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

Rao J, Qin J, Qian X, Lu L, Wang P, Wu Z, Zhai Y, Zhang F, Li G, Wang X - PLoS ONE (2013)

Bottom Line: 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.Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages.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.

View Article: PubMed Central - PubMed

Affiliation: Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing, PR China.

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.

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ATF4 knockdown protection against hepatocellular death induced by H2O2 in vitro.(A) Western-assisted analysis of ATF4, CHOP, Cleaved Caspase-12, Cleaved Caspase-3 and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4, CHOP, Cleaved Caspase-12 and Cleaved Caspase-3 to β-Actin, Mean±SD, **P<0.001 or *P<0.05 versus CONsiRNA. (C)The released LDH level of hepatocytes after H2O2 treatment, **P<0.001 versus CONsiRNA.
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pone-0065568-g005: ATF4 knockdown protection against hepatocellular death induced by H2O2 in vitro.(A) Western-assisted analysis of ATF4, CHOP, Cleaved Caspase-12, Cleaved Caspase-3 and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4, CHOP, Cleaved Caspase-12 and Cleaved Caspase-3 to β-Actin, Mean±SD, **P<0.001 or *P<0.05 versus CONsiRNA. (C)The released LDH level of hepatocytes after H2O2 treatment, **P<0.001 versus CONsiRNA.

Mentions: This work explored whether ATF4 expression manipulation using ATF4 siRNA provides protection against hepatocellular death induced by H2O2. Firstly, primary hepatocytes in ATF4 siRNA were transiently transfected. Second, ATF4 protein expression was significantly suppressed, compared with the cells of the negative control (Figs. 5A and B), indicating that the knockdown of ATF4 expression was successful in liver cells. Then, related protein expressions (CHOP, cleaved caspase-12, and cleaved caspase-3) were further examined, which showed that the knockdown of ATF4 also suppressed CHOP, cleaved caspase-12, and cleaved caspase-3 as induced by H2O2 treatment (Figs. 5A and B). Next, the released LDH level of hepatocytes was checked in the supernatant after H2O2 treatment for 24 h (Fig. 5C). Compared with the control siRNA, ATF4 knockdown significantly reduced the LDH level (36.66±6.08 and 18.18±0.52, respectively; P<0.05). The repression of ATF4 expression was observed to attenuate the induction of related protein apoptosis.


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

Rao J, Qin J, Qian X, Lu L, Wang P, Wu Z, Zhai Y, Zhang F, Li G, Wang X - PLoS ONE (2013)

ATF4 knockdown protection against hepatocellular death induced by H2O2 in vitro.(A) Western-assisted analysis of ATF4, CHOP, Cleaved Caspase-12, Cleaved Caspase-3 and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4, CHOP, Cleaved Caspase-12 and Cleaved Caspase-3 to β-Actin, Mean±SD, **P<0.001 or *P<0.05 versus CONsiRNA. (C)The released LDH level of hepatocytes after H2O2 treatment, **P<0.001 versus CONsiRNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672158&req=5

pone-0065568-g005: ATF4 knockdown protection against hepatocellular death induced by H2O2 in vitro.(A) Western-assisted analysis of ATF4, CHOP, Cleaved Caspase-12, Cleaved Caspase-3 and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4, CHOP, Cleaved Caspase-12 and Cleaved Caspase-3 to β-Actin, Mean±SD, **P<0.001 or *P<0.05 versus CONsiRNA. (C)The released LDH level of hepatocytes after H2O2 treatment, **P<0.001 versus CONsiRNA.
Mentions: This work explored whether ATF4 expression manipulation using ATF4 siRNA provides protection against hepatocellular death induced by H2O2. Firstly, primary hepatocytes in ATF4 siRNA were transiently transfected. Second, ATF4 protein expression was significantly suppressed, compared with the cells of the negative control (Figs. 5A and B), indicating that the knockdown of ATF4 expression was successful in liver cells. Then, related protein expressions (CHOP, cleaved caspase-12, and cleaved caspase-3) were further examined, which showed that the knockdown of ATF4 also suppressed CHOP, cleaved caspase-12, and cleaved caspase-3 as induced by H2O2 treatment (Figs. 5A and B). Next, the released LDH level of hepatocytes was checked in the supernatant after H2O2 treatment for 24 h (Fig. 5C). Compared with the control siRNA, ATF4 knockdown significantly reduced the LDH level (36.66±6.08 and 18.18±0.52, respectively; P<0.05). The repression of ATF4 expression was observed to attenuate the induction of related protein apoptosis.

Bottom Line: 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.Importantly, ATF4 siRNA is consistent with results of LPS preconditioning in macrophages.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.

View Article: PubMed Central - PubMed

Affiliation: Liver Transplantation Center, The First Affiliated Hospital of Nanjing Medical University, Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Nanjing, PR China.

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.

Show MeSH
Related in: MedlinePlus