Xu Mingcui, Zhang Feng, Wang Aixiu, Wang Chen, Cao Yu, Zhang Ming, Zhang Mingming, Su Min, Zou Xiaoping, Xu Guifang, Zhuge Yuzheng
Department of Gastroenterology, Affiliated Drum Tower Clinical Medical School of Nanjing Medical University, Nanjing, Jiangsu, China.
Department of Gastroenterology, Drum Tower Hospital, Affiliated to Medical School of Nanjing University, Nanjing, Jiangsu, China.
PLoS One. 2016 Dec 1;11(12):e0167658. doi: 10.1371/journal.pone.0167658. eCollection 2016.
In the liver, the signal and function of tumor necrosis factor-like weak inducer of apoptosis (TWEAK) have mainly been assessed in association with liver regeneration. However, the effects of TWEAK on liver fibrosis have not been fully elucidated. To investigate the effects of TWEAK on human hepatic stellate cells (HSCs) and to explore the relevant potential mechanisms, human HSCs line-LX-2 were cultured with TWEAK. Cell migration was detected by transwell assay; cell viability was evaluated by Cell Counting Kit-8; the expression of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13 gene was identified by quantitative real-time polymerase chain reaction and western blotting; the activity of matrix metalloproteinases (MMPs) was tested by enzyme-linked immuno sorbent assay; small interfering RNA transfection was applied for depletion of MMP9 and p65. The result of transwell assay revealed that TWEAK promoted LX-2 migration. Subsequently, our data testified that the expression and activity of MMP9 was induced by TWEAK in LX-2 cells, which enhanced the migration. Furthermore, our findings showed that TWEAK upregulated the phosphorylation of IκBα and p65 protein to increase MMP9 expression in LX-2 cells. Meanwhile, the alpha-smooth muscle actin, vimentin and desmin expression were upregulated following TWEAK treatment. The results in the present study revealed that TWEAK promotes HSCs migration via canonical NF-κB/MMP9 pathway, which possibly provides a molecular basis targeting TWEAK for the therapy of liver fibrosis.
在肝脏中,肿瘤坏死因子样凋亡微弱诱导剂(TWEAK)的信号和功能主要是在肝脏再生的背景下进行评估的。然而,TWEAK对肝纤维化的影响尚未完全阐明。为了研究TWEAK对人肝星状细胞(HSCs)的影响并探索相关潜在机制,将人HSCs系-LX-2用TWEAK进行培养。通过Transwell实验检测细胞迁移;用细胞计数试剂盒-8评估细胞活力;通过定量实时聚合酶链反应和蛋白质印迹法鉴定基质金属蛋白酶1(MMP1)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶3(MMP3)、基质金属蛋白酶7(MMP7)、基质金属蛋白酶8(MMP8)、基质金属蛋白酶9(MMP9)、基质金属蛋白酶10(MMP10)、基质金属蛋白酶11(MMP11)、基质金属蛋白酶12(MMP12)、基质金属蛋白酶13(MMP13)基因的表达;通过酶联免疫吸附测定法检测基质金属蛋白酶(MMPs)的活性;应用小干扰RNA转染来敲低MMP9和p65。Transwell实验结果显示TWEAK促进了LX-2细胞的迁移。随后,我们的数据证实TWEAK诱导了LX-2细胞中MMP9的表达和活性,从而增强了细胞迁移。此外,我们的研究结果表明,TWEAK上调IκBα和p65蛋白的磷酸化水平,以增加LX-2细胞中MMP9的表达。同时,TWEAK处理后α-平滑肌肌动蛋白、波形蛋白和结蛋白的表达上调。本研究结果表明,TWEAK通过经典的核因子κB(NF-κB)/MMP9途径促进肝星状细胞迁移,这可能为针对TWEAK治疗肝纤维化提供分子基础。