Remote ischemic preconditioning protects against liver ischemia-reperfusion injury via heme oxygenase-1-induced autophagy

Yun Wang, Jian Shen, Xuanxuan Xiong, Yonghua Xu, Hai Zhang, Changjun Huang, Yuan Tian, Chengyu Jiao, Xuehao Wang, Xiangcheng Li, Yun Wang, Jian Shen, Xuanxuan Xiong, Yonghua Xu, Hai Zhang, Changjun Huang, Yuan Tian, Chengyu Jiao, Xuehao Wang, Xiangcheng Li

Abstract

Background: Growing evidence has linked autophagy to a protective role of preconditioning in liver ischemia/reperfusion (IR). Heme oxygenase-1 (HO-1) is essential in limiting inflammation and preventing the apoptotic response to IR. We previously demonstrated that HO-1 is up-regulated in liver graft after remote ischemic preconditioning (RIPC). The aim of this study was to confirm that RIPC protects against IR via HO-1-mediated autophagy.

Methods: RIPC was performed with regional ischemia of limbs before liver ischemia, and HO-1 activity was inhibited pre-operation. Autophagy was assessed by the expression of light chain 3-II (LC3-II). The HO-1/extracellular signal-related kinase (ERK)/p38/mitogen-activated protein kinase (MAPK) pathway was detected in an autophagy model and mineral oil-induced IR in vitro.

Results: In liver IR, the expression of LC3-II peaked 12-24 h after IR, and the ultrastructure revealed abundant autophagosomes in hepatocytes after IR. Autophagy was inhibited when HO-1 was inactivated, which we believe resulted in the aggravation of liver IR injury (IRI) in vivo. Hemin-induced autophagy also protected rat hepatocytes from IRI in vitro, which was abrogated by HO-1 siRNA. Phosphorylation of p38-MAPK and ERK1/2 was up-regulated in hemin-pretreated liver cells and down-regulated after treatment with HO-1 siRNA.

Conclusions: RIPC may protect the liver from IRI by induction of HO-1/p38-MAPK-dependent autophagy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Hepatic inflow occlusion and reflow…
Figure 1. Hepatic inflow occlusion and reflow increased hepatic HO-1 and autophagic signaling.
(A) Western blotting for HO-1 and LC3-II in liver lysates from sham-operated or IR-treated mice shows increased HO-1 and LC3-II in the IR group, and the expression of HO-1, LC3-II peaked at 6–12 h and 12–24 h, respectively(*P

Figure 2. Remote ischemic preconditioning induced HO-1…

Figure 2. Remote ischemic preconditioning induced HO-1 to increase autophagy, but the inhibition of HO-1…

Figure 2. Remote ischemic preconditioning induced HO-1 to increase autophagy, but the inhibition of HO-1 by Znpp decreased autophagy.
(A) HO-1 was downregulated in the 12-h Znpp+IR group compared with the sham and 12-h IR groups (*P#P<0.05 compared with the IR group). (B) The data were quantified by counting the number of autophagosomes per cross-sectioned cell (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group, n = 15). (C) IR treatment increased autophagy compared to sham as observed by transmission electron microscopy and increased autophagosome formation in the RIPC+IR group, whereas autophagosomes were rare in the Znpp+IR group (black arrows). (D) Increased HO-1 and LC3-II staining was observed in the RIPC+IR group compared with the IR group, and LC3-II staining was decreased after inhibition of HO-1 (*P<0.05 compared with the IR group at the corresponding time point; #P<0.05 compared with the RIPC+IR group at the corresponding time point).

Figure 3. Increased HO-1 and autophagy alleviated…

Figure 3. Increased HO-1 and autophagy alleviated liver damage.

(A) IR treatment increases the histopathologic…

Figure 3. Increased HO-1 and autophagy alleviated liver damage.
(A) IR treatment increases the histopathologic changes of liver. Extensive areas of hepatocyte necrosis (N), vacuolation (V) and sinusoidal congestion were observed in the IR group. The Znpp+IR and Znpp+RIPC+IR group showed markedly higher hepatocyte necrosis accumulation (N. A) and vacuo(V), whereas the RIPC+IR group still showed large areas of normal liver architecture, similar to the sham group mice (original magnification 200×; insets 400×). (B) The mean injury score of the RIPC+IR group was significantly lower than the IR group according to Suzuki's histologic classification. The mean injury scores in the Znpp+IR and Znpp+RIPC+IR groups were significantly higher than in the IR and RIPC+IR groups (*P#P<0.05 compared with the RIPC+IR group). (C) IR treatment increased serum aminotransferase levels in mice compared with controls. Compared with the IR group, serum aminotransferase levels were lower in the RIPC+IR group. The mean serum levels of ALT and AST were highest in the Znpp+IR and Znpp+RIPC+IR groups. There were no significant differences in aminotransferase levels in the Znpp+IR and Znpp+RIPC+IR groups (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group).

Figure 4. Induction of autophagy was dependent…

Figure 4. Induction of autophagy was dependent on HO-1 in vivo.

(A) Increased HO-1 and…

Figure 4. Induction of autophagy was dependent on HO-1 in vivo.
(A) Increased HO-1 and LC3-II staining was observed in the RIPC+IR group compared with the Sham, IR, Znpp+IR, and Znpp+RIPC+IR groups, and there were no significant differences in the expression of HO-1 and LC3-II protein between the Znpp+IR and Znpp+RIPC+IR groups (*P#P<0.05 compared with the RIPC+IR group). (B) The data were quantified by counting the number of autophagosomes per cross-sectioned cell (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group, n = 15). (C) IR treatment increased autophagy as observed by transmission electron microscopy compared to the sham group and showed increased autophagosome formation in the RIPC+IR group, whereas autophagosomes were rare in the Znpp+IR and Znpp+RIPC+IR groups.

Figure 5. IR simulation in AML12 cells…

Figure 5. IR simulation in AML12 cells increased LC3-II expression; HO-1 siRNA effectively inhibited HO-1…

Figure 5. IR simulation in AML12 cells increased LC3-II expression; HO-1 siRNA effectively inhibited HO-1 expression.
(A) LC3-II staining increased in the IR cell model and peaked at 12–24 h (*P#P<0.05 compared with the SS group). (D) Western blotting showed lower expression of HO-1 protein in the S1 group compared with the SS and S2 groups (*P<0.05 compared with the sham group; #P<0.05 compared with the SS group).

Figure 6. Inhibition of HO-1 increased cell…

Figure 6. Inhibition of HO-1 increased cell death and decreased autophagy in AML12 cells.

(A)…

Figure 6. Inhibition of HO-1 increased cell death and decreased autophagy in AML12 cells.
(A) Immunofluorescence of AML12 cells demonstrated increased LC3-II staining in the HIR group; however, downregulated HO-1 decreased autophagy. (B) Increased photometric values of protein in the HIR group compared with the IR and HSIR groups are shown, and the lowest photometric values of protein were observed in the HSIR group (*P#P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (C) Induction of HO-1 and autophagy decreased biochemical enzyme levels, whereas inhibition of HO-1 and autophagy increased biochemical enzyme levels (*P<0.01 compared with the IR group; #P<0.05 compared with the HIR group). (D) The expression of HO-1 and autophagy proteins was upregulated by hemin, but downregulated by treatment with HO-1 siRNA (*P<0.01 compared with the sham group; #P<0.05 compared with the IR group; &P<0.05 compared with the HIR group).

Figure 7. HO-1 induced autophagy through activation…

Figure 7. HO-1 induced autophagy through activation of ERK1/2 and p38-MAPK.

(A) Apoptotic rates of…

Figure 7. HO-1 induced autophagy through activation of ERK1/2 and p38-MAPK.
(A) Apoptotic rates of the sham group and three treated groups are shown. Mineral oil treatment increased the rate of early and late apoptosis, and HO-1 siRNA treatment before IR increased the rate of apoptosis compared with the IR group. The HIR group had lower rates of apoptosis, similar to the sham group (*P#P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (B) Hemin treatment before IR increased phosphorylated ERK1/2 staining, as shown in immunoblotting, and HSIR group cells showed decreased phosphorylated ERK1/2 expression. Western blotting for phosphorylated p38-MAPK demonstrated increased phosphorylated p38-MAPK in the HIR group, but decreased signaling in the HSIR group (*P<0.01 compared with the sham group; #P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (C) HO-1 expression was decreased in the EIIR group compared with the IR group, and LC3-II expression was decreased in the PIIR group compared with the IR group (*P<0.05 compared with the sham group; #P<0.05 compared with the IR group). (D) HO-1 expression was nearly the same as in the PIIR and IR groups, but LC3-II expression was decreased in the EIIR group compared with the IR group (*P<0.05 compared with the sham group; #P<0.05 compared with the IR group).
All figures (7)
Figure 2. Remote ischemic preconditioning induced HO-1…
Figure 2. Remote ischemic preconditioning induced HO-1 to increase autophagy, but the inhibition of HO-1 by Znpp decreased autophagy.
(A) HO-1 was downregulated in the 12-h Znpp+IR group compared with the sham and 12-h IR groups (*P#P<0.05 compared with the IR group). (B) The data were quantified by counting the number of autophagosomes per cross-sectioned cell (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group, n = 15). (C) IR treatment increased autophagy compared to sham as observed by transmission electron microscopy and increased autophagosome formation in the RIPC+IR group, whereas autophagosomes were rare in the Znpp+IR group (black arrows). (D) Increased HO-1 and LC3-II staining was observed in the RIPC+IR group compared with the IR group, and LC3-II staining was decreased after inhibition of HO-1 (*P<0.05 compared with the IR group at the corresponding time point; #P<0.05 compared with the RIPC+IR group at the corresponding time point).
Figure 3. Increased HO-1 and autophagy alleviated…
Figure 3. Increased HO-1 and autophagy alleviated liver damage.
(A) IR treatment increases the histopathologic changes of liver. Extensive areas of hepatocyte necrosis (N), vacuolation (V) and sinusoidal congestion were observed in the IR group. The Znpp+IR and Znpp+RIPC+IR group showed markedly higher hepatocyte necrosis accumulation (N. A) and vacuo(V), whereas the RIPC+IR group still showed large areas of normal liver architecture, similar to the sham group mice (original magnification 200×; insets 400×). (B) The mean injury score of the RIPC+IR group was significantly lower than the IR group according to Suzuki's histologic classification. The mean injury scores in the Znpp+IR and Znpp+RIPC+IR groups were significantly higher than in the IR and RIPC+IR groups (*P#P<0.05 compared with the RIPC+IR group). (C) IR treatment increased serum aminotransferase levels in mice compared with controls. Compared with the IR group, serum aminotransferase levels were lower in the RIPC+IR group. The mean serum levels of ALT and AST were highest in the Znpp+IR and Znpp+RIPC+IR groups. There were no significant differences in aminotransferase levels in the Znpp+IR and Znpp+RIPC+IR groups (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group).
Figure 4. Induction of autophagy was dependent…
Figure 4. Induction of autophagy was dependent on HO-1 in vivo.
(A) Increased HO-1 and LC3-II staining was observed in the RIPC+IR group compared with the Sham, IR, Znpp+IR, and Znpp+RIPC+IR groups, and there were no significant differences in the expression of HO-1 and LC3-II protein between the Znpp+IR and Znpp+RIPC+IR groups (*P#P<0.05 compared with the RIPC+IR group). (B) The data were quantified by counting the number of autophagosomes per cross-sectioned cell (*P<0.05 compared with the IR group; #P<0.05 compared with the RIPC+IR group, n = 15). (C) IR treatment increased autophagy as observed by transmission electron microscopy compared to the sham group and showed increased autophagosome formation in the RIPC+IR group, whereas autophagosomes were rare in the Znpp+IR and Znpp+RIPC+IR groups.
Figure 5. IR simulation in AML12 cells…
Figure 5. IR simulation in AML12 cells increased LC3-II expression; HO-1 siRNA effectively inhibited HO-1 expression.
(A) LC3-II staining increased in the IR cell model and peaked at 12–24 h (*P#P<0.05 compared with the SS group). (D) Western blotting showed lower expression of HO-1 protein in the S1 group compared with the SS and S2 groups (*P<0.05 compared with the sham group; #P<0.05 compared with the SS group).
Figure 6. Inhibition of HO-1 increased cell…
Figure 6. Inhibition of HO-1 increased cell death and decreased autophagy in AML12 cells.
(A) Immunofluorescence of AML12 cells demonstrated increased LC3-II staining in the HIR group; however, downregulated HO-1 decreased autophagy. (B) Increased photometric values of protein in the HIR group compared with the IR and HSIR groups are shown, and the lowest photometric values of protein were observed in the HSIR group (*P#P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (C) Induction of HO-1 and autophagy decreased biochemical enzyme levels, whereas inhibition of HO-1 and autophagy increased biochemical enzyme levels (*P<0.01 compared with the IR group; #P<0.05 compared with the HIR group). (D) The expression of HO-1 and autophagy proteins was upregulated by hemin, but downregulated by treatment with HO-1 siRNA (*P<0.01 compared with the sham group; #P<0.05 compared with the IR group; &P<0.05 compared with the HIR group).
Figure 7. HO-1 induced autophagy through activation…
Figure 7. HO-1 induced autophagy through activation of ERK1/2 and p38-MAPK.
(A) Apoptotic rates of the sham group and three treated groups are shown. Mineral oil treatment increased the rate of early and late apoptosis, and HO-1 siRNA treatment before IR increased the rate of apoptosis compared with the IR group. The HIR group had lower rates of apoptosis, similar to the sham group (*P#P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (B) Hemin treatment before IR increased phosphorylated ERK1/2 staining, as shown in immunoblotting, and HSIR group cells showed decreased phosphorylated ERK1/2 expression. Western blotting for phosphorylated p38-MAPK demonstrated increased phosphorylated p38-MAPK in the HIR group, but decreased signaling in the HSIR group (*P<0.01 compared with the sham group; #P<0.05 compared with the IR group; &P<0.05 compared with the HIR group). (C) HO-1 expression was decreased in the EIIR group compared with the IR group, and LC3-II expression was decreased in the PIIR group compared with the IR group (*P<0.05 compared with the sham group; #P<0.05 compared with the IR group). (D) HO-1 expression was nearly the same as in the PIIR and IR groups, but LC3-II expression was decreased in the EIIR group compared with the IR group (*P<0.05 compared with the sham group; #P<0.05 compared with the IR group).

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