Lipopolysaccharide preconditioning protects hepatocytes from ischemia/reperfusion injury (IRI) through inhibiting ATF4-CHOP pathway in mice

PLoS One. 2013 Jun 4;8(6):e65568. doi: 10.1371/journal.pone.0065568. Print 2013.

Abstract

Background: Low-dose lipopolysaccharide (LPS) preconditioning-induced liver protection has been demonstrated during ischemia-reperfusion injury (IRI) in several organs but has not been sufficiently elucidated underlying causal mechanism. This study investigated the role of low-dose LPS preconditioning on ATF4-CHOP pathway as well as the effects of the pathway on tissue injury and inflammation in a mouse model of liver partial-warm IRI.

Methods: LPS (100 µg/kg/d) was injected intraperitoneally two days before ischemia. Hepatic injury was evaluated based on serum alanine aminotransferase levels, histopathology, and caspase-3 activity. The ATF4-CHOP pathway and its related apoptotic molecules were investigated after reperfusion. The role of LPS preconditioning on apoptosis and ATF4-CHOP pathway was examined in vitro. Moreover, the effects of the ATF4-CHOP pathway on apoptosis, Caspase-12, and Caspase-3 were determined with ATF4 small interfering RNA (siRNA). Inflammatory cytokine expression was also checked after reperfusion. Inflammatory cytokines and related signaling pathways were analyzed in vitro in macrophages treated by LPS preconditioning or ATF4 siRNA.

Results: LPS preconditioning significantly attenuated liver injury after IRI. As demonstrated by in vitro experiments, LPS preconditioning significantly reduced the upregulation of the ATF4-CHOP pathway and inhibited Caspase-12 and Caspase-3 activation after IRI. Later experiments showed that ATF4 knockdown significantly suppressed CHOP, cleaved caspase-12 and caspase-3 expression, as well as inhibited hepatocellular apoptosis. In addition, in mice pretreated with LPS, TNF-α and IL-6 were inhibited after reperfusion, whereas IL-10 was upregulated. Similarly, low-dose LPS significantly inhibited TNF-α, IL-6, ATF4-CHOP pathway, NF-κB pathway, and ERK1/2 in high-dose LPS-stimulated macrophages, whereas IL-10 and cytokine signaling (SOCS)-3 suppressor were induced. Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages.

Conclusions: This work is the first time to provide evidence for LPS preconditioning protects hepatocytes from IRI through inhibiting ATF4-CHOP pathway, which may be critical to reducing related apoptosis molecules and modulating innate inflammation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 4 / deficiency
  • Activating Transcription Factor 4 / genetics
  • Activating Transcription Factor 4 / metabolism*
  • Animals
  • Apoptosis / drug effects
  • Caspase 12 / metabolism
  • Caspase 3 / metabolism
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Gene Knockdown Techniques
  • Hepatocytes / drug effects*
  • Hepatocytes / immunology
  • Hepatocytes / metabolism
  • Hepatocytes / pathology
  • Hydrogen Peroxide / pharmacology
  • Ischemic Preconditioning*
  • Lipopolysaccharides / pharmacology*
  • Male
  • Mice
  • Mitogen-Activated Protein Kinases / metabolism
  • NF-kappa B / metabolism
  • Reperfusion Injury / immunology
  • Reperfusion Injury / metabolism
  • Reperfusion Injury / pathology
  • Reperfusion Injury / prevention & control*
  • Signal Transduction / drug effects*
  • Transcription Factor CHOP / metabolism*

Substances

  • Atf4 protein, mouse
  • Ddit3 protein, mouse
  • Lipopolysaccharides
  • NF-kappa B
  • Activating Transcription Factor 4
  • Transcription Factor CHOP
  • Hydrogen Peroxide
  • Mitogen-Activated Protein Kinases
  • Caspase 12
  • Caspase 3

Grants and funding

This study was supported by the International Collaboration Foundation of Jiangsu Province (BZ2011041, BK2009439, ZX05 200904, WS2011106, W201201, BZ2012058), Development of Innovative Research Team in the First Affiliated Hospital of NJMU and the National Nature Science Foundation of China (81100270, 81070380, 81310108001, 81210108017, 81273261, 81270583). Open project of the program of “Science and Education to Promote Health” from Health Department of Jiangsu Province (ZX05200903). First Innovation Team Foundation of Jiangsu province Hospital (for Sun BC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.