The liver protection of propylene glycol alginate sodium sulfate preconditioning against ischemia reperfusion injury: focusing MAPK pathway activity

Abstract

Hepatic ischemia reperfusion (IR) injury contributes to the morbidity and mortality associated with liver surgery. This study investigated the protective function and mechanism of propylene glycol alginate sodium sulfate (PSS), a sulfated polysaccharide, in a mouse hepatic IR injury model. PSS (25 or 50 mg/kg) or saline were injected intraperitoneally to male Balb/c mice 1 h before 45 min of 70% warm hepatic ischemia and 2, 8, and 24 h of reperfusion. Serum and liver tissue samples were collected for evaluation of hepatocellular damage, liver histology, and assay of inflammatory cytokines, apoptosis- and autophagy-related proteins, and proteins in the mitogen-activated protein kinase (MAPKs). Histological injury and release of transaminases, and inflammatory cytokine production were significantly reduced by PSS pretreatment. The expression of apoptosis- and autophagy-related proteins, and the activation of MAPK signal, including jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and P38 were all affected by PSS treatment compared with IR model controls. PSS protected the liver from IR injury by suppressing the MAPK signaling and down-regulating inflammation, apoptosis, and autophagy.

Figure 1

PSS (25 or 50 mg/kg) had effects on liver function as shown by (A) the serum ALT and AST levels in the NC, PSS (25), and PSS (50) groups 24 h after treatment. Data are means ± SD (n = 6, P > 0.05); (B) TNF-α, IL-6, Bax, Bcl-2, LC3, or Beclin-1 protein expression; or (C) liver histology following HE staining (Original magnification, ×200).

Figure 2

Assessment of PSS treatment in IR-induced liver injury showing (A) serum ALT and AST in the sham operation, IR, PSS (25) +IR, and PSS (50) +IR groups. Data are means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (25) +IR group vs. IR group, ⁺P < 0.05 PSS (50) +IR group vs. IR group. (B) the pathological changes in liver tissue (HE staining, original magnification ×200). Severity of liver injury at 8 h was scored using the Suzuki criteria. Data are means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (25) +IR group vs. IR group, ⁺P < 0.05 PSS (50) +IR group vs. IR group.

Figure 3

Anti-inflammatory activity of PSS in IR injury as shown by (A) expression of TNF-α, IL-6, and IFN-γ mRNA in liver tissue assayed by RT-PCR. Data are means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (25) +IR group vs. IR group, ⁺P < 0.05 PSS (50) +IR group vs. IR group. (B) serum TNF-α, IL-6, and IFN-γ levels by ELISA. Data are means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (25) +IR group vs. IR group, ⁺P < 0.05 PSS (50) +IR group vs. IR group. (C) expression of TNF-α, IL-6, and IFN-γ protein in liver tissue assayed by western blot. (D) TNF-α, IL-6, and IFN-γ immunostaining of liver tissue sections from sham operation, IR, PSS (25) +IR, and PSS (50) +IR groups 8 h after IR. Representative sections from six mice (Original magnification, ×200).

Figure 4

Anti-apoptotic activity of PSS treatment in hepatic IR injury as shown by (A) hepatocellular apoptosis 8 h after IR assayed by TUNEL staining. Both necrosis and apoptosis were found and the apoptotic cells with brown nuclei were counted. Data are means ± SD (n = 6). ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (50) +IR group vs. IR group). (B) expression of Bax, Bcl-2, and cleaved caspase 3 and 9 assayed by western blot. (C) expression of Bax and Bcl-2 mRNA was assayed by RT-PCR. Data are means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (50) +IR group vs. IR group. (D) Bax and Bcl-2 immunostaining in liver tissue from sham operated mice, IR, and PSS (50) +IR group 8 h after IR. Representative sections from six mice (Original magnification, ×200).

Figure 5

Anti-autophagy activity of PSS pretreatment in hepatic IR injury as shown by (A) expression of LC3 II, Beclin-1, and P62 protein assayed by western blotting. (B) expression of LC3 II, Beclin-1, and P62 mRNA by quantitative RT-PCR. Data expressed means ± SD (n = 6) ^#P < 0.05 IR group vs. sham operation group, *P < 0.05 PSS (50) +IR group vs. IR group). (C) LC3 II, Beclin-1 and P62 immunostaining of liver sections from sham operation, IR, and PSS (50) +IR groups 8 h after IR. Representative sections of tissue from six mice (Original magnification, ×200).

Figure 6

Effects of PSS treatment on MAPKs in hepatic IR injury as shown by (A) expression of total JNK, total ERK, total P38, phosphorylated JNK (p-JNK), phosphorylated ERK (p-ERK), phosphorylated P38 (p-P38) assayed by western blotting, (B) p-JNK, p-ERK, p-P38 immunostaining of liver sections from sham operation, IR, and PSS (50) +IR groups 8 h after IR. Representative sections of tissue from six mice (Original magnification, ×200).

Figure 7

Probable mechanisms of PSS pretreatment in protecting against hepatic IR injury. Inflammatory cytokines including TNF-α, IL-6, and IFN-γ produced by Kupffer cells contribute to liver injury and upregulate caspase 3 to induce cellular apoptosis. Activation of JNK and ERK by phosphorylated Bcl-2, an anti-apoptotic protein, to facilitate the hepatocellular apoptosis and autophagy. The P38 MAPK activated by IR downregulates the P62 expression, which in turn leads to an increase of LC3 and formation of autophagosomes that promote autophagic cellular death. PSS treatment significantly downregulated the release of inflammatory factors and activation of MAPKs, reduced apoptosis and autophagy.