Cold storage induces rat liver pyroptosis by activating endoplasmic reticulum stress response through the ATF6-CHOP pathway

Aims :Liver injury is a common complication of cold storage (CS), and often constitutes a direct cause for liver transplantation failure. The cellular and molecular mechanisms underlying CS-induced liver injury remain unclear. Recent evidence indicates that pyroptosis plays an important role in multiple pathophysiological processes. Using rat liver tissue and cells as a model, we identied a novel mechanism by which inammasome-dependent interleukin-1β (IL-1β) activation and hepatocyte pyroptosis mediate CS-induced liver injury. Methods :To induce CS, liver tissue and cells were subjected to storage at 4ºC for 12 and 24 h. Inhibition of endoplasmic reticulum (ER) stress was achieved by RNA silencing. Measurements of caspase-1, caspase-11, and IL-1β were performed. Results: Pyroptosis was activated in CS-treated livers, as evidenced by increased levels of caspase-1 and caspase-11 activity, and the elevated expression of IL-1β. ER stress response was activated as well. Inhibition of ER stress response prevented CS-induced liver pyroptosis and inammation. Conclusion : Our ndings suggest that pyroptosis might be playing an important role in the development of liver injury induced by CS. Overactivated ER stress response, followed by activation of the ATF6-CHOP signaling pathway, might be a novel molecular mechanism involved in CS-induced pyroptosis of liver tissue and cells.


Introduction
Liver transplantation is currently an established treatment for patients suffering from end-stage liver disease. Although signi cant progress in liver transplantation has been made over the past two decades, cold injury in cold-stored (CS) liver remains a major challenge to be overcome [1,2]. Extensive body of evidence indicates that apoptosis plays an important role in hepatic tissues injury [3][4][5]. Although a large number of experiments have been performed in an attempt to minimize apoptosis and its effects on transplanted organs, results are still not satisfactory. Whether other tissue-and cell-loss mechanisms are involved in transplanted organ injury requires further in-depth research.
Pyroptosis is a unique type of programmed cell death that is distinct from apoptosis and necrosis [6,7]. Pyroptosis depends on the activation of the caspase-1 cascade and the formation of in ammatory cytokines, including those of the IL-1 family. A recent study reported a link between cell pyroptosis and renal ischemia-reperfusion injury [8]. CS is a very important intermediate stage preceding organ transplantation, and organs often suffer from cold ischemic damage during storage. Whether pyroptosis is involved in liver cells death when stored at low temperatures, in this study we explored the effects of CS on liver tissue and cells.Whatever, pyroptosis and in ammatory cytokines are related to such damage remain unknown,So we rstly elucidated the association between CS and cells' pyroptosis.

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Animal care, rat model, and treatments All animal procedures were conducted in compliance with protocols approved by the local government ethical authorities. Eight to 12 weeks old male Sprague Dawley (SD) rats, weighing 250-300 g, were purchased from the Experimental Animal Center of Southwest Medical University (Luzhou, Sichuan, China). The rats were treated humanely, and all experiments were performed under conditions designed to minimize suffering. SD rats were randomly divided into three groups. Un-treated blank control (Group 1, n = 6), liver tissue CS for 12 h (Group 2, n = 6), liver tissue CS for 24 h (Group 3, n = 6). Brie y, rats were euthanized by intramuscular injection of sodium pentobarbital (30 mg kg − 1 of body weight). After opening the abdominal cavity, the livers were procured and ushed with 30 mL of University of Wisconsin (UW) solution. Livers of treatment groups 2 and 3 were then stored in the UW solution at 4ºC for 12 or 24 h, respectively.

Cell culture and intervention
Buffalo rat liver (BRL) cells from normal rat liver were obtained from the cell bank of the Chinese Academy of Sciences (Shanghai, China), and cultured in Dulbecco's modi ed Eagle medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco, Invitrogen), 100 U mL -1 penicillin, and 100 µg mL -1 streptomycin. For cold treatment, the cells were incubated at 4°C in an atmosphere of 5% CO 2 in air. The BRL cells were also treated with ATF6-speci c small interfering RNA (siRNA), using Lipofectamine 2000 (Invitrogen, Grand Island, NY) in the culture medium. Untreated cells acted as control. The cells were cultured at 4°C and an atmosphere of 5% CO 2 in air for 12 h and 24 h.

Histopathology and immunohistochemistry
Liver samples were xed in 4% neutral formaldehyde, dehydrated in a gradient series of ethyl alcohol, dealcoholized in xylene, and then embedded in para n. Liver tissue samples were sectioned at 4 µm, mounted on slides and stained with hematoxylin and eosin (HE). Severity of liver damage was estimated by evaluating ve randomly-selected low-magni cation (x100) elds in each HE-stained section. Liver oxidative stress was detected using gp91 phox immunohistochemical analysis. Brie y, after antigen retrieval, the sections were incubated with gp91 phox antibody (Abcam, Cambridge, USA) for 1 h at room temperature. The slides were then washed with 0.5% BSA and incubated with a secondary antibody (Abcam, Cambridge, USA) for 60 min. Subsequently, antigen-antibody complexes were detected using diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, St. Louis, MO, USA) as a chromogen. Nuclei were counterstained with hematoxylin.

Western blot analysis
Cells were harvested and homogenized with cell lysis buffer (Beyotime, China). Total protein in the lysates was quanti ed using BCA Protein Assay Kit (Beyotime, China). Equal quantities of protein samples were separated by denaturation with 10% SDS/PAGE and then transferred onto polyvinylidene di uoride (PVDF) membranes. Membranes were incubated in a 5% skim milk TBST blocking solution at room temperature (RT) for 1 h. The membranes were then incubated with primary antibody, followed by horseradish peroxidase-conjugated secondary antibody. Finally, protein bands were visualized using an enhanced chemiluminescence (ECL) western blotting detection system (GE Healthcare, Amersham, UK). Antibodies used in this study were: cleaved caspase-12, GRP78, ATF6, CHOP, caspase1, caspase-11, IL-1β, GAPDH, and β-actin. All antibodies were purchased from Abcam (Cambridge, MA, USA) or Cell Signaling Technology (Beverly, MA, USA).

Statistical analysis
Data are reported as mean + standard deviation (SD). Comparisons between groups were performed using the Student's t-test. Multiple groups were compared using ANOVA. The overall survival was analyzed by Pearson's correlation and Kaplan-Meier survival analysis. Differences were considered signi cant when P < 0.05.

Results
In this study, we found that the level of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1β, signi cantly increased after 12 h of liver tissue cold-storage and peaked after CS of 24 h.
Similarly, CS induced pyroptosis in BRL cells, as was evident from the increase in lactate dehydrogenase release and the pathological changes in the samples. In addition, clear upregulation of the endoplasmic reticulum (ER) stress biomarkers, activator of transcription factor 6 (ATF6) and C/EBP homologous protein (CHOP), preceded pyroptosis in cells treated with CS. Silencing of ATF6 with siRNA signi cantly decreased CS-induced pyroptosis of BRL cells, as evidenced by reduced caspase-1 and caspase-11 activity, and IL-1β generation. We therefore conclude that pyroptosis, resulting from ER stress and ATF6-CHOP activation, is an important process, contributing to the damage seen in CS-treated BRL cells.

Cold storage induces injury in rats' liver samples
To determine whether there is pyroptosis, and if so -what is its potential function in liver cold injury, SD Rats' livers were used as a CS model. As shown in Figure 1, after liver samples were maintained in UW solution at 4℃ for 12 h or 24 h, they showed CS-related injury. HE staining was used to evaluate embedded tissue slices. As shown in Fig 1A, focal loss of cells' integrity, karyopyknosis, and dilated sinusoids are evident in samples from both cold-storage groups, notably more after 24 h of cold storage.
The Suzuki scoring method was used to score liver pathology in each group (Fig 1B). Measurement of lactate dehydrogenase (LDH)| served as a marker for hepatocellular injury. As shown in Figure 1C, perfusate enzymes activity was markedly elevated in CS-treated livers when compared with sham-treated control livers. These pathophysiological changes indicate that the cold treatment was successful in inducing hepatic injury, justifying its use as a model in our study.
Cold storage induces pyroptosis and in ammation in liver tissues Pyroptosis was de ned by the presence of active caspase-1 or caspase-11. To determine whether activation of pyroptosis in hepatocytes is the result of CS, expression of pyroptosis-related proteins, namely caspase-1, caspase-11, and IL-1β was assessed. Western blot and immunohistochemistry analysis revealed that the levels of all three proteins were markedly elevated at 12 h, and peaked at 24 h of CS (Fig 2). These results suggest that CS induces liver tissues pyroptosis and in ammation.

Cold storage can trigger ER stress
It has been reported that damages to the ER and mitochondria are implicated in cold stress-induced apoptosis [9-10]. Furthermore, previous studies reported that ER stress protein ATF6 is activated and mediates apoptosis in liver cells during CS. In this experiment, expression of ER stress-related proteins was studied. Western blot results (Fig 3) show that the expression level of the ER stress-related proteins, GRP78, ATF6, and CHOP, has signi cantly increased following CS, as compared to the sham-treated control. Moreover, levels of these proteins showed a time-dependent expression. They were markedly elevated after 12 h of CS and peaked after 24 h of CS.

Cold storage induces pyroptosis and ER stress in BRL cells
It is imperative to also investigate the effect of CS of BRL cells on the expression of pyroptosis-related proteins, including those related to ER stress. As shown in Figure 4, GRP78, ATF6, CHOP, caspase-1, caspase-11, and IL-1β were all expressed at low levels in untreated BRL cells. Following 12 h of CS, expression of these proteins was signi cantly upregulated. Levels have further increased when the cells were treated with 24 h of CS. These results suggest that, just like with liver tissue, CS of BRL cells induces pyroptosis and ER stress.
The ATF6-CHOP pathway is involved in cold storage-induced pyroptosis of BRL cells To con rm the association between ATF6-CHOP and pyroptosis following CS of BRL cells, further experiments were carried out. Based on the results reported above, injury to hepatic cells and tissues was most severe following 24 h of CS. We therefore selected 24 h CS treatment to evaluate the signi cance of ATF6-mediated pyroptosis. After AFT6 silencing in BRL cells that were exposed to 24 h of CS, levels of ER stress-related proteins were investigated. As shown in Figure 5A, compared with the control group, no obvious changes took place in the levels of CHOP in the ATF6 siRNA-treated group.
Changes in the expression level of pyroptosis-related proteins after 24 h of CS were also evaluated. As shown in Figure 5A, levels of caspase-1, caspase-11, and IL-1β remained similar to those in the control group when the BRL cells were treated with ATF6 siRNA. The ATF6 siRNA-treated BRL cells markedly decreased the activation of in ammatory caspases as well as the levels of IL-1β. These results indicate that exposure of BRL cells to 24 h of CS results in activation and overexpression of GRP78, ATF6, and CHOP. These, in turn, activates hepatocyte pyroptosis. This conclusion is supported by the fact that ATF6 miRNA blocks this chain of events. Taken together, these results indicate that the ATF6-CHOP pathway is involved in CS-induced pyroptosis of BRL cells.

Discussion
At present, under normal conditions, livers are stored at 2-8ºC as whole organs, perfused in UW media or histidine-tryptophan-ketoglutarate solution (HTK) [11][12]. These solutions facilitate extending liver preservation time from several hours to a maximum of a few days. However, it has been shown that CS causes vasoconstriction, tubular and endothelial injury, and cell death [13]. These may lead to liver dysfunction or delayed graft function upon transplantation. Although the issue was discussed in many studies, the cellular and molecular mechanisms leading to hepatic cold-induced injury remain poorly characterized. Understanding the underlying mechanisms and devising ways to attenuate them could have a tremendous impact on the number of transplants performed, as well as on the outcome of liver transplantation procedures.
Pyroptosis is a programmed cell death characterized by rapid plasma membrane rupture and release of pro-in ammatory intracellular contents. The pathway is morphologically and mechanistically distinct from other forms of cell death. A de ning feature of pyroptosis is its dependence on caspase-1 [14]. Caspase-1 was rst recognized as a protease that cleaves the inactive precursors of IL-1β and IL-18 into mature in ammatory cytokines, and it was initially called IL-1β converting enzyme [15]. Activation of caspase-1 induces pore formation on the cell membrane and contributes to the generation and release of abundant in ammatory factors, thus contributing to pyroptosis [16][17]. Caspase-11, which is the upstream regulator of caspase-1, mediates its activity by direct cleavage of pro-caspase-1 [18]. Recent studies [19][20] have shown that caspase-11 can activate caspase-1 under the coordination of NLRP3 in ammasomes and can also induce caspase-1-independent pyroptosis. These ndings denote that caspase-11 mediates pyroptosis in a caspase-1-dependent or -independent manner; and, stemming from this,the potentially important role both caspase-1 and caspase-11 play in pyroptosis [21]. Our in vitro study has shown that pyroptosis is characterized by an increase in the expression of caspase-1 and caspase-11, and generation of IL-1β, that already appeared after 6 h and peaked after 24 h of CS. The altered levels of caspase-1, caspase-11, and IL-1β in liver tissues positively correlated with CS duration. Our in vitro study revealed that CS upregulated caspase-1 and caspase-11 in normal liver cells. This was accompanied by increased pore formation, LDH release, and IL-1β generation. These results suggest that when cells undergo pyroptosis, they lose membrane integrity, which leads to the release of cellular contents, including LDH and the in ammatory cytokine IL-1β.
Endoplasmic reticulum (ER) stress is an initiator of cell death and in ammatory mechanisms. A number of studies have shown that by activating ER stress, ischemia-reperfusion injury (IRI) can induce several cell death mechanisms, including autophagy, apoptosis, necroptosis, and mitochondria-mediated programmed necrosis [22][23][24]. A few studies on pancreatic β-cells [25][26] have demonstrated that ER stress can activate the NOD-like receptor (NLR) family, pyrin domain-containing 3 (NLRP3) in ammasome via protein kinase RNA-like ER kinase, and inositol-requiring enzyme 1 signaling pathways, thus triggering caspase-1 activity, and leading to cells death and IL-1β release. Yang and colleagues [8] were rst to demonstrate that the expression of GRP78 and CHOP was signi cantly elevated in renal IRI in mice. CHOP (C/EBP homologous protein) can induce the expression of casepase-11 and activate its effector, activator casepase-11. Casepase-11 overexpression promotes activation of the cytokines IL-1β and IL-18, and induces pyroptosis. Inhibiting the expression of CHOP would weaken the activity of caspase-11. Pretreatment with the ER stress inducer ionomycin can alleviate pyroptosis induced by IRI, suggesting that CHOP-caspase-11 is an important signaling pathway related to pyroptosis during renal ischemia-reperfusion. It is thus clear that over activated ER stress might be an important cause of pyroptosis in certain cell types.
The upregulation of GRP78 is a hallmark for the initiation of ER stress. Once initiated, ATF6 is an important signaling molecule associated with the process. Elevated expression of CHOP, a downstream molecule, indicates the activation of the ER-stress-mediated cell death signaling pathway. Hence, upregulation of GRP78,ATF6 and CHOP indicates over activated ER stress [27]. Our previous study [28] showed that ER stress is in an active state during CS of the liver, with GRP78, ATF6, and CHOP highly expressed. However, knowledge was lacking with respect to the involvement of pyroptosis in liver cell death following CS and its relationship with the release of in ammatory cytokines. Our objective in this study was thus to investigated the pyroptosis-related alterations that occur in tissues and cells of the liver when exposed to CS. Our results show that upregulation of GRP78, ATF6, and CHOP may precede pyroptosis, as indicated by increased pore formation, LDH release, elevated expression of caspase-1 and caspase-11, and increased production of IL-1β in liver tissue and cells treated with CS. Furthermore, our results show that pyroptosis-related proteins expression is positively correlated to the duration of CS treatment. Understanding the relationship between the duration of CS and liver damage would direct us to the realization that shortening the time of CS would help reduce pyroptosis. Our in vitro study demonstrated that silencing ATF6 signi cantly suppressed CS-induced BRL cell pyroptosis, supporting the notion that pyroptosis might be a downstream effector of overactivated ER stress.
Pyroptosis is a unique programmed cell death, distinct from apoptosis and necrosis. Here we showed that overactivated ER stress followed by activation of the ATF6-CHOP signaling pathway might be a novel molecular mechanism involved in CS-induced pyroptosis of hepatic tissue and BRL cells. Moreover, the severity of pyroptosis is closely related to hepatic tissue injury.

Declarations
Compliance with ethical standards Con ict of interest The authors declares that they have no con ict of interest. Characteristics of rat liver injury after cold-storage treatment. (A) Micrographs of representative HE stained rats' liver sections after 12 h and 24 h of cold storage. (B) Suzuki score of liver tissue in each experimental group. Data are presented as means + SD. Statistical analysis was performed by Student's ttest, n = 6 rats per group. (C) Perfusate LDH levels, analyzed as a measure of hepatocellular injury. *P < 0.05, **P < 0.01.

Figure 2
Cold storage altered the levels of pyroptosis-associated proteins. Levels of caspase-1 and caspase-11 in rat hepatic tissues characterized by immunohistochemistry assays after CS for speci c time durations (A). Protein expression level of caspase-1, caspase-11, IL-1β, and GAPDH in hepatic tissues examined by Western blot analysis (B, C). Images are true representation of the samples. Data shown are expressed as mean + SD of individual groups (n = 6 rats/group at each time point). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3
Cold storage-induced endoplasmic reticulum (ER) stress. Expression of glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) in rat hepatic tissues after cold storage for speci c time duration. Samples were characterized by Western blot. Images are true representation of the samples. Data are expressed as means + SD of individual groups (n = 6 rats/group at each time point). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4
Cold storage-induced ER stress and pyroptosis in BRL cells. BRL cells were cultured under normal conditions as healthy controls or at low temperature (4ºC) to induce injury for the indicated time periods.
After treatment, cells were harvested and evaluated by Western blot assay for the levels of GRP78, ATF6, CHOP, caspase-1, caspase-11, and IL-1β (A). Images are true representation of the samples. Data are expressed as means + SD of each group of cells from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. ATF6 siRNA reduces cells' cold-induced injury. Pretreatment with ATF6 siRNA mitigated cells' coldinduced injury by reducing ATF6 and its downstream effectors CHOP, caspase-1, caspase-11, and IL-1β in BRL. Groups of BRL cells were treated with ATF6 siRNA or control, and subjected to cold storage for 24h.