Cytochrome P450 2E1 predicts liver functional recovery from donation after circulatory death using air-ventilated normothermic machine perfusion

Abstract

The optimal oxygen concentration is unclear for normothermic machine perfusion (NMP) of livers from donation after circulatory death (DCD). Our purposes were to investigate the effect of air-ventilated NMP on the DCD liver, analyze the underlying mechanism and select the targets to predict liver functional recovery with NMP. NMP was performed using the NMP system with either air ventilation or oxygen ventilation for 2 h in the rat liver following warm ischemia and cold-storage preservation. Proteomics and metabolomics were used to reveal the significant molecular networks. The bioinformation analysis was validated by administering peroxisome proliferator activator receptor-γ (PPARγ) antagonist and agonist via perfusion circuit in the air-ventilated NMP. Results showed that air-ventilated NMP conferred a better functional recovery and a less inflammatory response in the rat DCD liver; integrated proteomics and metabolomics analysis indicated that intrahepatic docosapentaenoic acid downregulation and upregulation of cytochrome P450 2E1 (CYP2E1) expression and activity were associated with DCD liver functional recovery with air-ventilated NMP; PPARγ antagonist worsened liver function under air-oxygenated NMP whereas PPARγ agonist played the opposite role. In conclusion, air-ventilated NMP confers a better liver function from DCD rats through the DAP-PPARγ-CYP2E1 axis; CYP2E1 activity provides a biomarker of liver functional recovery from DCD.

Figure 1

Air-oxygenated normothermic machine perfusion (NMP) confers a better functional recovery in rat liver from donation after cardiac death (*denotes significant differences among control, air-oxygenated NMP and hyperoxygenated NMP, P < 0.05; n = 6). (A) Histology (hematoxylin and eosin staining) of the perfused livers (hematoxylin counterstaining, original magnification × 200, and scale bars 50 μm); (B) uzuki sinusoidal injury score of the perfused livers on a scale from 0 to 4; (C) Changes in levels of ALT and AST in the NMP perfusate; (D) Relative changes in SOD activity and MDA level in the perfused livers; (E) Relative levels of inflammation factors, TNF-α and IL-6, in the liver performed using ELISA.

Figure 2

The integrative analysis of proteomics and metabolomics-based detection in rat livers from donation after cardiac death following static cold storage and normothermic machine perfusion (NMP). Cyp2e1 represents cytochrome P450 2E1 protein; DPA represents docosapentaenoic acid. (A) The volcano plot represents the results of the proteomics (left) and metabolomics (right) analysis between air-oxygenated and hyperoxygenated NMP. Red spots represent the upregulated proteins/metabolites; blue spots represent the downregulated proteins/metabolites; and grey spots represent the unchanged proteins/metabolites. The differentially expressed protein (CYP2E1) and metabolite (DPA) were identified and encircled. (B) The KEGG enrichment bubble chart shows the differentially expressed proteins (triangle) and metabolites (bubble) in the corresponding KEGG pathway. The color represents the degree of statistical significance. The size represents the number of the differentially changed proteins/metabolites in the corresponding pathways. The encircled pathways of biosynthesis of unsaturated fatty acids were identified to relate to CYP2E1 and DPA. (C) Heatmap of Spearman correlation analysis of significant proteins (y-axis) and metabolites (x-axis). Each color cell on the map corresponds to a Spearman correlation coefficient between the identified proteins (y-axis) and metabolites (x-axis). Red denotes positive correlation, whereas blue denotes negative correlation, and the dark color denotes the value of the Spearman correlation coefficient. The encircled pathway was shown as negatively correlated to relate with P05182 (CYP2E1) and Met183 (DPA). (D) Identified from proteomics and metabolomics, the protein-metabolite interaction network provides a visualization of the interaction between functionally related proteins indicated by the red circle and metabolites by the blue circle. Cyp2e1 may react with metabolites (Octanoylcarnitine, Creatine, Adrenic acid, 12-Tridecenoic acid and DPA); DPA lies in the center of the reacted proteins including cyp2e1.

Figure 3

Expression of DPA, PPARγ and CYP2E1 in livers from donation after cardiac death using normothermic machine perfusion (NMP) (*denotes significant differences among control, hyperoxygenated NMP and air-oxygenated NMP, P < 0.05, n = 6). (A) Relative expression of DPA by metabolomics and CYP2E1 by proteomics; (B) Expressions of PPARγ and CYP2E1 by WB; (C) Semi-quantification of CYP2E1 by WB. (D) CYP2E1 activity quantified using the fluorescent probe.

Figure 4

Effect of PPARγ on functions of livers from donation of cardiac death under air-oxygenated normothermic machine perfusion (NMP) (*denotes significant differences among DMSO, Rosiglitazone and GW6471, P < 0.05, n = 4). (A) Histology (haematoxylin and eosin staining, original magnification × 200, and scale bars 50 μm); (B) Suzuki sinusoidal injury scores of the perfused livers on a scale from 0 to 4; (C) Changes in levels of ALT and AST in NMP perfusate; (D) Relative changes in MDA level and SOD activity in the perfused livers; (E) Relative levels of TNF-α and IL-6 in the perfusate using ELISA.