Chen Yi-xiong, Weng Zhi-hong, Qi Dan, Zhang Shu-ling
Department of Infectious Diseases, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhonghua Gan Zang Bing Za Zhi. 2012 Sep;20(9):677-82. doi: 10.3760/cma.j.issn.1007-3418.2012.09.008.
To investigate whether Notch signaling is activated in hepatic stellate cells (HSCs), and to determine whether manipulation of the Notch signaling pathway can effect the activation of HSCs.
The expression of Notch signaling components in unactivated or TGF-b1-activated HSC-T6 cells was detected by Taqman Probe-based gene expression analysis. Differential expression of Notch3 and Jagged1 was detected by immunofluorescence analysis. Notch3-mediated expression of the myofibroblastic markers, a-SMA and collagen I, was detected in HSC-T6 cells transfected with pcDNA3.1-N3ICD or Notch3 siRNA by Western blotting.
Notch signaling components were expressed in both unactivated and activated HSC-T6 cells, but the TGF-b1-treated cells showed significantly higher expression levels of Jagged1 (3.9-fold, F = 2543.482), Notch3 (4.2-fold, F = 287.982), and HES1 (3.2-fold, F = 1719.851). Transfection-mediated over-expression of Notch3 led to significantly increased expression of a-SMA (6.8-fold, t = 13.157) and collagen I (5.5-fold, t = 9.810) (both P less than 0.01). Transient knock-down of Notch3 expression by siRNA decreased expression of the myofibroblastic markers (a-SMA by approximately 90%, t = 19.863 and collagen I by 84%, t = 10.376; both, P less than 0.01). Moreover, knock-down of Notch3 antagonized the TGF-b1-induced expression of a-SMA and collagen I.
Notch signaling may participate in liver fibrogenesis by regulating HSC activation. Selective interruption of Notch3 may represent a new anti-fibrotic strategy to treat liver fibrosis.
研究Notch信号通路在肝星状细胞（HSCs）中是否被激活，并确定对Notch信号通路的调控是否会影响HSCs的激活。
采用基于Taqman探针的基因表达分析检测未激活或经转化生长因子-β1（TGF-β1）激活的HSC-T6细胞中Notch信号通路成分的表达。通过免疫荧光分析检测Notch3和Jagged1的差异表达。用蛋白质免疫印迹法检测转染pcDNA3.1-N3ICD或Notch3小干扰RNA（siRNA）的HSC-T6细胞中Notch3介导的肌成纤维细胞标志物α-平滑肌肌动蛋白（α-SMA）和I型胶原的表达。
Notch信号通路成分在未激活和激活的HSC-T6细胞中均有表达，但经TGF-β1处理的细胞中Jagged1（3.9倍，F = 2543.482）、Notch3（4.2倍，F = 287.982）和HES1（3.2倍，F = 1719.851）的表达水平显著更高。转染介导的Notch3过表达导致α-SMA（6.8倍，t = 13.157）和I型胶原（5.5倍，t = 9.810）的表达显著增加（均P < 0.01）。通过siRNA瞬时敲低Notch3表达可降低肌成纤维细胞标志物的表达（α-SMA降低约90%，t = 19.863；I型胶原降低84%，t = 10.376；均P < 0.01）。此外，敲低Notch3可拮抗TGF-β1诱导的α-SMA和I型胶原的表达。
Notch信号通路可能通过调节HSCs激活参与肝纤维化的发生。选择性阻断Notch3可能代表一种治疗肝纤维化的新的抗纤维化策略。
