In this study, we found that the level of the pyroptosis-related proteins, including caspase-1, caspase-11, and IL-1β, significantly 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 significantly 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 inflammation in liver tissues
Pyroptosis was defined 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
inflammation.
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 significantly 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 significantly 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 confirm 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 significance 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 inflammatory 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.