Wang Zhen, Xu Wei, Rong Ao, Lin Yan, Qiu Xu-Ling, Qu Shen, Lan Xian-Hai
Department of Ophthalmology, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
Department of Medical, Tongji Hospital, Tongji University School of Medicine Shanghai 200065, China.
Int J Clin Exp Pathol. 2015 Mar 1;8(3):3395-403. eCollection 2015.
The present study demonstrates the effect of neovibsanin B on the synthesis and deposition of ECM proteins and the signalling pathways used in optic nerve head (ONH) astrocytes and lamina cribrosa (LC) cells. For investigation of the signalling pathway used by neovibsanin B, ONH cells were treated with neovibsanin B. Western blot and immunostaining analyses were used to examine the phosphorylation of proteins involved in Smad and non-Smad signalling pathway. The results revealed that ONH cells on treatment with neovibsanin B showed enhanced synthesis of extracellular matrix (ECM) proteins. Neovibsanin B induced phosphorylation of canonical signalling proteins, Smad2/3. However phosphorylation of non-canonical signalling proteins, extracellular signal-regulated kinases, p38, and c-Jun N-terminal kinases (JNK) 1/2 remained unaffected. There was also increase in co-localization of pSmad2/3 with Co-Smad4 in the nucleus of ONH astrocytes and LC cells indicating activation of the canonical Smad signalling pathway. Treatment of ONH cells with SIS3, inhibitor of Smad3 phosphorylation reversed the neovibsanin B stimulated ECM expression as well as activation of canonical pathway signalling molecules. In addition, inhibition of Smad2 or Smad3 using small interfering RNA (siRNA) also suppressed neovibsanin B stimulated ECM protein synthesis in ONH astrocytes and LC cells. Thus neovibsanin B utilizes the canonical Smad signalling pathway to stimulate ECM synthesis in human ONH cells. The neovibsanin B induced ECM synthesis and activation of the canonical Smad signalling pathway may be due to its effect on transforming growth factor-β2 (TGF-β2). However, further studies are under process to understand the mechanism.
本研究证明了新维桑宁B对视神经乳头（ONH）星形胶质细胞和筛板（LC）细胞中细胞外基质（ECM）蛋白合成与沉积的影响以及所使用的信号通路。为了研究新维桑宁B所使用的信号通路，用新维桑宁B处理ONH细胞。采用蛋白质免疫印迹和免疫染色分析来检测参与Smad和非Smad信号通路的蛋白质磷酸化情况。结果显示，用新维桑宁B处理的ONH细胞表现出细胞外基质（ECM）蛋白合成增强。新维桑宁B诱导了经典信号蛋白Smad2/3的磷酸化。然而，非经典信号蛋白细胞外信号调节激酶、p38和c-Jun氨基末端激酶（JNK）1/2的磷酸化未受影响。在ONH星形胶质细胞和LC细胞核中，pSmad2/3与Co-Smad4的共定位也增加，表明经典Smad信号通路被激活。用Smad3磷酸化抑制剂SIS3处理ONH细胞可逆转新维桑宁B刺激的ECM表达以及经典信号通路信号分子的激活。此外，使用小干扰RNA（siRNA）抑制Smad2或Smad3也可抑制新维桑宁B刺激的ONH星形胶质细胞和LC细胞中ECM蛋白的合成。因此，新维桑宁B利用经典Smad信号通路来刺激人ONH细胞中的ECM合成。新维桑宁B诱导的ECM合成和经典Smad信号通路的激活可能是由于其对转化生长因子-β2（TGF-β2）的作用。然而，目前正在进行进一步研究以了解其机制。
