Figure 1.
Attenuating liver IRI through low-dosage LPS preconditioning.
Mice were subjected to 90 mim of partial liver ischemia, followed by 6 h and 24 h reperfusion. (A) and (B) Hepatocellular function evaluated by ALT (U/L) and LDH (U/L). Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group. (C) Histopathalogic analysis of livers harvested 6 hours after reperfusion: (a) Sham group: Normal hepatic architecture; (b) IR group: severe hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and massive necrosis; (c) LPS PC+IR group: mild vacuolization, punctate necrosis and edeman. (D) The severity of liver IRI by Suzuki’s histological grading.
Figure 2.
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.
Figure 3.
ATF4-CHOP pathway and related apoptotic pathway inhibition by LPS preconditioning after IR.
(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, Cleaved Caspase-3 to β-Actin, Mean±SD, **P<0.001 versus sham group; ##P<0.001 versus IR group; #P<0.05 versus IR group. (C) immunohistochmistry analysis of CHOP: (a) sham group; (b) IR group and (c) LPS PC+IR group. Positive cells were quantified in six high-power fields (400×), and expressed as percentages of positive cells among total cells. Mean±SD,**P<0.001 versus sham group; ##P<0.001 versus IR group.
Figure 4.
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.
Figure 5.
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.
Figure 6.
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.
Figure 7.
ATF4 knockdown inhibition of immune response in vitro.
(A) 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. (B) 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 or *P<0.05 versus CONsiRNA group.
Figure 8.
Knockdown of ATF4 improves hepatocellular function and ameliorates liver IRI.
Mice were subjected to 90 mim of partial liver ischemia, followed by 6 h reperfusion. (A) Hepatocellular function evaluated by ALT (U/L). Mean±SD, **P<0.001; ##P<0.001. (B) Histopathalogic analysis: (a) Sham group: normal hepatic architecture; (b) CON group: severe hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and massive necrosis; (c) ATF4 siRNA group: mild vacuolization, punctate necrosis and edeman; and (d) Ns siRNA: severe hepatic lobule distortion, sinusoidal congestion, apparent edema, vacuolization and massive necrosis. (C) The severity of liver IRI by Suzuki’s histological grading. Mean±SD, **P<0.001; ##P<0.001.