Hepatic 31 P-magnetic resonance spectroscopy identified the impact of melatonin-pretreated mitochondria in acute liver ischaemia-reperfusion injury

Abstract

Acute liver ischaemia-reperfusion injury (IRI), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. However, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. This study tested the hypothesis that ³¹ P-magnetic resonance spectroscopy (³¹ P-MRS) findings could provide reliable living images to accurately identify the degree of acute liver IRI and melatonin-pretreated mitochondria was an innovative treatment for protecting the liver from IRI in rat. Adult male SD rats were categorized into group 1 (sham-operated control), group 2 (IRI only) and group 3 (IRI + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). By the end of study period at 72 hours, ³¹ P-MRS showed that, as compared with group 1, the hepatic levels of ATP and NADH were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. The liver protein expressions of mitochondrial-electron-transport-chain complexes and mitochondrial integrity exhibited an identical pattern to ³¹ P-MRS finding. The protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial-damaged biomarkers displayed an opposite finding of ³¹ P-MRS, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. Microscopic findings showed that the fibrotic area/liver injury score and inflammatory and DNA-damaged biomarkers exhibited an identical pattern of cellular stress signalling. Melatonin-pretreated mitochondria effectively protected liver against IRI and ³¹ P-MRS was a reliable tool for measuring the mitochondrial/ATP consumption in living animals.

Keywords: 31P-magnetic resonance spectroscopy; liver ischaemia-reperfusion injury; melatonin; mitochondria.

FIGURE 1

Circulatory levels of liver enzyme and inflammatory biomarkers by day 0 and 3 after liver ischaemia‐reperfusion injury induction. A, ELISA result of circulating level of tumour necrosis factor (TNF)‐α, * vs other groups with different symbols (†, ‡), P < 0.0001. B, ELISA result of circulating level of interleukin (IL)‐6, * vs other groups with different symbols (†, ‡), P < 0.0001. C, ELISA result of circulating level of myeloperoxidase (MPO), * vs other groups with different symbols (†, ‡), P < 0.0001. D, Serum level of aspartate aminotransferase (AST) at day 0, P = 1.0. E, Serum level of AST at day 3, * vs other groups with different symbols (†, ‡), P < 0.0001. F, Serum level of alanine Aminotransferase (ALT) at day 0, P = 1.0. G, Serum level of ALT at day 3, * vs other groups with different symbols (†, ‡), P < 0.001. All statistical analyses were performed by one‐way ANOVA, followed by Bonferroni's multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). IRI, ischaemia‐reperfusion injury; Mito, mitochondria; SC, sham control

FIGURE 2

MRI findings of hepatic phosphorylated metabolism by 72 h after liver ischaemia‐reperfusion injury induction. A, Illustrating the hepatic ³¹P‐magnetic resonance spectra (ie ³¹P‐MRS) examination for identifying the ATP (an indicator of energy storage) in hepatocytes of SC, IRI and IRI + Mito animals, respectively. B‐G, The relative level of each metabolites was subtracted from the β‐ATP value. The data are expressed as mean ± SD (n = 8 per group). One‐way ANOVA followed by post hoc Tukey‐Kramer test was used for statistical analysis. Symbols (*, †, ‡) indicate significant differences (at 0.05 level). ATP, adenosine triphosphate; IRI, ischaemia‐reperfusion injury; NADH, nicotinamide adenine dinucleotide; PDE, phosphodiester; Pi, inorganic phosphate; PME, phosphomonoesters; SC, sham control

FIGURE 3

Protein expressions of mitochondrial integrity, oxidative phosphorylation, oxidative stress, inflammation and cellular stress signalling by 72 h after liver ischaemia‐reperfusion injury induction. A, Protein expression of complex I, * vs other groups with different symbols (†, ‡), P < 0.0001. B, Protein expression of complex II, * vs other groups with different symbols (†, ‡), P < 0.001. C, Protein expression of complex III, * vs other groups with different symbols (†, ‡), P < 0.0001. D, Protein expression of complex V, * vs other groups with different symbols (†, ‡), P < 0.001. E, Protein expression of dynamin‐related protein 1 (DRP1), * vs other groups with different symbols (†, ‡), P < 0.0001. F, Protein expression of cyclophilin (Cyc‐D), * vs other groups with different symbols (†, ‡), P < 0.0001. G, Protein expression of cytosolic cytochrome C (cyto‐Cyt C), * vs other groups with different symbols (†, ‡), P < 0.0001. H, Protein expression of mitochondrial cytochrome C (mito‐Cyt C), * vs other groups with different symbols (†, ‡), P < 0.0001. I, Protein expression of NOX‐1, * vs other groups with different symbols (†, ‡), P < 0.0001. J, Protein expression of NOX‐2, * vs other groups with different symbols (†, ‡), P < 0.0001. K, Protein expression of haeme oxygenase (HO)‐1, * vs other groups with different symbols (†, ‡), P < 0.0001. L, Protein expression of NAD(P)H dehydrogenase (quinone) 1 (NQO1), * vs other groups with different symbols (†, ‡), P < 0.001. M, Protein expression of nuclear factor erythroid 2‐related factor 2 (Nrf2), * vs other groups with different symbols (†, ‡), P < 0.01. N, Protein expression of interleukin (IL)‐1β, * vs other groups with different symbols (†, ‡), P < 0.0001. O, Protein expression of tumour necrosis factor (TNF)‐α, * vs other groups with different symbols (†, ‡), P < 0.0001. P, Protein expression of phosphorylated (p)‐nuclear factor (p‐NF)‐κB, * vs other groups with different symbols (†, ‡), P < 0.0001. Q, Protein expression of matrix metalloproteinase (MMP)2, * vs other groups with different symbols (†, ‡), P < 0.0001. R, Protein expression of MMP9, * vs other groups with different symbols (†, ‡), P < 0.0001. S, Protein expression of PI3K, * vs other groups with different symbols (†, ‡), P < 0.0001. T, Protein expression of p‐Akt, * vs other groups with different symbols (†, ‡), P < 0.0001. U, Phosphorylated mammalian target of rapamycin (p‐mTOR), * vs other groups with different symbols (†, ‡), P < 0.0001. All statistical analyses were performed by one‐way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). IRI, ischaemia‐reperfusion injury; Mito, mitochondria; SC, sham control

FIGURE 4

Histopathological assessment of liver parenchyma by 72 h after liver ischaemia‐reperfusion injury induction. A‐C, Illustrating microscopic finding (200×) of haematoxylin and eosin stain for identification of liver injury area (ie ischaemic/infarct area) indicated by dotted lines. D, Analytical result of liver injury score, * vs other groups with different symbols (†, ‡), P < 0.0001. E‐G, Illustrating the immunofluorescent microscopic finding (100×) of Masson's trichrome stain for identification of fibrotic area (blue colour). H, Analytical result of liver injury score, * vs other groups with different symbols (†, ‡), P < 0.0001. Scale bar in right lower corner represents 100 µm. All statistical analyses were performed by one‐way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). IRI, ischaemia‐reperfusion injury; Mito, mitochondria; SC, sham control

FIGURE 5

Inflammatory cell infiltration in liver parenchyma by 72 h after liver ischaemia‐reperfusion injury induction. A‐C, Illustrating the immunofluorescent (IF) microscopic finding (400×) for identification of CD14⁺ cells (ie green colour indicated by red arrows) in liver parenchyma. D, Analytical result of number of positively stained CD14 cells, * vs other groups with different symbols (†, ‡), P < 0.0001. E‐G, Illustrating the IF microscopic finding (400×) for identification of CD68 cells (ie green colour indicated by red arrows) in liver parenchyma. H, Analytical result of number of positively stained CD68 cells, * vs other groups with different symbols (†, ‡), P < 0.0001. Red colour in (C) and (G) (yellow arrows) indicated the dye stained exogenous mitochondria. Scale bar in right lower corner represents 100 µm. All statistical analyses were performed by one‐way ANOVA, followed by Bonferroni's multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). IRI, ischaemia‐reperfusion injury; Mito, mitochondria; SC, sham control

FIGURE 6

Cellular expressions of γ‐H2AX and MMP‐9 in liver parenchyma by 72 h after liver ischaemia‐reperfusion injury induction. A‐C, Illustrating the immunofluorescent microscopic finding (400×) for identification of cellular expressions of γ‐H2AX in liver ischaemic area (ie green colour indicated by red arrows). D, Analytical result of number of γ‐H2AX+ cells, * vs other groups with different symbols (†, ‡), P < 0.0001. Red colour in (C) (yellow arrows) indicated the dye stained exogenous mitochondria. Scale bar in right lower corner represents 20 µm. E‐G, Illustrating the microscopic finding of immunohistochemical stain for identification of matrix metalloproteinase (MMP)‐9 (brown colour indicated by red arrows). H, Analytical result of number of MMP‐9+ cells per high‐power field, * vs other groups with different symbols (†, ‡), P < 0.0001. HPF, high‐power field. Scale bar in right lower corner represents 50 µm. All statistical analyses were performed by one‐way ANOVA, followed by Bonferroni's multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance (at 0.05 level). IRI, ischaemia‐reperfusion injury; Mito, mitochondria; SC, sham control

FIGURE 7

Schematically proposed mechanism of mitochondrial transfusion for reducing acute liver ischaemia‐reperfusion injury in rat. ³¹P‐MRS, ³¹P‐magnetic resonance spectroscopy; ATP, adenosine triphosphate; IL‐6, interleukin 6; IRI, ischaemia‐reperfusion injury; Mito, mitochondria; MPO, myeloperoxidase; ROS, reactive oxygen species; SC, sham control; TNF‐α, tumour necrosis factor alpha