Abstract
BackgroundHepatic stellate cell (HSC) plays a key role in pathogenesis of liver fibrosis. During liver injury, hypoxia in local micro-environment is inevitable. Hif-1α is the key transcriptional regulation factor that induces cell’s adaptive responses to hypoxia. Recently, it was reported that MAPK is involved in regulation of Hif-1α activity.AimsTo explore whether Hif-1α regulates HSC activation upon hypoxia, and whether MAPK affects Hif-1α-regulated signaling cascades, thus providing new targets for preventing liver fibrosis.MethodsHif-1α expression in livers of Schistosoma japonicum infected BALB/c mice was detected with western blot and immunohistochemistry. A rat cell line of HSC, HSC-T6, was cultured in 1% oxygen. HSC activation, including F-actin reorganization, increase of vimentin and α-SMA, was detected with western blot or immunocytochemistry. Cells were transfected with specific siRNA to Hif-1α, expression of activation markers, transcription of fibrosis-promoting cytokines, secretion of collagen I were detected with western blot, Real Time PCR and ELISA. Lysate from HSC-T6 cells pretreated with PD98059, a specific MEK1 pharmacological inhibitor, was subjected to detect Hif-1α ubiquitination and nuclear translocation with western blot and immunoprecipitation.Results and ConclusionsHif-1α apparently increased in liver tissues of Schistosoma japonicum infected mice. 1% O2 induced F-actin reorganization, increase of Hif-1α, vimentin and α-SMA in HSC-T6 cells. Hif-1α Knockdown inhibited HSC-T6 activation, transcription of IL-6, TGF-β and CTGF and secretion of collagen I from HSC-T6 cells upon hypoxia. Inhibition of MAPK phosphorylation enhanced Hif-1α ubiquitination, and inhibited Hif-1α translocation into nucleus. Conclusively, Hif-1α and MAPK participate in HSC activation upon hypoxia.
Highlights
Liver fibrosis is an important pathological feature of various chronic liver diseases, and is characterized by excessive deposition of extracellular matrix (ECM), especially collagen, in the liver [1,2]
Quiescent hepatic stellate cells are vitamin A and lipid-storing cells, once activated, Hepatic stellate cell (HSC) are transformed into myofibroblast-like cells (MFC), which leads to the loss of fat vacuoles and vitamin A, reorganization of cytoskeleton proteins, expression of α-smooth-muscle actin and vimentin, acquire the ability to synthesize plenty of collagen [3], secrete fibrosis-promoting cytokines such as transforming growth factor (TGF)-β, CTGF and IL-6 and so on [4,5,6,7]
In order to determine whether Hif-1α was induced in liver fibrotic tissues as liver was injured by infection, we firstly detected Hif-1α expression in liver tissues of Schistosoma japonicum infected mice, which is recognized as a good model for infectious liver fibrosis [11]
Summary
Liver fibrosis is an important pathological feature of various chronic liver diseases, and is characterized by excessive deposition of extracellular matrix (ECM), especially collagen, in the liver [1,2]. Hypoxiainducible factor 1 (Hif-1) is the key transcriptional regulation factor which induces cell’s adaptive responses to hypoxic micro-environment and activates a number of hypoxia responsive genes. Aims: To explore whether Hif-1α regulates HSC activation upon hypoxia, and whether MAPK affects Hif-1αregulated signaling cascades, providing new targets for preventing liver fibrosis. Methods: Hif-1α expression in livers of Schistosoma japonicum infected BALB/c mice was detected with western blot and immunohistochemistry. HSC activation, including Factin reorganization, increase of vimentin and α-SMA, was detected with western blot or immunocytochemistry. Cells were transfected with specific siRNA to Hif-1α, expression of activation markers, transcription of fibrosis-promoting cytokines, secretion of collagen I were detected with western blot, Real Time PCR and ELISA. Hif-1α and MAPK participate in HSC activation upon hypoxia
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