Liraglutide Attenuates Hepatic Ischemia-Reperfusion Injury by Modulating Macrophage Polarization

Abstract

Ischemia-reperfusion injury (IRI) is a common complication associated with liver surgery, and macrophages play an important role in hepatic IRI. Liraglutide, a glucagon-like peptide-1 (GLP-1) analog primarily used to treat type 2 diabetes and obesity, regulates intracellular calcium homeostasis and protects the cardiomyocytes from injury; however, its role in hepatic IRI is not yet fully understood. This study aimed to investigate whether liraglutide can protect the liver from IRI and determine the possible underlying mechanisms. Our results showed that liraglutide pretreatment significantly alleviated the liver damage caused by ischemia-reperfusion (I/R), as evidenced by H&E staining, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and TUNEL staining. Furthermore, the levels of inflammatory cytokines elicited by I/R were distinctly suppressed by liraglutide pretreatment, accompanied by significant reduction in TNF-α, IL-1β, and IL-6 levels. Furthermore, pretreatment with liraglutide markedly inhibited macrophage type I (M1) polarization during hepatic IRI, as revealed by the significant reduction in CD68⁺ levels in Kupffer cells (KCs) detected via flow cytometry. However, the protective effects of liraglutide on hepatic IRI were partly diminished in GLP-1 receptor-knockout (GLP-1R^-/-) mice. Furthermore, in an in vitro study, we assessed the role of liraglutide in macrophage polarization by examining the expression profiles of M1 in bone marrow-derived macrophages (BMDMs) from GLP-1R^-/- and C57BL/6J mice. Consistent with the results of the in vivo study, liraglutide treatment attenuated the LPS-induced M1 polarization and reduced the expression of M1 markers. However, the inhibitory effect of liraglutide on LPS-induced M1 polarization was largely abolished in BMDMs from GLP-1R^-/- mice. Collectively, our study indicates that liraglutide can ameliorate hepatic IRI by inhibiting macrophage polarization towards an inflammatory phenotype via GLP-1R. Its protective effect against liver IRI suggests that liraglutide may serve as a potential drug for the clinical treatment of liver IRI.

Keywords: acute liver injury; glucagon-like peptide-1 receptor; ischemia-reperfusion; liraglutide; macrophage polarization.

Figure 1

Liraglutide pretreatment ameliorates I/R-induced liver damage. Mice were subjected to 60 min of hepatic ischemia, followed by 2, 6, and 24 h of reperfusion. (A) H&E staining was performed to assess live injury in mice (original magnification 200×). (B, C) Serum ALT and AST levels of the control and liraglutide-treated mice were detected after hepatic IRI (n = 4–6 per group). (D) The degree of liver injury was assessed by Suzuki’s injury score (n = 3 per group). These results were obtained from at least three independent experiments. Values are presented as mean ± SD. **P < 0.01, ^***P < 0.001 vs. the control group.

Figure 2

Liraglutide pretreatment attenuates the apoptosis of liver cells caused by I/R. Mice were subjected to 60 min of hepatic ischemia, followed by 6 h of reperfusion. (A) TUNEL staining was performed, and TUNEL-positive cells were calculated (original magnification 400×). (B) The expression of Bax and Bcl-2 was determined via western blotting. Densitometric analysis of the Bax/β-actin (C), Bcl-2/β-actin (D) ratios is shown. All the results were obtained from at least three independent experiments. Data are presented as mean ± SD, n = 3 per group. ^**P < 0.01, ^***P < 0.001 vs. the Sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. the I/R group.

Figure 3

The protective effect of liraglutide on hepatic IRI may be realized through its action on macrophages. NKs, NKTs, neutrophils, and macrophages were eliminated before liver IRI. Serum ALT and AST levels of each group after NK (A), NKT (B), neutrophil (C), and macrophage (D) depletion. Similar results were obtained from at least three independent experiments. Data are shown as mean ± SD, n = 3–6 per group. ^**P < 0.01, ^***P < 0.001 vs. the I/R+Lira group; ^##P < 0.01 vs. the I/R+Lira group. NKs, natural killer cells; NKTs, natural killer T cells.

Figure 4

Liraglutide pretreatment inhibits I/R-elicited M1 polarization of KCs in the liver. Mice underwent 60 min of liver ischemia and 6 h of reperfusion; liver tissues were harvested for immunofluorescence staining (A) and MPO staining (B) (original magnification 400×), and the number of F4/80⁺/iNOS⁺ positive cells and MPO-positive cells was calculated. The expression of pro-inflammatory cytokines, including TNF-α (C), IL-1β (D), and IL-6 (E), in the liver was detected via qRT-PCR. (F) The number of CD68⁺-positive cells of KCs under F4/80⁺ and CD11b⁺ subsets was calculated via flow cytometry. The results represent the means from at least 3 independent experiments. Values are shown as mean ± SD, n = 3 per group. ^*P < 0.05, ^***P < 0.001 vs. the Sham group; ^#P < 0.05, ^###P < 0.001 vs. the I/R group. ^&&P < 0.01 vs. the I/R+Lira group. KCs, resident macrophages; MPO, myeloperoxidase.

Figure 5

Liraglutide pretreatment reduces LPS-induced M1 polarization in vitro. BMDMs from GLP-1R^-/- and C57BL/6J mice were isolated and cultured for in vitro study. The cells were pretreated with liraglutide (50 µm) for 24 h, stimulated with LPS (100 nm) for 24 h, and the expression profiles of M1 in BMDMs in each group were analyzed via flow cytometry, RT-PCR, and western blotting. (A) The number of CD86⁺-positive cells of BMDMs under F4/80⁺ and CD11b⁺ subsets was calculated via flow cytometry. The mRNA levels of iNOS (B), TNF-α (C), IL-1β (D), and IL-6 (E) were measured via qRT-PCR. (F) The phosphorylation of STAT1 was analyzed via western blotting. Similar results were obtained from at least three independent experiments. Data are expressed as mean ± SD, n = 3 per group. ^**P < 0.01, ^***P < 0.001 vs. the Normal group; ^##P < 0.01 vs. the LPS group. ^&P < 0.05, ^&&P < 0.01 vs. the LPS+Lira group. BMDMs, bone marrow derived macrophages.

Figure 6

The protective effects of liraglutide on hepatic IRI are partly diminished in GLP-1R- knockout mice. A mouse liver partial IRI model was established using C57BL/6J mice and GLP-1R^-/- mice. (A)qRT-PCT was performed to detect GLP-1R expression in KCs from sham-operated, Lira-treated, I/R, and I/R+ Lira-treated groups (n = 3 per group). Serum levels of ALT (B) and AST (C) in each group (n = 4–6 per group). (D) Liver injury, neutrophil infiltration, hepatocyte apoptosis, and macrophage phenotyping were assessed via H&E staining, MPO, TUNEL assays, and immunofluorescence staining, respectively. Quantitation of liver injury (E), neutrophil infiltration (F), hepatocyte apoptosis (G), and macrophage phenotyping (H) in each group (n = 3 per group). These results were obtained from at least three independent experiments. All values are expressed as mean ± SD. ^**P < 0.01, ^***P < 0.001 vs. the Sham group; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. the I/R group. ^&P < 0.05, ^&&P < 0.01 vs. the I/R+Lira group.