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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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Role of ATF4-CHOP pathway in modulating immune responses of LPS preconditioning.(A) Cytokine gene (TNF-α, IL-6 and IL-10) expression in livers harvested 6 hours after reperfusion by Quantitative RT-PCR analysis. Mean ± SD, **P<0.001 versus sham group; ##P<0.001 versus IR group; #P<0.05 versus IR group. (B) Cytokine gene (TNF-α, IL-6 and IL-10)expression in BM-macrophage stimulated by 1 µg/ml LPS treatment for 3 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (C) Cytokine secretion (TNF-α, IL-6 and IL-10) by ELISA in BM-macrophage stimulated by 1 µg/ml LPS treatment for 24 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (D) Western-assisted analysis of ATF4, CHOP, P-NF-κB p65, IκBα, SCOS3, p-ERK1/2 and β-Actin. Representative of three experiments. The relative quantities of protein to β-Actin, Mean±SD, **P<0.001 versus CON group, #P<0.05 versus LPS PC+LPS group.
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pone-0065568-g006: Role of ATF4-CHOP pathway in modulating immune responses of LPS preconditioning.(A) Cytokine gene (TNF-α, IL-6 and IL-10) expression in livers harvested 6 hours after reperfusion by Quantitative RT-PCR analysis. Mean ± SD, **P<0.001 versus sham group; ##P<0.001 versus IR group; #P<0.05 versus IR group. (B) Cytokine gene (TNF-α, IL-6 and IL-10)expression in BM-macrophage stimulated by 1 µg/ml LPS treatment for 3 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (C) Cytokine secretion (TNF-α, IL-6 and IL-10) by ELISA in BM-macrophage stimulated by 1 µg/ml LPS treatment for 24 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (D) Western-assisted analysis of ATF4, CHOP, P-NF-κB p65, IκBα, SCOS3, p-ERK1/2 and β-Actin. Representative of three experiments. The relative quantities of protein to β-Actin, Mean±SD, **P<0.001 versus CON group, #P<0.05 versus LPS PC+LPS group.

Mentions: A large quantity of inflammatory cytokines is involved in hepatocellular apoptosis and necrosis after IR. TNF-α and IL-6 showed a proapoptotic role during liver IR. On the other hand, IL-10, an anti-inflammatory cytokine, was demonstrated to reduce liver IRI. In further assessing the hepatoprotective effects of LPS preconditioning, mRNA expressions of TNF-α, IL-6, and IL-10 were analyzed in ischemic liver after 6 h of reperfusion by real-time polymerase chain reaction. Figure 6A shows a significantly lower level of TNF-α (0.62±0.10 and 2.47±0.28, respectively; P<0.001) and IL-6 (0.13±0.05 and 0.37±0.06, respectively; P<0.05) in the LPS preconditioning group compared with that in the IR group. By contrast, IL-10 expression was significantly higher in the LPS pretreatment group than in the IR group (0.67±0.08 and 3.51±0.58, respectively; P<0.001). These data indicated that LPS preconditioning inhibited the expression of proapoptotic cytokines and induced the expression of anti-inflammatoy cytokines during liver IR. BM-macrophages were previously treated by 1 ng/ml LPS for 8 h, then by 1 µg/ml LPS treatment for 3 h for real-time polymerase chain reaction and 24 h for ELISA to assess the modulating role of LPS pretreatment on immune responses. Fig. 6B shows that LPS preconditioning significantly reduced TNF-α and IL-6 expression as well as upregulated IL-10 expression. The secretion of these cytokines in the supernatant was in line with cell mRNA expression (Fig. 6C).


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)

Role of ATF4-CHOP pathway in modulating immune responses of LPS preconditioning.(A) Cytokine gene (TNF-α, IL-6 and IL-10) expression in livers harvested 6 hours after reperfusion by Quantitative RT-PCR analysis. Mean ± SD, **P<0.001 versus sham group; ##P<0.001 versus IR group; #P<0.05 versus IR group. (B) Cytokine gene (TNF-α, IL-6 and IL-10)expression in BM-macrophage stimulated by 1 µg/ml LPS treatment for 3 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (C) Cytokine secretion (TNF-α, IL-6 and IL-10) by ELISA in BM-macrophage stimulated by 1 µg/ml LPS treatment for 24 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (D) Western-assisted analysis of ATF4, CHOP, P-NF-κB p65, IκBα, SCOS3, p-ERK1/2 and β-Actin. Representative of three experiments. The relative quantities of protein to β-Actin, Mean±SD, **P<0.001 versus CON group, #P<0.05 versus LPS PC+LPS group.
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getmorefigures.php?uid=PMC3672158&req=5

pone-0065568-g006: Role of ATF4-CHOP pathway in modulating immune responses of LPS preconditioning.(A) Cytokine gene (TNF-α, IL-6 and IL-10) expression in livers harvested 6 hours after reperfusion by Quantitative RT-PCR analysis. Mean ± SD, **P<0.001 versus sham group; ##P<0.001 versus IR group; #P<0.05 versus IR group. (B) Cytokine gene (TNF-α, IL-6 and IL-10)expression in BM-macrophage stimulated by 1 µg/ml LPS treatment for 3 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (C) Cytokine secretion (TNF-α, IL-6 and IL-10) by ELISA in BM-macrophage stimulated by 1 µg/ml LPS treatment for 24 hours. Mean ± SD, **P<0.001 versus CON group; *P<0.05 versus CON group, ##P<0.001 versus LPS PC+LPS group. (D) Western-assisted analysis of ATF4, CHOP, P-NF-κB p65, IκBα, SCOS3, p-ERK1/2 and β-Actin. Representative of three experiments. The relative quantities of protein to β-Actin, Mean±SD, **P<0.001 versus CON group, #P<0.05 versus LPS PC+LPS group.
Mentions: A large quantity of inflammatory cytokines is involved in hepatocellular apoptosis and necrosis after IR. TNF-α and IL-6 showed a proapoptotic role during liver IR. On the other hand, IL-10, an anti-inflammatory cytokine, was demonstrated to reduce liver IRI. In further assessing the hepatoprotective effects of LPS preconditioning, mRNA expressions of TNF-α, IL-6, and IL-10 were analyzed in ischemic liver after 6 h of reperfusion by real-time polymerase chain reaction. Figure 6A shows a significantly lower level of TNF-α (0.62±0.10 and 2.47±0.28, respectively; P<0.001) and IL-6 (0.13±0.05 and 0.37±0.06, respectively; P<0.05) in the LPS preconditioning group compared with that in the IR group. By contrast, IL-10 expression was significantly higher in the LPS pretreatment group than in the IR group (0.67±0.08 and 3.51±0.58, respectively; P<0.001). These data indicated that LPS preconditioning inhibited the expression of proapoptotic cytokines and induced the expression of anti-inflammatoy cytokines during liver IR. BM-macrophages were previously treated by 1 ng/ml LPS for 8 h, then by 1 µg/ml LPS treatment for 3 h for real-time polymerase chain reaction and 24 h for ELISA to assess the modulating role of LPS pretreatment on immune responses. Fig. 6B shows that LPS preconditioning significantly reduced TNF-α and IL-6 expression as well as upregulated IL-10 expression. The secretion of these cytokines in the supernatant was in line with cell mRNA expression (Fig. 6C).

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