PMID- 28539891
OWN - NLM
STAT- PubMed-not-MEDLINE
LR  - 20200930
IS  - 1664-042X (Print)
IS  - 1664-042X (Electronic)
IS  - 1664-042X (Linking)
VI  - 8
DP  - 2017
TI  - Double Knockdown of PHD1 and Keap1 Attenuated Hypoxia-Induced Injuries in 
      Hepatocytes.
PG  - 291
LID - 10.3389/fphys.2017.00291 [doi]
LID - 291
AB  - Background and Aims: Hypoxia and oxidative stress contribute toward liver 
      fibrosis. In this experiment, we used small hairpin RNA (shRNA) to interfere with 
      the intracellular oxygen sensor-prolyl hydroxylase 1 (PHD1) and the intracellular 
      oxidative stress sensor-kelch-like ECH associated protein 1 (Keap1) in the 
      hypoxic hepatocytes in order to investigate the function of PHD1and Keap1. 
      Methods: We first established the CCl(4)-induced liver fibrosis model, 
      subsequently, the levels of the PHD1, hypoxia-inducible factor-1alpha (HIF-1alpha), 
      hypoxia-inducible factor-2alpha (HIF-2alpha), Keap1, and nuclear factor-erythroid 2 
      p45-related factor 2 (Nrf2) were detected in liver tissues. Simultaneously, AML12 
      cells co-transfected with PHD1 and Keap1shRNAs were constructed in vitro, then 
      the intracellular oxidative stress, the proportion of cells undergoing apoptosis, 
      and cell viability were measured. The expression of pro-fibrogenic molecules were 
      analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) and 
      western blot. The level of alpha-1 type I collagen (COL1A1) was determined using 
      an enzyme-linked immunosorbent assay (ELISA). Finally, serum-free "conditioned 
      medium" (CM) from the supernatant of hypoxic AML12 hepatocytes was used to 
      culture rat hepatic stellate cells (HSC-T6), and the levels of fibrosis-related 
      molecules, apoptosis, and cell proliferation were determined. Results: The marker 
      of hypoxia-HIF-1alpha and HIF-2alpha in the livers with fibrosis were upregulated, 
      however, the increase in PHD1 expression was not statistically significant in 
      comparison to the control group. Sign of oxidative stress-Keap1 was increased, 
      while the expression of Nrf2, one of the Keap1 main downstream molecules, was 
      reduced in the hepatocytes. And in vitro, the double-knockdown of PHD1 and Keap1 
      in AML12 hepatocytes presented with decreased hypoxia-induced oxidative stress 
      and apoptosis, furthermore, these hypoxic AML12 cells showed the increased cell 
      viability and the doweregulated expression of pro-fibrogenic molecules. In 
      addition, HSC-T6 cells cultured in the hypoxic double-knockdown CM demonstrated 
      the downregulation of fibrosis-related molecules, diminished cell proliferation, 
      and enhanced apoptosis. Conclusions: Our study demonstrated that double-knockdown 
      of PHD1 and Keap1 attenuated hypoxia and oxidative stress induced injury in the 
      hepatocytes, and subsequently inhibited HSC activation, which offers a novel 
      therapeutic strategy in the prophylaxis and treatment of liver fibrosis.
FAU - Liu, Jing
AU  - Liu J
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Li, Yiping
AU  - Li Y
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Liu, Lei
AU  - Liu L
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Wang, Zhi
AU  - Wang Z
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Shi, Chuanbing
AU  - Shi C
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Cheng, Zhengyuan
AU  - Cheng Z
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Zhang, Xiaoyi
AU  - Zhang X
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Ding, Fengan
AU  - Ding F
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
FAU - Chen, Ping Sheng
AU  - Chen PS
AD  - Department of Pathology and Pathophysiology, School of Medicine, Southeast 
      UniversityNanjing, China.
LA  - eng
PT  - Journal Article
DEP - 20170510
PL  - Switzerland
TA  - Front Physiol
JT  - Frontiers in physiology
JID - 101549006
PMC - PMC5423937
OTO - NOTNLM
OT  - Keap1
OT  - PHD1
OT  - hepatocytes
OT  - hypoxia
OT  - liver fibrosis
OT  - oxidative stress
EDAT- 2017/05/26 06:00
MHDA- 2017/05/26 06:01
PMCR- 2017/05/10
CRDT- 2017/05/26 06:00
PHST- 2016/10/22 00:00 [received]
PHST- 2017/04/21 00:00 [accepted]
PHST- 2017/05/26 06:00 [entrez]
PHST- 2017/05/26 06:00 [pubmed]
PHST- 2017/05/26 06:01 [medline]
PHST- 2017/05/10 00:00 [pmc-release]
AID - 10.3389/fphys.2017.00291 [doi]
PST - epublish
SO  - Front Physiol. 2017 May 10;8:291. doi: 10.3389/fphys.2017.00291. eCollection 
      2017.