title: Indole-3-Carbinol Induces Apoptosis of Hepatic Stellate Cells through K63 De-Ubiquitination of RIP1 in Rats.
abstract: OBJECTIVE
  The apoptosis of activated hepatic stellate cells (HSCs) is the central event in the reversal of liver fibrosis. K63 de-ubiquitinated receptor-interacting protein (RIP)1 promotes apoptosis in tumor necrosis factor (TNF)-α signaling pathway. In the previous study, we have proved that indole-3-carbinol (I3C) could reverse different models of liver fibrosis in rats, but the mechanism is still unclear. Thus, the present research aimed to demonstrate the induction of I3C on apoptosis of HSCs and the underlying molecular mechanism.
  
  
  METHODS
  HSC-T6, an immortalized rat liver stellate cell line, was treated for 24 hours with 25, 50 and 100 µM of I3C. The apoptosis of HSC-T6 was analyzed by flow cytometric analysis, acridine orange staining and RT-PCR, respectively. K63 de-ubiquitination of RIP1 and the expression of ubiquitin ligases and deubiquitinases were analyzed by Co-IP assay and western blot. Knockdown of deubiquitinases was undertaken by small interfering RNA (siRNA).
  
  
  RESULTS
  The results of flow cytometric analysis indicated that the apoptotic rate of HSC-T6 was induced by I3C in a dose-dependent manner. Observation by acridine orange staining exhibited nuclear condensation and apoptotic bodies in I3C treated cells. Consistently, the expression ratio of Bax/bcl-2 was markedly increased by I3C. These results indicated that I3C could significantly induce apoptosis of HSC-T6 cells. Furthermore, Co-IP assay revealed K63 de-ubiquitination of RIP1 by I3C, associated with the induction of caspase 8. Although I3C had no effect on the expression of ubiquitin ligases cellular inhibitor of apoptosis 1/2 (cIAP1/2), the protein level of deubiquitinase cylindromatosis (CYLD) was significantly induced by I3C. Moreover, CYLD silencing reversed the pro-apoptosis induction effect of I3C and reduced the expression ratio of Bax/bcl-2 in HSC-T6 cells.
  
  
  CONCLUSIONS
  These results demonstrated that I3C could induce apoptosis of HSC through RIP1 K63 de-ubiquitination by upregulating deubiquitinase CYLD.
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.|Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Cong
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Yanan
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Xiong
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Jin
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wan
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.
authors.forename: Yunjiao
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.|Hubei Provincial Key Laboratory of Developmentally Originated Disorder, Wuhan, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ao
authors.affiliation: Department of Pharmacology, School of Basic Medical Science of Wuhan University, Wuhan, China.|Hubei Provincial Key Laboratory of Developmentally Originated Disorder, Wuhan, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor; D000072017:Deubiquitinating Enzymes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055166:Hepatic Stellate Cells / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D054875:Ubiquitination / Q000187:drug effects*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
chemical_list: C494828:Bax protein, rat; C000617196:Bcl2 protein, rat; D007211:Indoles; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; C016517:indole-3-carbinol; D017346:Protein Serine-Threonine Kinases; C586536:RIPK1 protein, rat; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D000072017:Deubiquitinating Enzymes
keywords: Cylindromatosis; Hepatic stellate cells; Indole-3-carbinol; K63 de-ubiquitination; Receptor-interacting protein 1