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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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Reduction of hepatocellular apoptosis after liver IR through LPS preconditioning.(A) Liver apoptosis by TUNEL stainning: (a) sham group; (b) IR group and (c) LPS PC+IR group. (B) Apoptotic cells were quantified in six high-power fields (400×), and expressed as percentages of apoptotic cells among total cells. Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C) Caspase-3 activity, Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group.
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pone-0065568-g002: Reduction of hepatocellular apoptosis after liver IR through LPS preconditioning.(A) Liver apoptosis by TUNEL stainning: (a) sham group; (b) IR group and (c) LPS PC+IR group. (B) Apoptotic cells were quantified in six high-power fields (400×), and expressed as percentages of apoptotic cells among total cells. Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C) Caspase-3 activity, Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group.

Mentions: Hepatocellular apoptosis was analyzed by TUNEL assay 24 h after reperfusion. TUNEL-positive cells were significantly lower in LPS preconditioned IR liver compared with those in IR control (Fig. 2A). TUNEL-positive cells in the total hepatocytes of three groups were (0.60±0.25)%, (10.20±1.28)%, and (4.00±0.63)%, respectively, indicating that hepatocyte apoptosis was significantly inhibited by LPS preconditioning (Fig. 2B). Apoptotic active caspase-3 directly caused hepatocellular apoptosis after liver IR and reflected the status of apoptosis. Along with the TUNEL assay, Fig. 2C shows that the activity of caspase-3 is significantly inhibited after LPS preconditioning in ischemic liver tissue compared with the IR group (1.38±0.08 and 4.44±0.289, respectively; P<0.001).


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)

Reduction of hepatocellular apoptosis after liver IR through LPS preconditioning.(A) Liver apoptosis by TUNEL stainning: (a) sham group; (b) IR group and (c) LPS PC+IR group. (B) Apoptotic cells were quantified in six high-power fields (400×), and expressed as percentages of apoptotic cells among total cells. Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C) Caspase-3 activity, Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group.
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Related In: Results  -  Collection

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

pone-0065568-g002: Reduction of hepatocellular apoptosis after liver IR through LPS preconditioning.(A) Liver apoptosis by TUNEL stainning: (a) sham group; (b) IR group and (c) LPS PC+IR group. (B) Apoptotic cells were quantified in six high-power fields (400×), and expressed as percentages of apoptotic cells among total cells. Mean±SD, **P<0.001 versus sham group; #P<0.05 versus IR group. (C) Caspase-3 activity, Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group.
Mentions: Hepatocellular apoptosis was analyzed by TUNEL assay 24 h after reperfusion. TUNEL-positive cells were significantly lower in LPS preconditioned IR liver compared with those in IR control (Fig. 2A). TUNEL-positive cells in the total hepatocytes of three groups were (0.60±0.25)%, (10.20±1.28)%, and (4.00±0.63)%, respectively, indicating that hepatocyte apoptosis was significantly inhibited by LPS preconditioning (Fig. 2B). Apoptotic active caspase-3 directly caused hepatocellular apoptosis after liver IR and reflected the status of apoptosis. Along with the TUNEL assay, Fig. 2C shows that the activity of caspase-3 is significantly inhibited after LPS preconditioning in ischemic liver tissue compared with the IR group (1.38±0.08 and 4.44±0.289, respectively; P<0.001).

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