Protective effect of lipopolysaccharide preconditioning in hepatic ischaemia reperfusion injury

Abstract

Background: Preconditioning using lipopolysaccharide (LPS), a toll-like receptor 4 (TLR4) ligand, has been demonstrated to reduce ischaemia/reperfusion injury (IRI) in some organs, but its effect in the liver has not been elucidated. We examined the liver protective mechanism and correlated signalling pathway of LPS preconditioning in mice.

Methods: BALB/c and TLR4 mutant mice underwent 90 min of 70% hepatic ischaemia. Lipopolysaccharide (100 µg/kg) was injected intraperitoneally 20 h or 30 min before ischaemia. Liver damage after reperfusion was examined using serum samples and liver specimens. To analyse the mechanism of preconditioning in detail, phosphorylation of representative signalling mediators to nuclear factor-κB (NF-κB) activation, Akt and interleukin-1 receptor-associated kinase-1 (IRAK-1), and expression of a negative feedback inhibitor, suppressor of cytokine signalling-1 (SOCS-1), were evaluated by Western blotting.

Results: Pretreatment with LPS only 20 h before ischaemia elicited a preconditioning effect; however, preconditioning was absent in TLR4 mutant mice. Lipopolysaccharide significantly decreased serum alanine aminotransferase, tumour necrosis factor-α, hepatocyte necrosis and NF-κB activity after reperfusion. Phosphorylated IRAK-1 was suppressed by LPS, whereas no difference was observed in phosphorylated Akt. Pre-ischaemic LPS provided early induction of SOCS-1.

Discussion: Late-phase LPS preconditioning provided liver protection against IRI through the downregulation of the TLR4 cascade derived from early induction of SOCS-1 during ischaemia/reperfusion.

Figure 1

Lipopolysaccharide (LPS) pretreatment reduced serum levels of alanine aminotransferase (ALT) after ischaemia/reperfusion, but in late-phase treatment only. (A) LPS (100 µg/kg) was administered 20 h or 30 min before ischaemia (n = 6). Mice underwent 90 min of ischaemia and subsequent reperfusion. Serum levels of ALT in each sample were measured 6 h after reperfusion. (B) Expression of tumour necrosis factor-α (TNF-α) was examined for evaluation of the pre-ischaemic inflammatory response by reverse-transcription polymerase chain reaction (n = 6). As an endogenous control, the amplification of 18S rRNA was used. Results are expressed as mean ± standard error of the mean. *P < 0.05, compared with the LPC(−) group and the Early-LPC group. LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group; Early-LPC, early-phase LPS preconditioning group

Figure 2

Late-phase lipopolysaccharide (LPS) pretreatment attenuated liver parenchymal necrosis after ischaemia/reperfusion, whereas early-phase pretreatment was unable to prevent liver damage. Mice were subjected to LPS pretreatment, ischaemia and reperfusion as described above. Liver specimens were sampled 6 h after perfusion (n = 6). (A) Effect of LPS pretreatment on necrosis of hepatocytes following ischaemia/reperfusion was evaluated according to the pathological damage score. (B) Liver sections of each group (haematoxylin and eosin stain; original magnification × 100). Results are expressed as mean ± standard error of the mean. *P < 0.05, compared with the LPC(−) group and the Early-LPC group. LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group; Early-LPC, early-phase LPS preconditioning group

Figure 3

Late-phase lipopolysaccharide (LPS) preconditioning made no difference in the phosphorylation of Akt, but inhibited the activation of transcription factor nuclear factor (NF)-κB. Mice underwent 100 µg/kg LPS pretreatment 20 h before 90 min ischaemia and reperfusion. Samples were obtained 1 h after reperfusion (n = 6). (A) Effect of LPS preconditioning on the phosphorylation of Akt was examined by Western blotting. (B) NF-κB binding activity was investigated by electrophoretic mobility shift assay using nuclear extracts prepared from liver samples. LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group; Phospho-, phosphorylated

Figure 4

Toll-like receptor 4 (TLR4) signalling pathway was associated with hepatic protection provided by lipopolysaccharide (LPS) preconditioning. TLR4-mutant mice (C3H/HeJ) and wild-type mice for TLR4-mutant (C3H/HeN) were subjected to ischaemic procedures for 90 min with or without LPS pretreatment. After 6 h of reperfusion, both strains of mice were killed and liver specimens were obtained. Liver sections of (A) mutant mice and (B) wild-type mice (haematoxylin and eosin stain; original magnification × 100). LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group

Figure 5

Lipopolysaccharide (LPS) preconditioning inhibited inflammatory response after ischaemia/reperfusion via the toll-like receptor 4 (TLR4) signalling pathway. Mice underwent LPS pretreatment and ischaemic procedures as described above. Serum and liver samples were obtained 1 h and 6 h after reperfusion, respectively (n = 6). (A) Western blotting showed the difference between the LPC(−) group and the Late-LPC group in phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). (B) Serum levels of tumour necrosis factor-α (TNF-α) were measured for evaluation of the inflammation after reperfusion (n = 6). Results are expressed as mean ± standard error of the mean. *P < 0.05, compared with the LPC(−) group. LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group; Phospho-, phosphorylated

Figure 6

Lipopolysaccharide (LPS) preconditioning induced suppressor of cytokine signalling-1 (SOCS-1) in the earlier post-ischaemic period. Mice were subjected to pretreatment, ischaemia and reperfusion as described above. Protein extracts were prepared from liver parenchyma sampled after reperfusion (n = 5). Expression of SOCS-1 was investigated using (A) reverse transcription polymerase chain reaction and (B) Western blotting. As an endogenous control, the amplification of 18S rRNA was used. Results are expressed as mean ± standard error of the mean. *P < 0.05, compared with the LPC(−) group. LPC(−), LPS-non-treated group; Late-LPC, late-phase LPS preconditioning group