Limits...
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.

Show MeSH

Related in: MedlinePlus

ATF4-CHOP pathway inhibited by low-dose LPS preconditioning in hepatocytes.(A) Western-assisted analysis of ATF4, CHOP and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4 and CHOP to β-Actin, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C)The released LDH level of hepatocytes after TM or H2O2 treatment, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672158&req=5

pone-0065568-g004: ATF4-CHOP pathway inhibited by low-dose LPS preconditioning in hepatocytes.(A) Western-assisted analysis of ATF4, CHOP and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4 and CHOP to β-Actin, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C)The released LDH level of hepatocytes after TM or H2O2 treatment, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group.

Mentions: In addressing the role of low-dose LPS preconditioning on ATF4-CHOP pathway in hepatocytes, primary hepatocytes were previously treated by 10 ng/ml LPS for 8 h, then by 1 µg/ml TM for 6 h. The expression of ATF4 and CHOP was then analyzed by Western blot, which showed that LPS preconditioning significantly repressed ATF4 and TM-induced CHOP (Figs. 4A and B). Furthermore, the direct role of LPS preconditioning on hepatocellular death was studied by the released LDH level of hepatocytes induced by TM or H2O2 in vitro. Figure 4C shows that LPS preconditioning significantly reduced the released LDH level of hepatocytes after TM (18.01±0.60 and 27.13±2.26, respectively; P<0.05) or H2O2 treatment (36.60±1.37 and 46.93±2.25, respectively; P<0.05).


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-CHOP pathway inhibited by low-dose LPS preconditioning in hepatocytes.(A) Western-assisted analysis of ATF4, CHOP and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4 and CHOP to β-Actin, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C)The released LDH level of hepatocytes after TM or H2O2 treatment, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065568-g004: ATF4-CHOP pathway inhibited by low-dose LPS preconditioning in hepatocytes.(A) Western-assisted analysis of ATF4, CHOP and β-Actin. Representative of three experiments. (B) Relative quantities of protein of ATF4 and CHOP to β-Actin, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C)The released LDH level of hepatocytes after TM or H2O2 treatment, Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group.
Mentions: In addressing the role of low-dose LPS preconditioning on ATF4-CHOP pathway in hepatocytes, primary hepatocytes were previously treated by 10 ng/ml LPS for 8 h, then by 1 µg/ml TM for 6 h. The expression of ATF4 and CHOP was then analyzed by Western blot, which showed that LPS preconditioning significantly repressed ATF4 and TM-induced CHOP (Figs. 4A and B). Furthermore, the direct role of LPS preconditioning on hepatocellular death was studied by the released LDH level of hepatocytes induced by TM or H2O2 in vitro. Figure 4C shows that LPS preconditioning significantly reduced the released LDH level of hepatocytes after TM (18.01±0.60 and 27.13±2.26, respectively; P<0.05) or H2O2 treatment (36.60±1.37 and 46.93±2.25, respectively; P<0.05).

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