Hypothermic oxygenated perfusion attenuates DCD liver ischemia-reperfusion injury by activating the JAK2/STAT3/HAX1 pathway to regulate endoplasmic reticulum stress

Abstract

Background: Hepatic ischemia-reperfusion injury (IRI) in donation after cardiac death (DCD) donors is a major determinant of transplantation success. Endoplasmic reticulum (ER) stress plays a key role in hepatic IRI, with potential involvement of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway and the antiapoptotic protein hematopoietic-lineage substrate-1-associated protein X-1 (HAX1). In this study, we aimed to investigate the effects of hypothermic oxygenated perfusion (HOPE), an organ preservation modality, on ER stress and apoptosis during hepatic IRI in a DCD rat model.

Methods: To investigate whether HOPE could improve IRI in DCD livers, levels of different related proteins were examined by western blotting and quantitative real-time polymerase chain reaction. Further expression analyses, immunohistochemical analyses, immunofluorescence staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and transmission electron microscopy were conducted to analyze the effects of HOPE on ER stress and apoptosis. To clarify the role of the JAK2/STAT3 pathway and HAX1 in this process, AG490 inhibitor, JAX1 plasmid transfection, co-immunoprecipitation (CO-IP), and flow cytometry analyses were conducted.

Results: HOPE reduced liver injury and inflammation while alleviating ER stress and apoptosis in the DCD rat model. Mechanistically, HOPE inhibited unfolded protein responses by activating the JAK2/STAT3 pathway, thus reducing ER stress and apoptosis. Moreover, the activated JAK2/STAT3 pathway upregulated HAX1, promoting the interaction between HAX1 and SERCA2b to maintain ER calcium homeostasis. Upregulated HAX1 also modulated ER stress and apoptosis by inhibiting the inositol-requiring enzyme 1 (IRE1) pathway.

Conclusions: JAK2/STAT3-mediated upregulation of HAX1 during HOPE alleviates hepatic ER stress and apoptosis, indicating the JAK2/STAT3/HAX1 pathway as a potential target for IRI management during DCD liver transplantation.

Keywords: DCD; Endoplasmic reticulum stress; HAX1; HOPE; IRI; JAK2/STAT3; Liver transplantation.

Fig. 1

HOPE improves liver function and reduces inflammation in a rat model of DCD. A, B The levels of AST and ALT in the perfusate (n = 6 for each group). C, D H&E staining and histological injury score in rat liver tissue (n = 6 for each group). Scale = 200 μm (top panel) and 50 μm (bottom panel). E–L mRNA expression levels of IL-1β, IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and HMGB1 (n = 3 for each group). ^*p < 0.05 and ^** p < 0.01

Fig. 2

HOPE alleviates hepatic ER stress in a rat model of DCD. A–C Western blotting and statistical analyses of BIP, CHOP, ATF6, ATF4, IRE1, p-IRE1, XBP-1s, and β-actin proteins (n = 3 for each group). p-IRE1 indicates phospho-IRE1. D IF staining of BIP and CHOP proteins in rat liver tissues (n = 6 for each group). Scale = 100 μm. E The microstructure of hepatocytes as observed by TEM (n = 3 for each group). The red arrow indicates the ER, and the blue arrow indicates the mitochondria. Scale = 500 nm. ^*p < 0.05 and ^**p < 0.01

Fig. 3

HOPE alleviates hepatic apoptosis in a rat model of DCD. A, B TUNEL staining and scoring of rat liver tissue (n = 6 for each group). Scale = 200 μm. C–F Western blotting and statistical analyses of BCL2, BAX, PUMA, NOXA, GADD45A, caspase-9, caspase-3, and β-actin proteins (n = 3 for each group). T-caspase indicates total caspase, C-caspase indicates cleaved caspase. G Caspase-3 activity detection (n = 6 for each group). H, I IHC staining and statistical analysis of C-caspase-3 protein in rat liver tissue (n = 6 for each group). Scale = 100 μm. ^*p < 0.05 and ^**p < 0.01

Fig. 4

HOPE attenuates liver injury by activating JAK2/STAT3 pathway in a rat model of DCD. A–F Western blotting and statistical analyses of JAK2, p-JAK2, STAT3, p-STAT3, and β-actin proteins (n = 3 for each group). p-JAK2 indicates phospho-JAK2, p-STAT3 indicates phospho-STAT3. G, H The levels of AST and ALT in the perfusate (n = 6 for each group). I, J H&E staining and histological injury score in rat liver tissue (n = 6 for each group). Scale = 200 μm (top panel) and 50 μm (bottom panel). ^*p < 0.05 and ^**p < 0.01

Fig. 5

HOPE attenuates ER stress and apoptosis by activating JAK2/STAT3 pathway in a rat model of DCD. A–F Western blotting and statistical analyses of BIP, CHOP, ATF6, ATF4, IRE1, p-IRE1, XBP-1s, BCL2, BAX, PUMA, NOXA, GADD45A, caspase-9, caspase-3, and β-actin proteins (n = 3 for each group). p-IRE1 indicates phospho-IRE1, T-caspase indicates total caspase, C-caspase indicates cleaved caspase. G Caspase-3 activity detection (n = 6 for each group). H, I TUNEL staining and scoring of rat liver tissue (n = 6 for each group). Scale = 200 μm. ^*p < 0.05 and ^**p < 0.01

Fig. 6

JAK2/STAT3 pathway regulates the interaction between HAX1 and SERCA2b. A, B Western blotting and statistical analyses of HAX1, SERCA2b, and β-actin proteins in rat liver tissues (n = 3 for each group). C, D Western blotting and statistical analyses of HAX1, SERCA2b, and β-actin proteins after AG490 pretreatment in rat liver tissues (n = 3 for each group). E–H Western blotting and statistical analyses of JAK2, p-JAK2, STAT3, p-STAT3, HAX1, SERCA2b, and β-actin proteins after AG490 and OE-HAX1 treatment in H/R cell model of BRL-3A (n = 3 for each group). p-JAK2 indicates phospho-JAK2, p-STAT3 indicates phospho-STAT3, OE-HAX1 indicates overexpressed HAX1. I SERCA activity analysis after AG490 and OE-HAX1 treatment in H/R cell model of BRL-3A (n = 6 for each group). J CO-IP analysis of the interaction between HAX1 and SERCA2b in rat liver tissues (n = 3 for each group). K Co-staining of HAX1 (green) with SERCA2b (red) in BRL-3A cell model (n = 6 for each group). Scale = 20 μm. ^*p < 0.05 and ^**p < 0.01

Fig. 7

JAK2/STAT3 pathway regulates ER stress and apoptosis through HAX1. A–D Western blotting and statistical analyses of BIP, CHOP, ATF6, ATF4, IRE1, p-IRE1, XBP-1s, BCL2, BAX, PUMA, NOXA, GADD45A, caspase-9, caspase-3, and β-actin proteins after AG490 and OE-HAX1 treatment in H/R cell model of BRL-3A (n = 3 for each group). p-IRE1 indicates phospho-IRE1, C-caspase indicates cleaved caspase, OE-HAX1 indicates overexpressed HAX1. E Caspase-3 activity detection (n = 6 for each group). F, G Flow cytometric analysis used for the detection of apoptosis after AG490 and OE-HAX1 treatment in H/R cell model of BRL-3A (n = 3 for each group). H, I Flow cytometric analysis used for the detection of mitochondrial membrane potential in H/R cell model of BRL-3A (n = 3 for each group). ^*p < 0.05 and ^**p < 0.01

Fig. 8

Schematic showing proposed mechanism. HOPE inhibits the three branches of UPR (PERK, ATF6, and IRE1 pathways) by activating JAK2/STAT3 signal pathway, thus alleviating ER stress and apoptosis. In addition, activated JAK2/STAT3 signal pathway can maintain ER calcium homeostasis by upregulating HAX1 and promoting the interaction of HAX1/SERCA2b. Upregulated HAX1 also regulates ER stress and apoptosis by inhibiting the IRE1 pathway