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. 2025 Dec 8;110(3):e583–e594. doi: 10.1097/TP.0000000000005594

Role of Histamine in Liver Ischemia-Reperfusion Injury—Implication for H1 Receptor Antagonist in the Clinical Application

Hiroshi Kawamoto 1,2, Yoichiro Uchida 1,2,✉, Hirofumi Hirao 1, Kenichi Saga 1, Junya Kawasoe 2, Kentaro Kadono 1, Masaaki Ito 1, Yuki Kidoguchi 1, Kosuke Tanaka 1, Shoichi Kageyama 1, Kojiro Nakamura 1, Kojiro Taura 2, Hiroaki Terajima 2, Takeshi Watanabe 3, Etsuro Hatano 1
PMCID: PMC12908635  PMID: 41358797

Abstract

Background.

Liver ischemia-reperfusion injury (IRI) is a critical complication of liver resection and transplantation. Our previous research demonstrated that preoperative fasting regimen significantly mitigated liver IRI in mice, whereas how fasting alleviates liver IRI remains largely unknown. In this study, we aimed to identify the metabolites that contribute to the pathology of liver IRI and to investigate their roles in modulating liver damage.

Methods.

Comprehensive metabolomic analysis was performed on liver tissues obtained from wild-type (WT) mice subjected to partial warm liver ischemia and reperfusion with and without preoperative fasting. To investigate the role of histamine in liver IRI, histamine H1 receptor knockout (Hrh1KO) and WT mice were exposed to liver IRI. Some WT and Hrh1KO mice were pretreated with H1-antihistamines before the ischemia challenge.

Results.

Among the identified metabolites, histamine displayed a distinctive pattern, with significantly elevated levels in the ischemia and reperfusion insult group without preoperative fasting and substantially reduced levels in the preoperative fasting group. Significant amelioration of liver IRI was observed in Hrh1KO mice, accompanied by a profound reduction in the release of high-mobility group box 1 into the bloodstream. Furthermore, administration of H1-antihistamines, which block histamine binding to the histamine H1 receptor (H1R) and suppress basal H1R activity, significantly inhibited high-mobility group box 1 release and ameliorated liver IRI.

Conclusions.

Histamine is a key metabolite that exacerbates liver IRI via H1R-mediated signaling pathways. These findings highlight the potential of H1-antihistamines as a promising therapeutic approach for mitigating liver injury associated with liver surgery and liver transplantation.

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INTRODUCTION

Liver ischemia-reperfusion injury (IRI) poses a significant challenge in cases of shock, trauma, and liver surgeries such as hepatectomy and transplantation. In liver transplantation, IRI is related to early allograft dysfunction,1 responsible for approximately 10% of early graft failures, and contributes to an increased risk of both acute and chronic rejection.2 Ischemic stress causes hepatic glycogen consumption, oxygen deprivation, and adenosine triphosphate depletion. These changes provoke the production of reactive oxygen species and promote organelle damage, initiating hepatic cell death or injury, which ultimately releases damage-associated molecular patterns (DAMPs) from the liver. After reperfusion, released DAMPs, such as the intracellular nuclear protein high-mobility group box 1 (HMGB1),3 activate host innate immune cells and evoke inflammatory cascades, further accelerating hepatocellular damage,4-6 and in severe cases, leading to liver failure.7,8 Although it is essential to mitigate liver IRI for better clinical outcomes, the underlying mechanisms are not fully understood, and the development of novel therapeutic approaches is warranted.

A growing body of evidence suggests that preoperative fasting is a noninvasive strategy to attenuate liver IRI.9 Indeed, several studies have reported that a 2–3-d fasting regimen before ischemia could significantly mitigate liver IRI in mice.10,11 In contrast, our previous research revealed that even a short-duration fasting intervention of 12 h, recognized as the minimum effective duration,9 before ischemic insult, markedly attenuated liver IRI in mice.12 This study found that fasted mice exhibited significantly reduced serum HMGB1 levels at multiple time points (0, 1, 3, 6, and 12 h) after reperfusion compared with non-fasted controls. Additionally, we identified beta-hydroxybutyrate, a ketone body generated through the ketogenic pathway, as a fasting-related metabolite with potential protective effects against liver IRI. Since fasting alters metabolomic profiles significantly,13 we assumed that additional metabolites might contribute to the attenuation of liver IRI observed in the study mentioned above. Hence, we sought to uncover fasting-related metabolites that could be pivotal in modulating liver IRI. In this study, we first analyzed the metabolome of the livers of mice exposed to warm IRI with or without fasting. We found that histamine is 1 of the candidates that might be involved in the pathogenesis of IRI. Second, we investigated the impact of histamine on liver IRI using histamine H1 receptor (H1R)-null mice. Finally, we tested S-(+)- chlorpheniramine (CPM), a clinically existing pharmacological intervention against allergy, to determine whether this H1-antihistamine might alleviate liver IRI.

MATERIALS AND METHODS

Animals

Wild-type (WT) and histamine H1 receptor knockout (Hrh1KO) mice14,15 on a C57BL/6 background were purchased from Oriental Bioservice, Inc. (Kyoto, Japan). All mice were housed under specific pathogen-free conditions with a 12:12 h light/dark cycle. Food and water were provided ad libitum. Animal experimental procedures were approved by the Institutional Animal Welfare and Animal Care Committee of Kyoto University (approval number: Med kyo 24173) and complied with the Guide for the Care and Use of Laboratory Animals (Eighth edition).

Reagents

CPM, an H1-antihistamine, and compound 48/80 (C48/80) were purchased from Sigma Chemical Co. (St. Louis, MO). CPM was dissolved in saline (NaCl 0.9%) and administered to mice intraperitoneally at a dose of 20 mg/kg body weight 1 h before the ischemic insult. C48/80, which selectively activates mast cells to release histamine,16 was injected intravenously at a dose of 10 mg/kg body weight.

Preoperative Short-term Fasting

The control-fed group was provided food and sterile water ad libitum. In contrast, the fasting group was deprived of food but was given free access to water for 12 h before the ischemia and reperfusion (IR) procedure.

Liver IR Injury Model

We used a partial (70%) liver warm IR model, as previously described.12 Mice were euthanized 6 h after reperfusion, and the left lobe of the liver and serum samples were collected for further analysis.

Metabolome Analysis

A comprehensive metabolome analysis of the mouse liver was performed to measure the metabolite levels. Twenty mice were divided equally into 4 groups (control diet without IR, 12-h fasting without IR, control diet with IR, and 12-h fasting with IR), and liver samples were collected from each group. The details of the analysis method are provided in the Supplemental Materials (SDC, https://links.lww.com/TP/D335).

Assessment of Hepatocyte Injury

Serum alanine aminotransferase levels were measured using a standard spectrophotometric method with an automated clinical analyzer (JCA-BM9030, JEOL, Ltd., Tokyo, Japan).

Histology

Liver paraffin sections (5 µm thick) were stained with hematoxylin and eosin. The severity of liver IRI (necrosis, sinusoidal congestion, and centrilobular ballooning) was graded from 0 to 4 by an investigator blinded to the experimental conditions, using modified Suzuki’s criteria.17

Immunohistochemistry/Immunofluorescence

Antigen retrieval (citrate, pH 6) was performed on paraffin-embedded liver sections (5 μm). The sections were incubated overnight at 4 °C with primary antibodies against H1R (Proteintech), lymphocyte antigen 6 complex locus G (B&D), and CD11b (Abcam). Then, a biotinylated secondary antibody was applied. After incubation, immunoperoxidase (VECTASTAIN ABC Kit; Vector Labs) was applied to the sections and developed using 3,3′-diaminobenzidine. Frozen mouse liver samples (5 μm) were stained with rabbit anti-heme oxygenase-1 (HO-1) antibody. Signals were visualized using a secondary Alexa Fluor antibody.

ELISA

Serum HMGB1 and histamine levels were quantified with HMGB1 ELISA Kit Exp (Shino-Test) and Histamine ELISA kit (ab213975; Abcam), respectively.

Apoptosis Assay

Apoptosis in 5 µm liver paraffin sections was detected using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method with an in situ Apoptosis Detection Kit (Takara Bio) according to the manufacturer’s instructions.

Quantitative Reverse Transcription-polymerase Chain Reaction

Total RNA was extracted from the liver tissues using the RNeasy Mini Kit (QIAGEN). Complementary DNA was prepared using the PrimeScript RT Reagent Kit (Takara Bio). Quantitative reverse transcription-polymerase chain reaction was performed using a StepOnePlus Real-Time Polymerase Chain Reaction System (Life Technologies). The primers used to amplify specific gene fragments are listed in Table S1 (SDC, https://links.lww.com/TP/D335). All samples were analyzed in triplicate, and glyceraldehyde-3-phosphate dehydrogenase served as the internal control. Gene expression was analyzed using the comparative threshold cycle method.

Western Blotting

Proteins were extracted from the liver tissue samples, and their concentrations were measured using a BCA Assay Kit (Thermo Fisher Scientific). Equal amounts of protein lysate (20 μg for liver samples) were electrophoresed under denaturing conditions, blotted, incubated with primary antibodies and secondary horseradish peroxidase-conjugated antibodies, and developed. Relative values were normalized to beta-actin. The antibodies used in this study are listed in Table S2 (SDC, https://links.lww.com/TP/D335).

Statistical Analysis

All statistical analyses were performed using Prism 9 (GraphPad Software Inc., La Jolla, CA). The Shapiro-Wilk test was performed to confirm whether the data for continuous variables were normally distributed. The significance of the difference was calculated using Welch’s t test for 2 experimental groups or 1-way ANOVA for multiple groups, and results with P < 0.05 were regarded as statistically significant. All data are presented as mean ± SD.

RESULTS

Fasting Changes Hepatic Metabolite Profiles During IRI

To systematically investigate the alterations in the liver metabolome induced by IR insult and preoperative fasting, we obtained liver samples from 4 groups of C57BL/6J mice (5 mice per group) as outlined in our previous study12: (1) mice provided ad libitum access to food and sterile water without exposure to IR insult (control), (2) mice subjected to a 12-h food deprivation with free access to water without IR insult (fasting), (3) mice offered ad libitum access to food and sterile water for 12 h followed by IR insult (control IRI), and (4) mice deprived of food for 12 h with free access to water, followed by IR insult (fasting IRI).

Principal component analysis revealed distinct segregation among the 4 experimental groups (Figure 1A). The principal component analysis score plot showed that the first principal component score (PC1), the highest proportion of variance, comprised 34.2% of all detected metabolites. The second principal component score (PC2), the second highest proportion of variance, included 19.8% of all metabolites detected (Figure 1A). Hierarchical cluster analysis was performed to further elucidate the differential metabolic profiles (Figure 1B), revealing clear distinctions in the metabolite profiles among the 4 groups. Finally, 581 metabolites were annotated from candidate metabolites. To identify metabolites exhibiting substantial alterations in response to IR insult and preoperative fasting, we calculated the fold changes for each metabolite between the control and control IRI groups and between the control IRI and fasting IRI groups. We found that 60 metabolites were significantly upregulated in the control IRI group compared with the control group, whereas 222 metabolites were significantly downregulated (Figure S1A, SDC, https://links.lww.com/TP/D335). In contrast, 56 metabolites were significantly upregulated in the control IRI group compared with the fasting IRI group, whereas 168 metabolites were significantly downregulated (Figure S1B, SDC, https://links.lww.com/TP/D335).

FIGURE 1.

FIGURE 1.

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Metabolic landscape of the liver of mice receiving liver ischemia and reperfusion (IR) insult and treated with preoperative fasting. A, Score plot of principal component analysis. B, Heatmap displaying sample clusters (n = 5/group). C, The relative quantity of histamine in the liver (n = 5 mice per group; 1-way ANOVA; P = 0.0033, Sidak’s multiple comparisons test: *P < 0.05, **P < 0.01, and ns). IRI, ischemia-reperfusion injury; ns, no significant difference; PC, principal component.

Hepatic Histamine Is Upregulated During IRI but Dampened by Fasting

Among the metabolites, histamine was significantly upregulated in the control IRI group compared with that in the control group (Figure S1C, SDC, https://links.lww.com/TP/D335), suggesting that IR stress augmented intrahepatic histamine levels. In contrast, the fasting intervention profoundly suppressed hepatic histamine levels compared with those in the control IRI group (Figure 1C). Therefore, we hypothesized that histamine might play a crucial role in liver IRI, as its expression is positively correlated with the severity of IRI-related liver damage in clinical settings.18

Liver IRI Is Attenuated in Hrh1KO Mice

Having demonstrated that hepatic histamine levels were positively correlated with the severity of IRI in mice, we then focused on the H1R, which plays a pivotal role in allergic and inflammatory responses.19 First, we performed immunohistochemistry to examine the expression of H1Rs in the liver. As shown in Figure 2A, H1Rs were expressed on the membranes of cholangiocytes (a) and vascular endothelial cells (b), consistent with a previous report.20 To assess the impact of H1R signaling on liver IRI, we used Hrh1KO mice.14 Serum alanine aminotransferase levels were markedly alleviated in Hrh1KO mice compared with those in WT mice (Figure 2B), suggesting that H1R signaling contributes to the exacerbation of liver IRI. Histological analysis using hematoxylin and eosin staining revealed more extensive necrotic areas in WT than in Hrh1KO mice (Figure 2C). Suzuki’s histological score was lower in Hrh1KO mice than in WT mice (Figure 2D). TUNEL staining was performed to investigate the extent of cell death 6 h after reperfusion (Figure 2E). The number of TUNEL-positive cells was significantly reduced in Hrh1KO mice compared with that in WT mice (Figure 2F).

FIGURE 2.

FIGURE 2.

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IR-induced hepatocellular damage in WT and Hrh1KO mice. A, Representative immunohistochemistry of histamine H1 receptors (dark brown) in the WT liver (magnification ×400). B, Serum alanine transferase (sALT) levels 6 h after IRI (n = 6 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001, and ns). C, Representative hematoxylin and eosin (H&E) staining of the liver following IR. The black arrow indicates necrotic parenchyma and sinusoidal congestion. D, Suzuki’s histological score after IR (n = 6 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001, and ns). E, TUNEL-assisted detection of hepatic cell death after IR. F, Quantification of TUNEL-positive cells (n = 6 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001, and ns). ALT, alanine transaminase; HPF, high-power field; Hrh1KO, histamine H1 receptor knockout; IR, ischemia and reperfusion; IRI, ischemia-reperfusion injury; ns, no significant difference; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling; WT, wild-type.

The Ablation of H1R Suppresses the HMGB1 Release and Proinflammatory Cytokines/Chemokines Response and Infiltration of Immune Cells

HMGB1, one of the DAMPs that provoke an inflammatory response,21 has been reported to exert a detrimental effect on liver IRI3 in the mechanism that HMGB1 activates inflammatory cells through Toll-like receptor 4 and induces the production of proinflammatory cytokines that lead to the development of liver IRI.22 Serum HMGB1 levels were elevated in WT mice subjected to IRI, whereas Hrh1KO mice exhibited a significant reduction in serum HMGB1 levels 6 h after reperfusion (Figure 3A). In line, the expression of proinflammatory genes, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, IL-6, C-X-C motif chemokine ligand (CXCL) 1, and CXCL2, was significantly downregulated in the liver tissue of Hrh1KO mice compared with that in WT controls (Figure 3B). Notably, the infiltration of neutrophils (lymphocyte antigen 6 complex locus G-positive) and macrophages (CD11b-positive) into the liver tissue following IR was significantly diminished in Hrh1KO mice (Figure 3C–F). These findings underscore the critical role of H1R signaling in HMGB1 release and promotion of the inflammatory response in the liver during IRI.

FIGURE 3.

FIGURE 3.

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Hrh1KO mice exhibited lower serum HMGB1 levels, downregulated expression of cytokines/chemokines, and lower Ly6G and CD11b positive cells in the post-IR livers. A, Serum HMGB1 levels (sham, n = 3 per sham group; n = 6 mice per IR group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: *P < 0.05, ****P < 0.0001, and ns). B, Quantitative PCR detection of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines (CXCL1, CXCL2). Data were normalized to GAPDH gene expression (sham, n = 4; IR 6 h, n = 6 mice per group; 1-way ANOVA; P < 0.01 [TNF-α, IL-6], P < 0.001 [IL-1β], P < 0.0001 [CXCL1, CXCL2], Sidak’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns). C, Representative immunostaining of Ly6G at 6 h of reperfusion. D, Quantification of Ly6G positive cells (sham, n = 4; IR 6 h, n = 6 mice per group; 1-way ANOVA; P < 0.001, Sidak’s multiple comparisons test: **P < 0.01, ***P < 0.001, and ns). E, Representative immunohistochemistry of CD11b at 6 h of reperfusion. F, Quantification of CD11b positive cells (sham, n = 4; IR 6 h, n = 6 mice per group; 1-way ANOVA; P < 0.01, Sidak’s multiple comparisons test: *P < 0.05, **P < 0.01, and ns). CXCL, C-X-C motif chemokine ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMGB1, high-mobility group box 1; HPF, high-power field; Hrh1KO, histamine H1 receptor knockout; IL-1β, interleukin-1 beta; IL-6, interleukin-6; Ly6G, lymphocyte antigen 6 complex locus G; ns, no significant difference; PCR, polymerase chain reaction; TNF-α, tumor necrosis factor alpha; WT, wild-type.

H1-Antihistamines Dampen Liver IRI Through the Suppression of Immune Responses and HMGB1 Secretion

Having demonstrated that genetic depletion of H1R in mice had a protective effect against liver IRI, we sought to validate whether the clinically relevant pharmacological blockade of H1R with CPM effectively attenuated liver IRI. Consistent with the observations in Hrh1KO mice subjected to liver IR, CPM treatment alleviated liver injury, as evidenced by lower sALT levels (Figure 4A), attenuated histological findings (Figure 4B), and suppressed Suzuki’s score (Figure 4C). Serum HMGB1 levels were significantly decreased following H1R antagonism (Figure 5A). Additionally, the hepatic expression of proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, CXCL1, and CXCL2, was markedly suppressed in the CPM-treated group (Figure 5B). The number of infiltrating neutrophils and macrophages in the liver tissues was also significantly decreased in CPM-treated mice (Figure 5C–F). These findings demonstrate that CPM, an H1-antihistamine, robustly ameliorates liver IRI when administered prophylactically, primarily by inhibiting HMGB1 release and the production of inflammatory cytokines.

FIGURE 4.

FIGURE 4.

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Protective effect of H1-antihistamine against IR-related hepatic damage. A, sALT levels 6 h after reperfusion (sham and control IR mice, n = 6; CPM-treated IR mice, n = 4; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001, and ns). B, Representative hematoxylin and eosin (H&E) staining of the liver following IR. Necrotic parenchyma and sinusoidal congestion are indicated (arrows). C, Suzuki’s histological score after IR (sham and control IR mice, n = 6; CPM-treated IR mice, n = 4; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: **P < 0.01, ****P < 0.0001). ALT, alanine transaminase; CPM, chlorpheniramine; IR, ischemia and reperfusion; ns, no significant difference; NS, normal saline; sALT, serum alanine transferase; WT, wild-type.

FIGURE 5.

FIGURE 5.

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CPM-treated mice exhibited lower serum HMGB1 levels, downregulated expression of cytokines/chemokines, and lower Ly6G and CD11b positive cells in the post-IR livers. A, Serum HMGB1 levels (sham, n = 3; NS IR 6 h, n = 6; CPM-treated IR 6 h, n = 4 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001). B, Quantitative PCR detection of proinflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokines (CXCL1, CXCL2) at 6 h of reperfusion. Data were normalized to GAPDH gene expression (sham, n = 4; control IR mice, n = 6; CPM-treated IR mice, n = 4; 1-way ANOVA; P = 0.0059 [TNFα], P = 0.0025 [IL-6], P = 0.0044 [IL-6], P = 0.0008 [CXCL1], P < 0.0001 [CXCL2], Sidak’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001, and ns). C, Representative immunohistochemistry of Ly6G at 6 h of reperfusion. D, Quantification of Ly6G-positive cells (sham, n = 4 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ****P < 0.0001, and ns). E, Representative immunohistochemistry of CD11b at 6 h of reperfusion. F, Quantification of CD11b-positive cells (sham, n = 4 mice per group; 1-way ANOVA; P < 0.0001, Sidak’s multiple comparisons test: ***P < 0.001, ****P < 0.0001, and ns). CPM, chlorpheniramine; CXCL, C-X-C motif chemokine ligand; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMGB1, high-mobility group box 1; HPF, high-power field; IL-1β, interleukin-1 beta; IL-6, interleukin-6; Ly6G, lymphocyte antigen 6 complex locus G; ns, no significant difference; NS, normal saline; PCR, polymerase chain reaction; TNF-α, tumor necrosis factor alpha; WT, wild-type.

Histamine Promotes HMGB1 Release via H1R In Vivo

To confirm whether H1 receptor signaling is involved in HMGB1 secretion, we treated WT and Hrh1KO mice with C48/80, an agent that promotes mast cell degranulation and histamine release. Serum HMGB1 levels were elevated in WT mice 10 min after C48/80 administration, while the HMGB1 levels were unresponsive to C48/80 stimulation in Hrh1KO mice (Figure 6A). This result aligns with a previous report that H1R is crucial for HMGB1 release by histamine stimulation.23

FIGURE 6.

FIGURE 6.

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Mechanistic insight into the attenuation of liver IRI in CPM-treated mice. A, Serum HMGB1 levels 10 min after intravenous administration of C48/80 in WT and Hrh1KO mice. (sham, n = 3 mice per group; C48/80-treated mice, n = 4 mice per group; 1-way ANOVA; P = 0.0002, Sidak’s multiple comparisons test: **P < 0.01, ***P < 0.001, and ns). B, Western blot analysis of Akt, p-Akt, and HO-1 in liver tissue, with beta-actin serving as the loading control (n = 3 mice per group; 1-way ANOVA; P = 0.9977 [Akt], P = 0.0100 [p-Akt], P = 0.0008 [HO-1]; Sidak’s multiple comparisons test: *P < 0.05, **P < 0.01, and ns). C, Representative immunofluorescence staining of HO-1 (green) and merged images of sham (upper panel), NS IR 6 h (middle), and CPM-treated IR 6 h mice (lower panel; ×400). Akt, the Ser and Thr kinase AKT; C48/80, compound 48/80; CPM, chlorpheniramine; DAPI, 4’,6-diamidino-2-phenylindole; HMGB1, high-mobility group box 1; HO-1, heme oxygenase 1; Hrh1KO, histamine H1 receptor knockout; IR, ischemia and reperfusion; IRI, ischemia-reperfusion injury; ns, no significant difference; NS, normal saline; p-Akt, phospho-Akt; WT, wild-type.

The Blockade of the H1R Signal Suppresses Hepatic Akt Phosphorylation and Enhances HO-1 in Hepatocytes In Vivo

To explore how CPM attenuates liver IRI, we investigated the downstream signaling pathways of H1R. As shown in Figure 6B, the expression of phospho-the Ser and Thr kinase AKT (p-Akt) was significantly lower in CPM-treated WT mice than that in nontreated WT mice. These findings are inconsistent with the previous report that documented the protective role of the Ser and Thr kinase AKT (Akt) in liver IRI.24 A recent study reported that an H1R inverse agonist suppressed Akt phosphorylation, thereby promoting the transcription of forkhead box O (FOXO) transcription factors in experimental osteoarthritis.25 At the same time, FOXO is closely related to HO-1 induction,12 which is well known for its antioxidant properties against IR stress.26 Hence, we analyzed the expression of HO-1 in post-IR livers and found that HO-1 expression was significantly upregulated in the CPM group. Hepatic HO-1 expression after IRI is predominantly derived from macrophages in both mice and humans.27 However, hepatic HO-1 expression was higher in the CPM group, despite the small number of infiltrating CD11b-positive cells in the liver (Figure 5E and F). To investigate this discrepancy, we performed HO-1 immunostaining of liver tissue and found that HO-1 expression tended to be higher in hepatocytes of the CPM group (Figure 6C). Thus, CPM may serve as an inverse agonist to induce hepatic HO-1 expression and exert the ultimate protection against IR stress.

DISCUSSION

Liver IRI, which is reported to be a major contributor to postoperative liver dysfunction and graft failure in liver transplantation,28 remains a significant clinical challenge because of the lack of definitive therapeutic strategies. Based on our previous study, which demonstrated the beneficial effect of preoperative short-term (12 h) fasting on murine liver IRI model,12 we sought to (1) elucidate the comprehensive metabolic changes induced by preoperative fasting and liver IR insult, (2) identify the metabolites involved in the pathogenesis of liver IRI, and (3) explore their mechanisms of action.

In the present study, we conducted a comprehensive metabolomic analysis of >580 metabolites in liver samples and found profound metabolic alterations in the histidine/histamine pathway induced by IR combined with preconditioned short-term fasting (Figure 1A–C; Figure S2A, SDC, https://links.lww.com/TP/D335). These results coincide with those of a previous study, which documented that enhanced histidine metabolism in human donor livers positively correlated with the incidence of early allograft dysfunction.18 As demonstrated in Figure 1C, the histamine levels in the liver were markedly elevated after the IR insult. Given that histamine is synthesized from histidine via histidine decarboxylase (HDC), the upregulation of HDC by IR stress is likely to enhance the enzymatic conversion of histidine to histamine during hepatic IR (Figure S2B, SDC, https://links.lww.com/TP/D335). Previous reports suggest that HDC expression is induced by lipopolysaccharides29 or inflammatory cytokines such as IL-130 or TNF-α,29 which is consistent with our findings of elevated levels of inflammatory cytokines in IR-treated mice (Figure 3B). Interestingly, fasting mice exhibited low hepatic histamine levels and high histidine reserves before and after IR (Figure 1C; Figure S2A, SDC, https://links.lww.com/TP/D335). The reduction in hepatic histamine levels in fasting mice may be attributed to the upregulation of diamine oxidase, which degrades histamine, as shown in Figure S2C (SDC, https://links.lww.com/TP/D335). This metabolic fluctuation in histidine and histamine, driven by hepatic IR insult and preoperative fasting, accentuates the crucial role of histamine in the pathogenesis of liver IRI.

Histamine receptors are G protein–coupled receptors that bind histamine as the primary ligand. To date, 4 types of receptors have been identified to date.31 Several reports have documented the involvement of histamine receptors in IRI of the brain,32 heart,33 kidney,34 and skin.35 In the context of liver IRI, Wu et al36 reported that histamine aggravated oxidative stress via histamine H2 receptors in buffalo rat liver-3A cells in vitro, while Adachi et al37 showed that engagement of histamine H4 receptors attenuated liver IRI in rat. Histamine H3 receptors are mainly expressed on histaminergic neurons,38 and thioperamide, a histamine H3/H4 antagonist, was reported to exacerbate liver IRI.37 Thus, little is known about the role of H1R in liver IRI, although the expression of H1R is relatively abundant in the liver,39 as shown in Figure 2A. Therefore, we interrogated the impact of H1R on liver IRI using H1R-deficient mice. Notably, H1R plays a pivotal role in liver IRI, as evidenced by reduced liver injury (Figure 2B–F), diminished proinflammatory cytokine/chemokine expression, and lower serum HMGB1 levels in Hrh1KO mice (Figure 3).

In the present study, we tested CPM, a widely used H1-antihistamine, and revealed that this clinically applicable medicine significantly mitigated liver IRI in WT mice (Figure 4). CPM and other H1 antagonists are known to exert off-target effects. To assess whether off-target effects were elicited in the alleviation of liver IRI in mice administered 20 mg/kg CPM, we confirmed that pretreatment with H1-antihistamine failed to attenuate liver IRI in Hrh1KO mice (Figure S3, SDC, https://links.lww.com/TP/D335). These findings, substantiated by both Hrh1KO mice and H1-antihistamine treatment, highlight the critical role of the histamine - H1R interaction in liver IRI.

One of the potential reasons why H1R signal blockade attenuates liver IRI may be the suppression of HMGB1 release from endothelial cells in the liver. A previous in vitro study showed that the interaction between histamine and H1R on endothelial cells induced HMGB1 release.23 In line with this report, C48/80 stimulation provoked HMGB1 release in WT but not in Hrh1KO mice, indicating that histamine stimulates HMGB1 release via an H1R-dependent mechanism (Figure 6A). Serum levels of HMGB1 increase during IR, recruiting immune cells to the damage site and accelerating inflammatory responses via Toll-like receptor 4.12,40 In contrast, HMGB1 released into the bloodstream can serve as a chemotactic factor for immune cell recruitment to damaged organs.41 Although histamine is known to exert similar effects,42,43 no significant differences in serum concentrations were observed after IRI, regardless of H1-antihistamine therapy (Figure S4, SDC, https://links.lww.com/TP/D335), suggesting that HMGB1, rather than histamine, is more likely to contribute to the infiltration of immune cells into hepatic tissue (Figures 3 and 5). Another possible mechanism may involve the inverse agonist activity of CPM. H1-antihistamines function as inverse agonists, stabilizing the inactive state of the receptor and downregulating its constitutive activity, even in the absence of histamine.44 In light of the previously reported FOXO-inducing effect of H1R inverse agonism,25 together with our findings regarding the relationship between HO-1 and FOXO,12 it appears plausible to consider that CPM may have induced hepatic HO-1, given that H1R is also expressed in hepatocytes.45 Thus, CPM appears to exert dual actions: as an antagonist, it suppresses the release of HMGB1, and as an inverse agonist, it induces HO-1, thereby potentially conferring protective effects against IRI (Figure 6B; Figure S5, SDC, https://links.lww.com/TP/D335).

Mast cells, which are a major source of histamine, reside in the liver. The number of mast cells is relatively low in the steady state but increases with the progression of liver disease, including metabolic dysfunction-associated fatty liver disease.46 In the current experiment, it was challenging to detect mast cells in the liver, whereas they were successfully stained in the small intestine (Figure S6, SDC, https://links.lww.com/TP/D335). These results imply that the origin of histamine may be derived from other liver-resident immune cells or the small intestine via the portal vein. Nevertheless, fat accumulation may lead to increased intrahepatic histamine levels because of cell proliferation and mast cell activation, which may be 1 of the reasons why so-called marginal grafts are vulnerable to IRI. Investigating the histamine/H1R axis using experimental models of marginal grafts (eg, high-fat diet) awaits future studies.

Although several reports have documented the benefits of preoperative fasting in experimental IRI models, it is unclear whether preoperative fasting is effective in reducing IRI in clinical settings. The necessity and effectiveness of improving the nutritional status of patients in poor general condition before surgery has attracted much attention in recent years.47 Indeed, fasting may be harmful, particularly in patients with liver cirrhosis because of impaired glycogen storage and release capacity.48 Hence, perioperative H1-antihistamine treatment could be an alternative therapy for fasting to prevent IR-related liver damage.

One of the limitations of our study was that the metabolic changes observed in IR-insulted livers between fasted and non-fasted mice resulted not only from metabolic changes in liver cells but also from alterations in the cellular composition of the livers. The number of T cells, neutrophils, and macrophages infiltrating liver tissue after IR treatment was significantly lower in the 12-h fasted mice than in non-fasted mice.12 Therefore, future studies are warranted to investigate how these immune cells affect the metabolomic changes caused by fasting and liver IR insult.

Another limitation was the CPM dosage. We decided on the administration route by considering the pharmacokinetic characteristics of intraperitoneal injection, such as rapid systemic delivery and high effective bioavailability,49 but at the same time, intraperitoneal injection demonstrates different pharmacokinetics from intravenous injection, which is a common way of introducing H1-antihistamines perioperatively. In terms of dose, we referred to a report on the murine septic model administering 10 mg/kg of CPM intraperitoneally,50 which is relatively higher than that in clinical settings. Hence, the timing and dosage of H1-antihistamines effective for mitigating liver IRI without causing side effects need to be scrutinized before their application in human clinical settings.

In conclusion, H1R blockade with H1-antihistamine CPM effectively suppressed inflammatory responses and mitigated liver IRI in mice. Clinical trials on CPM for therapeutic or preventative applications in liver surgery and transplantation are warranted.

ACKNOWLEDGMENTS

The authors thank Koichi Hirano (Kitano Hospital Medical Research Institute) for his technical assistance.

Supplementary Material

tpa-110-e583-s001.pdf (1.1MB, pdf)

Footnotes

This work was supported by Grant-in-Aid for Scientific Research B: 20H03743 and Grant-in-Aid for Early-Career Scientists: 23K15463, 24K19318, 24K19333 from the Ministry of Education, Culture, Science and Sports, Japan.

The authors declare no conflicts of interest.

H.K., Y.U., K.S., J.K., and T.W. participated in research design. H.K., Y.U., H.H., and T.W. participated in the writing of the article. H.K. and K.S. participated in the performance of the research. H.K., Y.U., H.H., K.S., J.K., K.K., M.I., Y.K., K.T., S.K., K.N., K.T., H.T., T.W., and E.H. participated in data analysis.

Supplemental digital content (SDC) is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.transplantjournal.com).

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Supplementary Materials

tpa-110-e583-s001.pdf (1.1MB, pdf)

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