS可抑制Forskolin和人参环氧炔醇促轴突生长的作用，ERK抑制剂U0126也可抑制这3种因子的促轴突生长的作用；PKA抑制剂H89可明显抑制Forskolin促轴突生长的作用，但不能抑制人参环氧炔醇促轴突生长的作用。实验进一步通过Western blot检测了3种刺激因子和抑制剂对ERK磷酸化水平的影响，发现神经生长因子通过络氨酸受体通路活化ERK通路从而诱导PC12细胞轴突生长，而人参环氧炔醇和forskolin会升高细胞cAMP水平并被腺苷酸环化酶抑制剂所抑制。PKA抑制剂H89完全抑制forskolin引起的轴突生长和ERK磷酸化，但不能抑制人参环氧炔醇诱导的轴突生长和ERK磷酸化，说明人参环氧炔醇是通过不同的cAMP下游通路促进PC12细胞轴突生长。鉴于Epac1在介导cAMP信号通路中的起重要调节作用，设计了针对Epac1基因的RNA干扰实验，验证了Epac可介导人参环氧炔醇诱导的轴突生长信号通路。上述数据说明，与神经生长因子和forskolin相比，人参环氧炔醇主要通过cAMP-Epac1-Rap1-MEK-ERK-CREB通路发挥促轴突生长的作用，而该作用不依赖PKA。

orcid:0000-0002-8343-0262(Bao-Ming Nie)

 

关键词: 三七, 聚乙炔, 人参醇, NGF, PC 12细胞, 轴突生长, cAMP, PKA, Epac1, ERK, CREB, 神经再生

Abstract:

Nerve growth factor (NGF) promotes axonal growth in PC12 cells primarily by regulating the RTK-RAS-MEK-ERK pathway. Panaxydol, a polyacetylene isolated from Panax notoginseng, can mimic the effects of NGF. Panaxydol promotes neurite outgrowth in PC12 cells, but its molecular mechanism remains unclear. Indeed, although alkynol compounds such as panaxydol can increase intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) levels and the ERK inhibitor U0126 inhibits alkynol-induced axonal growth, how pathways downstream of cAMP activate ERK have not been investigated. This study observed the molecular mechanism of panaxydol-, NGF- and forskolin-induced PC12 cell axon growth using specific signaling pathway inhibitors. The results demonstrated that although the RTK inhibitor SU5416 obviously inhibited the growth-promoting effect of NGF, it could not inhibit the promoting effect of panaxydol on axonal growth of PC12 cells. The adenylate cyclase inhibitor SQ22536 and cAMP-dependent protein kinase inhibitor RpcAMPS could suppress the promoting effect of forskolin and panaxydol on axonal growth. The ERK inhibitor U0126 inhibited axonal growth induced by all three factors. However, the PKA inhibitor H89 inhibited the promoting effect of forskolin on axonal growth but could not suppress the promoting effect of panaxydol. A western blot assay was used to determine the effects of stimulating factors and inhibitors on ERK phosphorylation levels. The results revealed that NGF activates the ERK pathway through tyrosine receptors to induce axonal growth of PC12 cells. In contrast, panaxydol and forskolin increased cellular cAMP levels and were inhibited by adenylyl cyclase inhibitors. The protein kinase A inhibitor H89 completely inhibited forskolin-induced axonal outgrowth and ERK phosphorylation, but could not inhibit panaxydol-induced axonal growth and ERK phosphorylation. These results indicated that panaxydol promoted axonal growth of PC12 cells through different pathways downstream of cAMP. Considering that exchange protein directly activated by cAMP 1 (Epac1) plays an important role in mediating cAMP signaling pathways, RNA interference experiments targeting the Epac1 gene were employed. The results verified that Epac1 could mediate the axonal growth signaling pathway induced by panaxydol. These findings suggest that compared with NGF and forskolin, panaxydol elicits axonal growth through the cAMP-Epac1-Rap1-MEK-ERK-CREB pathway, which is independent of PKA.

Key words: nerve regeneration, panax notoginseng, polyacetylene, panaxydol, nerve growth factor, PC12 cells, neurite outgrowth, cAMP, PKA, Epac1, ERK, CREB, neural regeneration