Blocking CIRP protects against acute pancreatitis by improving mitochondrial function and suppressing pyroptosis in acinar cells

Acute pancreatitis (AP) continues to pose a major challenge as targeted therapeutic interventions are absent. Mitochondrial dysfunction and inflammasome-dependent pyroptosis are involved in the pathogenic mechanisms of AP. CIRP is a stress-response protein and a damage-associated molecular pattern (DAMP) molecule. In our previous studies, we discovered that excessive CIRP can directly damage pancreatic acinar cells. Nonetheless, the precise involvement of CIRP in AP is still unexplored. The primary aim of this study was to examine the potential involvement of CIRP in the development of pyroptosis and mitochondrial dysfunction in AP. To study this, an L-arginine-induced AP mouse model was used. Our results showed that Caspase-1-mediated pyroptosis and mitochondria-derived reactive oxygen species (ROS) were crucial factors in the occurrence of tissue damage and inflammation in AP. A substantial increase in the CIRP serum levels was observed in AP mice. Blocking CIRP by either CIRP gene knockout or systemic administration of C23, a competing inhibitor of CIRP, reduced ROS accumulation and pyroptosis in AP mice. These effects were associated with attenuated pancreatic injury and inflammation. In addition, CIRP-triggered mitochondrial dysfunction, autophagy impairment, and pyroptosis in pancreatic acinar cells were prevented by TAK242, an inhibitor of CIRP receptor TLR4. In conclusion, CIRP can induce mitochondrial dysfunction and pyroptosis in pancreatic acinar cells, and blocking CIRP may be a valuable approach to treating patients with AP.


INTRODUCTION
Acute pancreatitis (AP), a common inflammation disorder affecting the digestive system, is hallmarked by rapid development and high mortality [1].AP is a prominent contributor to hospitalizations in the United States, accounting for an estimated 300,000 visits to emergency rooms annually [2].The annual incidence of AP globally ranges between 30 and 40 cases per 100,000 individuals, resulting in significant financial burdens for those affected [3].In particular, severe AP not only leads to necrosis of pancreatic tissue but also results in systemic inflammatory response syndrome (SIRS) and even multiple organ dysfunction syndrome (MODS) [4].Severe AP is one of the main etiological factors for clinical pancreatitis-related mortality [1,5].Currently, effective targeted therapeutics are not available for treating pancreatitis in clinical settings, and AP is managed using supporting treatment.
Pyroptosis is an inflammation-triggered mode of programmed cell death [6].In contrast to classical apoptosis and necrosis, pyroptosis is characterized by the formation of cell membrane pores, the swelling of the cytoplasm, the rupture of the cell membranes, the release of cytokines, and exacerbated systemic inflammatory responses [7].Pyroptosis is Caspase-1-dependent and mediated mainly by the NLPR3 pathway.The recruitment of an apoptosis-related speck-like protein that binds to the cysteine protease Caspase-1 and the caspase recruitment domain (ASC) by inflammasome results in the formation of the NLRP3 inflammasome complex.This leads to the activation of GSDMD and interleukin (IL)-1β, which promotes several pathological processes [6].Furthermore, pyroptosis is involved in the pathogenic mechanisms of several diseases, including some autoimmune diseases, tumors [8,9], liver fibrosis [10], and atherosclerosis [11].
Cold-inducible RNA binding protein (CIRP) is an intracellular RNA chaperone protein produced from the cell when exposed to external stimuli, like low temperature or hypoxia [12].Additionally, necrotic cells are capable of passively releasing CIRP [13].Multiple studies have shown evidence that extracellular CIRP is involved in the pathogenesis of inflammatory disorders and functions as a damage-associated molecular pattern (DAMP) [12,14].
Mitochondria, which are the energy stations of cells, are the primary source of ATP production.Mitochondrial dysfunction can affect energy (ATP) metabolism, promote reactive oxygen species (ROS) production, and as a result, trigger oxidative stress [16,17].Oxidative stress can damage mitochondrial DNA and membrane structures and impair mitochondrial function [18].Mitochondrial dysfunction is a pathological factor in AP [19].Thus, AP can be mitigated by protecting mitochondrial function [19,20].ROS accumulation can also induce pyroptosis by activating NLRP3 [21,22].Mitochondrial dysfunction and impaired autophagy promote ROS accumulation [23].Previously, we demonstrated that extracellular CIRP can directly damage pancreatic acinar cells [24].Nonetheless, the precise role of CIRP in the pathogenesis of AP is unclear.This study hypothesized that CIRP mediates AP pathogenesis by promoting ROS accumulation, impairing mitochondrial function and autophagy, and upregulating pancreatic acinar cell pyroptosis.
Therefore, our primary focus is to study whether CIRP can cause excessive accumulation of ROS by damaging mitochondrial function and mitochondrial autophagy, thus activating the pyroptosis pathway in L-arginine-induced AP.

Inhibition of pyroptosis alleviated pancreatic tissue damage in the L-arginine-induced AP model
An L-arginine-induced AP mouse model was established to examine the involvement of CIRP and pyroptosis in the pathogenesis of AP.Previous studies have reported that pancreatic tissue damage peaked at 72 h post-L-arginine intraperitoneal injection [20].Immunohistochemical (IHC) analysis revealed that the expression of Caspase-1, a pyroptosis marker protein, was remarkably (p < 0.05) upregulated in AP mice (Fig. 1A, B).Additionally, the serum concentration of IL-1β, an inflammatory effector of the pyroptosis pathway, was remarkably upregulated in AP mice (Fig. 1C).These findings indicate that the occurrence of pancreatitis was accompanied by pyroptosis induction.Mice were intraperitoneally administered with VX-765 (200 mg/kg body weight), a Caspase-1 selective inhibitor, which is a key pyroptosis-related protein.In AP animals, the extent of necrosis, infiltration of inflammatory cells, and pancreatic tissue edema was increased relative to that in the sham group, as shown by HE staining (Fig. 1D).Nevertheless, VX765 administration mitigated pancreatic tissue damage and markedly decreased injury scores and necrosis area in AP mice (Fig. 1E, F).Similarly, VX765 administration mitigated the release of necrosis factor LDH and proinflammatory markers TNF-α and IL-6 in mice serum (Fig. 1G-I).Furthermore, the VX765 treatment alleviated the elevated serum pancreatic amylase level (Fig. 1J).

Inhibition of mitochondrion-derived ROS suppressed pancreatic pyroptosis and tissue damage in the AP model
To further investigate the correlation between ROS and pyroptosis in AP, mice were administered with a specific scavenger of mitochondrial superoxide (MitoTEMPO; 20 mg/kg body weight; Sigma) 1 h before L-arginine injection.HE staining revealed that the pancreatic damage and necrotic area in the MitoTEMPOtreated group were considerably ameliorated in comparison to those in the vehicle group (Fig. 2A-C).The results of serum LDH analysis validated these findings (Fig. 2M).IHC analysis revealed that the Caspase-1 level was substantially upregulated in AP mice but was downregulated upon MitoTEMPO treatment (Fig. 2D, E).Western blotting analysis revealed that MitoTEMPO treatment markedly downregulated Caspase-1 expression and other pyroptosis activation-associated proteins, NLRP3, ASC, and GSDMD in AP mice (Fig. 2F-I).Additionally, MitoTEMPO mitigated the upregulated serum levels of IL-1β and systemic proinflammatory indicators (TNF-α and IL-6), suggesting that ROS suppression alleviated pyroptosis in the L-arginine-induced AP mouse model (Fig. 2J-L).
Pancreatic AR42J cells (5 × 10 5 cells/well) were pretreated with the ROS scavenger n-acetylcysteine (NAC, Sigma-Aldrich, 5 nM) for 2 h and treated with cerulein (10 nmol/L) + LPS (10 µg/mL) for 24 h to verify the correlation between ROS and pyroptosis in vitro.Western blotting analysis revealed that the Caspase-1, ASC, NLRP3, and GSDMD expression were remarkably upregulated in the cerulein + LPS-treated group, suggesting that pyroptosis was activated in the in vitro AP model.However, these proteins were shown to be downregulated considerably with the intervention of NAC, implying that suppressing ROS can reduce the pyroptosis level of pancreatic acinar cells (Supplementary Fig. 2A-D).ROS are crucial in triggering pyroptosis during the pathogenesis of AP, as validated by our findings.

CIRP deficiency attenuated pancreatic pyroptosis and tissue injury in the L-arginine-induced AP mouse model
The serum CIRP levels in a mouse model of AP triggered by L-arginine were measured to investigate the function of CIRP in AP.The serum CIRP level was remarkably upregulated in AP mice (Fig. 3A).Subsequently, pancreatic tissues were subjected to Caspase-1 immunohistochemical analysis to additionally examine the involvement of CIRP in pyroptosis in AP.The pancreatic levels of Caspase-1 were significantly upregulated in AP mice.However, the upregulation of Caspase-1 was significantly suppressed in CIRP −/− mice (Fig. 3B, C).Similarly, the expression levels of other pyroptosis activation-associated proteins ASC, GSDMD, and NLRP3 were significantly higher in the acute pancreatitis model.Interestingly, the above phenomena were reduced in CIRP -/-mice This suggests that CIRP promotes pyroptosis in the L-arginineinduced AP model (Fig. 3D-G).NLRP3 can also recruit Caspase-1 through ASC.Activated Caspase-1 enhances the maturation of IL-1β by cleaving pro-IL-1β.The IL-1β levels were remarkably elevated in AP mice, and this upregulation was mitigated in CIRP −/− mice (Fig. 3H).HE staining revealed that CIRP KO significantly alleviated pancreatic tissue damage in AP mice (Fig. 3I-K), as well as suppressed the upregulated serum levels of LDH, pro-inflammatory indicators (TNF-α and IL-6) (Fig. 3L-N), and amylase (Fig. 3O).Please note that serum amylase levels in CIRP KO mice induced by L-Arginine were consistent with the control mice, but HE staining still showed significant damage in the pancreatic tissue of these animals.These findings are consistent with pathophysiological changes of L-arginine-induced AP models.Following L-arginine injection, amylase concentrations in the serum peaked between 12 and 24 h but recovered to normal between 24 and 48 h later.After 72 h, however, the degree and severity of necrotic alterations in the exocrine tissue of the pancreas accompanied by infiltration of inflammatory cells peaked [25].

CIRP deficiency improved mitochondrial function and suppressed ROS accumulation in the L-arginine-induced AP mouse model
The ROS levels were examined in AP mice to ascertain the potential ROS function in pyroptosis in AP.DHE staining revealed that the pancreatic ROS levels were substantially upregulated in AP mice, and this upregulation was mitigated in CIRP −/− mice (Fig. 4B, C).Consistently, the pancreatic levels of SOD and GSH were markedly downregulated in AP mice, and this downregulation was mitigated in CIRP −/− mice (Fig. 4G, H).Mitochondrial dysfunction has been observed to be linked to excessive oxidative stress triggered by ROS [16].Thus, maintaining physiological mitochondrial function requires healthy mitochondrial dynamics, which include fission, biogenesis, and fusion.Notably, the levels of Tfam and PINK1 (two critical regulators of mitochondrial biogenesis), as well as those of Mfn2 (a regulator of mitochondrial fusion and mitophagy), were significantly downregulated in AP mice, as evidenced by Western blotting analysis.Transmission electron microscopy (TEM) revealed that following the induction of AP in mice, there was an increase in the swelling of mitochondria, as well as condensation, disruption, or cristae loss (Fig. 4A).These L-arginine-induced changes were suppressed in CIRP −/− mice (Fig. 4D, E).Furthermore, mitochondrial dysfunction can be alleviated by suppressing the accumulation of ROS via mitophagy [26].The LAMP2A (autophagy function-related protein) expression was remarkably downregulated in AP mice, whereas that of LC3-II was significantly upregulated, indicating that autophagy function was impaired in AP mice.However, these changes were mitigated in CIRP −/− mice, suggesting that the CIRP KO alleviated mitochondrial autophagy dysfunction (Fig. 4D-F).C23 administration improved mitochondrial function and suppressed ROS accumulation in the L-arginine-induced AP mouse model DHE staining revealed that C23 mitigated the L-arginine-induced upregulation of pancreatic ROS levels (Fig. 6B, C) and downregulation of pancreatic SOD and GSH levels, suggesting that the levels of antioxidants in pancreatic tissue were restored by C23 (Fig. 6G, H).TEM also revealed that mitochondrial swelling and condensation, disruption, or loss of cristae were mitigated by C23 administration (Fig. 6A).Western blotting analysis revealed that C23 suppressed the L-arginine-triggered downregulation of Tfam, PINK1, and Mfn2 (Fig. 6D, E), upregulation of LAMP2A and LC3-II, and impairment of mitochondrial autophagy function (Fig. 6D, F).
C23 mitigated cerulein + LPS-induced mitochondrial dysfunction, autophagy impairment, ROS accumulation, and pyroptosis in cultured pancreatic acinar cells Next, in vitro experiments were performed with pancreatic AR42J cells.Subsequently, pancreatic AR42J cells (5 × 10 5 cells/well) were subjected to cerulein (10 nmol/L) + LPS (10 µg/mL) for 24 h and used to simulate AP in vitro.Cerulein is a cholecystokinin (CCK) analog, which provokes digestive enzyme secretion.Both cerulein alone and cerulein + LPS are widely used methods to induce pancreatitis [25].Cerulein alone usually induces mild acute pancreatitis, while cerulein + LPS can induce severe acute pancreatitis under both in vivo and in vitro conditions.Here, we found that the CIRP level in the supernatant of AR42J cells was significantly increased after cerulein + LPS treatment, which was in agreement with the findings of investigations conducted in vivo (Supplementary Fig 1).Additionally, the Tfam, PINK1, and Mfn2 expressional levels were remarkably downregulated in the cerulein + LPS-treated group (Fig. 7A, B).Meanwhile, the expression levels of autophagy function-related protein LAMP2A were significantly downregulated, whereas those of LC3-II were considerably upregulated in the cerulein + LPS-treated group (Fig. 7A, C).Treatment with C23 alleviated cerulein + LPS-induced mitochondrial dysfunction and autophagy impairment in acinar cells (Fig. 7A-C).The ROS levels in pancreatic AR42J cells were examined using the ROS assay kit.
Treatment with C23 significantly mitigated the cerulein + LPSinduced upregulation of ROS accumulation in AR42J cells (Fig. 7D, E).Moreover, the level of pyroptosis-related proteins Caspase-1, ACS, NLRP3, and GSDMD remarkably increased in vitro and alleviated by using CIRP's competing inhibitor C23 (Fig. 7F-I),  which agrees with the in vivo finding.Next, pancreatic AR42J cells were pretreated with different cell death inhibitors and stimulated with recombinant CIRP.The MTT assay illustrated that treatment with the pyroptosis inhibitor VX765 significantly increased cell survival rates (Fig. 7J).Based on these findings, it is evident that CIRP mediates pyroptosis in the in vitro AP model.
CIRP directly induced mitochondrial dysfunction, autophagy impairment, ROS accumulation, and pyroptosis in cultured pancreatic acinar cells TAK242 was administered to the AR42J cells to better explore the specific mechanisms that are involved in CIRP.In particular, 1.5 µg/ mL recombinant CIRP was administered to AR42J cells (5 × 10 5 cells/well) for 72 h after pretreatment with or without TAK242 (500 µM) for 24 h.Notably, TAK242 upregulated the Tfam, Mfn2, and PINK1 expression levels in AR42J cells, as shown by Western blotting analysis results (Fig. 8A, B).Additionally, TAK242 alleviated autophagy impairment by upregulating the LAMP2A levels and downregulating the LC3-II levels (Fig. 8A, C).Furthermore, TAK242 significantly downregulated ROS accumulation in AR42J cells (Fig. 8D, E).These results suggest that CIRP promotes ROS accumulation, mitochondrial dysfunction, and autophagy injury in the in vitro AP model through its receptor, TLR4.
Pancreatic AR42J cells (5 × 10 5 cells/well) were pretreated with NAC (Sigma-Aldrich; 5 nM) for 2 h followed by subsequent treatment with 1.5 µg/mL recombinant CIRP for 24 h to verify the correlation between ROS and pyroptosis in vitro.Accordingly, the expression levels of related proteins Caspase-1, ASC, NLRP3, and GSDMD were substantially upregulated in pancreatic AR42J cells treated with recombinant CIRP as shown by Western blotting, suggesting that CIRP directly activated pyroptosis in pancreatic acinar cells.Treatment with NAC significantly mitigated the recombinant CIRP-induced upregulation of Caspase-1, ASC, NLRP3, and GSDMD, indicating that the inhibition of ROS can suppress pyroptosis in pancreatic AR42J cells (Fig. 8F-I).These findings indicate that ROS promotes pyroptosis in AP.

DISCUSSION
This study demonstrated that pyroptosis is activated in AP. injury, the injury score and necrotic area, and the release of injuryrelated factor LDH and other pro-inflammatory factors to alleviate the progression of AP.Inhibition of CIRP in various ways alleviates pyroptosis in both in vitro and in vivo AP models by relieving mitochondrial dysfunction, mitochondrial autophagy damage, and reactive oxygen species accumulation.CIRP can not only aggravate tissue injury by damaging the mitochondrial function but also hinder the clearance of ROS by damaging the autophagy function of mitochondria so that excessive ROS can further activate NLRP3 to mediate the pyroptosis in AP.These results reveal a novel CIRP-mediated acute pancreatitis injury pathway and provide a new target for future treatment.

Inhibition of pyroptosis can significantly reduce pancreatic tissue
AP, an inflammatory disease without specific treatment, is characterized by pancreatic tissue damage and necrosis as well as the secretion of several inflammatory cytokines into the bloodstream, leading to a cascade reaction, which, in extreme circumstances, may lead to multiple organ failure and systemic inflammatory response syndrome [27,28].Existing research has shown that serum CIRP levels are substantially upregulated after the onset of AP and exhibit a favorable correlation with the severity of AP [29].This was confirmed in this study using the Larginine-induced AP murine model.According to Linders J et al., the extracellular CIRP is an essential factor in the formation of neutrophil extracellular traps in AP [30].Xu Q et al. demonstrated that emodin alleviates lung injury in AP by blocking the CIRPmediated NLRP3 pathway [31].CIRP, an intracellular RNA chaperone protein, is transferred from the intracellular environment to the extracellular environment when exposed to external stimuli, such as hypoxia and low temperature [12].CIRP can also be passively released extracellularly from necrotic cells [13].Extracellular CIRP functions as a DAMP and regulates the pathogenesis of various inflammatory diseases through TLR4.Previous studies have reported that TLR4 expression is significantly upregulated in acinar cells during AP [32].Thus, CIRP may mediate damage by binding to TLR4 expressed on acinar cells.C23, an oligopeptide derived from CIRP, competitively inhibits CIRP binding to TLR4 [33,34].In this study, CIRP KO and C23 were used to block CIRP to elucidate its action mechanisms.The findings of this study demonstrated that CIRP inhibition significantly alleviates AP.
Mitochondrial damage is a critical pathogenic process in AP [19].Recently, Biczo et al. reported that mitochondrial dysfunction mediates hyperamylase, trypsinogen activation, necrosis, vacuolization, and inflammation in L-arginine-mediated AP [19].CIRP is also recognized for its involvement in promoting mitochondrial dysfunction.Zheng X et al. demonstrated that CIRP mediates mitochondrial dynamics disorder, exacerbates intracellular oxidative stress, upregulates NADPH oxidase via the TLR-4/MyD88 pathway, and consequently promotes apoptotic activity in HK-2 cells [35].Li Z et al. demonstrated that coldinducible RNA-binding proteins induce mitochondrial DNA breakage through the TLR4 signaling pathway and regulate macrophage death after trauma [15].Consistent with the results of our previous studies, the expression of the mitochondrial function-associated proteins Tfam and Mfn2 was significantly downregulated in the L-arginine-induced AP mouse model [36], indicating that mitochondrial function was significantly impaired in AP.The L-arginine-induced effects were significantly mitigated in CIRP −/− and C23-treated mice.Similar findings were obtained in the in vitro AP model.Treatment with recombinant CIRP significantly downregulated the expression of mitochondrial function-related proteins in acinar cells.TAK242, a specific inhibitor of TLR4, significantly suppressed the effect of recombinant CIRP, suggesting that CIRP promotes mitochondrial dysfunction in pancreatic acinar cells through TLR4.Furthermore, ROS synthesis primarily occurs in mitochondria.The dysfunction of mitochondria promotes ROS production, exacerbating mitochondrial dysfunction [37].
Mitochondrial autophagy is a cellular response that is both conserved and adaptable and is responsible for the selective elimination of mitochondria that are defective or damaged through the autophagosome [38].Autophagy in mitochondria is essential for the removal of ROS since mitochondria are the principal generator of ROS [39].Damaged mitochondria can lead to impaired ROS clearance [26].Liu Z et al. demonstrated that the overexpression of mitochondrial ROS promotes pyroptosis in hepatocytes by impairing mitochondrial autophagy [40].AP is often accompanied by impaired autophagy [41,42].A previous study reported that the dysregulation of the autophagic pathway in AP contributes to the downregulation of LAMP2A, a marker of enzyme-body formation [43].The lack of lysosomal involvement inhibits the autophagy pathway and the clearance of autophagosomes, resulting in the upregulation of LC3-II [44].In this study, the expression of Lamp2a was significantly downregulated, whereas that of LC3-II was significantly upregulated in the in vivo and in vitro AP models, suggesting that the autophagy function of mitochondria was impaired.However, autophagy was significantly downregulated in CIRP KO and C23-treated mice.Thus, it is evident from these findings that CIRP promotes autophagy injury in AP.Additionally, treatment with TAK242 mitigates recombinant CIRP-induced autophagy injury in pancreatic acinar cells, suggesting that CIRP promotes autophagy injury through TLR4.As demonstrated in existing literature, the PINK1 expression is upregulated after autophagy activation [45].Based on these findings, it seems that the PINK1 and Mfn2 expression levels are particularly significant for the activation of mitophagy [46,47].In this study, the PINK1 and Mfn2 expression levels were remarkably downregulated in the in vivo and in vitro AP models.
CIRP is reported to promote ROS accumulation.Sakurai T et al. demonstrated that the lack of CIRP downregulates ROS accumulation and consequently suppresses the onset of liver cancer [48].Additionally, Liu W et al. found that rhCIRP up-regulated the expression of gp91phox and P47phox in HL-7702 cells, caused apoptosis, elevated ROS levels, and stimulated the secretion of inflammatory factors [49].Immunofluorescence analysis revealed that the ROS levels were significantly upregulated in the Larginine-induced AP mouse model, while the antioxidant indices (SOD and GSH) were significantly downregulated, suggesting that AP was accompanied by oxidative stress induction.CIRP KO or C23 treatment significantly mitigated the effects of L-arginine in mice.C23 treatment suppressed ROS accumulation in the in vitro AP model.TAK242 suppressed recombinant CIRP-induced ROS accumulation in pancreatic acinar cells.Based on these findings, CIRP promotes ROS accumulation in AP through Tlr4.The formation of ROS is a typical upstream process that is involved in the activation of the NLRP3 inflammasome.Previous studies have reported that ROS promotes pyroptosis.Wu X et al. demonstrated that nicotine is responsible for the promotion of atherosclerosis via the ROS-NLRP3-elicited pyroptosis of endothelial cells [21].Liu Z et al. reported that the accumulation of mitochondrial ROS resulting from impaired mitophagy enhances pyroptosis in hepatocytes [40].In this study, the marker proteins of the pyroptosis pathway Caspase-1, NLRP3, and GSDMD, the marker proteins of the pyroptosis pathway, were significantly downregulated in the in vitro and in vivo AP models upon treatment with a ROS scavenger.Additionally, the ROS scavenger significantly mitigated recombinant CIRP-induced pyroptosis in AR42J cells.These findings indicate that ROS can activate the Caspase1-dependent pyroptosis pathway through NLRP3 in AP.The excessive accumulation of ROS may be caused by the impaired autophagy function of mitochondria is mediated by CIRP.Thus, CIRP released to the extracellular environment in AP serves as a DAMP to mediate mitochondrial dysfunction in pancreatic acinar cells, exacerbating tissue damage and promoting ROS accumulation.
Pyroptosis, a novel mode of programmed cell death that is distinct from traditional cell necrosis and apoptosis, is distinguished by the development of cell membrane pores and the secretion of the effector cytokine IL-1β [7].Caspase-1, which has been activated, is involved in cleaving the GSDMD protein molecule and initiating the oligomerization of the amino terminus (GSDMD-N), thereby promoting the formation of plasma membrane pores, the secretion of IL-1β and IL-18, and consequently inducing pyroptosis, which is implicated in the pathogenesis of systemic inflammation [50].It has been illustrated in a series of studies that pyroptosis which is reliant on the NLRP3 inflammasome contributes to the pathophysiology of AP.Gao L et al. demonstrated that AP involves NLRP3 inflammasome and GSDMD activation-mediated pyroptosis and systemic inflammation [51].Wang J et al. reported that cathepsin B exacerbates the severity of AP through the activation of NLRP3 inflammasome and enhancement of Caspase-1-elicited pyroptosis [52].Herein, the expression of Caspase-1, a pyroptosis-related marker protein, was significantly upregulated in the L-arginine-induced AP mouse model.Treatment with pyroptosis inhibitors alleviated AP-related tissue damage, indicating that pyroptosis is involved in the pathogenic mechanisms of AP.However, CIRP KO and C23 treatment significantly alleviated pyroptosis in AP.Similarly, C23 treatment suppressed pyroptosis in the in vitro AP models.As demonstrated by the MTT assay, the pyroptosis inhibitor VX765 significantly increased the survival rate of recombinant CIRP-treated pancreatic acinar cells.These results suggest that CIRP induces acinar cell damage by promoting pyroptosis, suggesting that CIRP suppression alleviates pyroptosis in AP.
Our research has a few drawbacks.First, the CIRP-deficient mice employed in this study are global knockout.Although we proved the key role of CIRP in the pathogenic mechanism of AP by using these mice, the source of extracellular CIRP could not be identified.A recent review article suggested that both activated macrophages and necrotic cells can be the sources of extracellular CIRP [13].In the future, macrophage-specific CIRP knockout mice and pancreatic acinar cell-specific CIRP knockout mice should be used to pinpoint the major source of CIRP in acute pancreatitis.Second, the C23 dosage utilized in this study (8 mg/kg body weight) was chosen based on the effective dose of C23 in animal models of sepsis and ischemia-reperfusion injury [33].The optimal dose of C23 in acute pancreatitis remains to be determined.Third, C23 was administered at 2 h post-last L-arginine injection in this study.The therapeutic window of C23 in acute pancreatitis also warrants further investigation.
In summary, CIRP upregulation in AP promotes mitochondrial dysfunction and impairs mitochondrial autophagy through TLR4.This results in ROS accumulation, NLRP3-Caspase-1 pyroptosis signaling pathway activation, tissue damage, and systemic inflammation in AP.CIRP inhibition exerts protective effects on AP by improving mitochondrial function and suppressing pyroptosis in acinar cells.Based on our understanding, this is the first study to show that CIRP can directly induce pyroptosis in pancreatic acinar cells.Blocking CIRP could be a promising strategy idea for treating AP in the future.

MATERIALS AND METHODS Experimental animals and L-arginine AP model
The Experimental Animal Center of Xi'an Jiaotong University provided male wild-type C57BL/6J mice.Subsequently, CIRP knockout (KO) mice, which were purchased from the Shanghai Model Organisms Center, were established by removing CIRP on a C57BL/6J background using the clustered regularly interspaced short palindrome repeat/caspase 9 technique.Animals were maintained at the Specific Pathogen-Free Center of Laboratory Animals, Xi'an Jiaotong University.
All mice (age, 8-10 weeks; bodyweight, 20-22 g) were allowed to fast for 12 h before the experiments.Two intraperitoneal doses of 4.0 g/kg L-arginine (A0013; Solarbio, Beijing, China) were utilized to induce L-Arginine-AP.In the pyroptosis inhibition group, a specific inhibitor for Caspase-1 (VX-765;200 mg/kg body weight, Selleck, S2228) was intraperitoneally injected into mice before the L-arginine injection and 1 h following the last L-arginine dose, respectively.In the ROS inhibition experiment, a specific scavenger of mitochondrial superoxide (MitoTEMPO, 20 mg/kg bodyweight; SML0737, Sigma, USA), 1 h before L-arginine injection.Furthermore, mice in the C23-treated group were administered with C23 (8 mg/kg bodyweight) at 2 h post-last L-arginine injection.An equivalent volume of physiological saline was administered intraperitoneally to the control animals.
Before the trials, all animals were kept for one week under standard parameters to achieve acclimation to the environment.Randomization was used to operate on all animals in an identical treatment.For this study, 18 mice (n = 6 per group) in total were utilized in the pyroptosis inhibition group.With six mice per group, the animals were categorized as indicated: We euthanized the animals 72 h after the initial injection of L-arginine.At 72 h following the initial injection of L-arginine, all mice were put under anesthesia by inhaling isoflurane.Furthermore, samples of blood and pancreatic tissue were collected.The Xi'an Jiaotong University Health Science Center Institutional Animal Care and Use Ethics Committee approved the research procedure.

Fig. 1
Fig. 1 Pyroptosis suppression mitigated L-arginine-induced acute pancreatitis injury.L-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine.A specific inhibitor for Caspase-1 (VX-765; 200 mg/kg) was intraperitoneally injected before the L-arginine injection and 1 h after the last L-arginine injection, respectively.The animals were sacrificed 72 h after the first injection of L-arginine.A, B Immunohistochemical analysis of the Caspase-1 expression in the pancreas.Scale bar: 20 µm.C Serum interleukin (IL)-1β levels.D Representative images of pancreatic sections subjected to hematoxylin and eosin (HE) staining.Scale bar: 20 µm.E Pancreatic injury scores.F Percentages of necrotic areas.G Serum interleukin (IL)-6 levels.H Serum tumor necrosis factor (TNF)-α levels.I Serum lactate dehydrogenase (LDH) levels.J Serum amylase levels.n = 6/group.*p < 0.05 versus sham group; #p < 0.05 versus vehicle group.Data are expressed as means ± SEM.

Fig. 2
Fig. 2 Pancreatic tissue injury and pyroptosis were alleviated by ROS inhibition in L-arginine-induced acute pancreatitis.L-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine.A specific scavenger of mitochondrial superoxide (MitoTEMPO,20 mg/kg, Sigma) was intraperitoneally injected 1 h before L-arginine injection.The animals were sacrificed 72 h after the first injection of L-arginine.A Representative images of pancreatic sections subjected to hematoxylin and eosin (HE) staining.Scale bar: 20 µm.B Pancreatic injury scores.C Percentages of necrotic areas.D, E Immunohistochemical analysis of the Caspase-1 expression in the pancreas.Scale bar: 20 µm.F−I Western blot analysis of Caspase-1, ASC, NLRP3, and GSDMD in the pancreas.J Serum interleukin (IL)-1β levels.K Serum interleukin (IL)-6 levels.L Serum tumor necrosis factor (TNF)-α levels.M Serum lactate dehydrogenase (LDH) levels.n = 6/group.*p < 0.05 versus L-arginine-AP with MitoTEMPO-untreated group.Data are expressed as means ± SEM.

Fig. 3
Fig. 3 CIRP KO attenuated pancreatic pyroptosis and tissue damage in the L-arginine-induced AP model.L-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine.The animals were sacrificed 72 h after the first injection of L-arginine.A Serum CIRP levels.B, C Immunohistochemical analysis of pancreatic Caspase-1 expression in wild-type and CIRP −/− mice.Scale bar: 20 µm.D−G Western blotting analysis of Caspase-1, ASC, NLRP3, and GSDMD in the pancreas.H Serum interleukin (IL)-1β levels.I Representative images of pancreatic tissues subjected to hematoxylin and eosin (HE) staining.Scale bar: 20 µm.J Pancreatic injury scores.K Percentages of necrotic areas.L Serum interleukin (IL)-6 levels.M Serum tumor necrosis factor (TNF)-α levels.N Serum lactate dehydrogenase (LDH) levels.O Serum amylase levels.n = 6/group.*p < 0.05 versus sham group; #p < 0.05 versus WT-AP group.Data are expressed as means ± SEM.

Fig. 4
Fig. 4 CIRP knockout improved mitochondrial function and reduced ROS accumulation in the L-arginine-induced AP model.A Ultrastructural alterations in the pancreas (Transmission electron microscopy).Scale bars:2 µm and 5 µm.B Representative images of pancreatic tissues subjected to dihydroethidium (DHE) immunofluorescence staining.Scale bar: 20 µm.C DHE relative fluorescence density analysis.D-F Western blotting analysis of Tfam, PINK1, Mfn2, LAMP2A, and LC3-II in the pancreas; (G) Superoxide dismutase (SOD) levels in the pancreatic tissue.H Glutathione (GSH) levels in the pancreatic tissue.n = 6/group.*p < 0.05 versus sham group; #p < 0.05 versus WT-AP group.Data are expressed as means ± SEM.

Fig. 5
Fig. 5 C23 administration attenuated pancreatic pyroptosis and tissue damage in the L-arginine-induced AP model.L-Arginine-AP was induced by two hourly intraperitoneal injections of 4.0 g/kg L-arginine.The animals were sacrificed 72 h after the first injection of L-arginine.C23 (8 mg/kg) was administered 2 h after the last L-arginine injection was administered by intraperitoneal injection.Mice in the sham group were intraperitoneally administered with the same volume of physiological saline.A, B Immunohistochemical analysis of the Caspase-1 expression in the pancreas.Scale bar: 20 µm.C−F Western blotting analysis of Caspase-1, ASC, NLRP3, and GSDMD in the pancreas.G ©(IL)-1β levels.H Representative images of pancreatic sections subjected to hematoxylin and eosin (HE) staining.Scale bar: 20 µm.I Pancreatic injury scores.J Percentages of necrotic areas.K Serum interleukin (IL)-6 levels.L Serum tumor necrosis factor (TNF)-α levels.M Serum lactate dehydrogenase (LDH) levels.N Serum amylase levels.n = 6/group.*p < 0.05 versus sham group; #p < 0.05 versus vehicle group.Data are expressed as means ± SEM.

Fig. 6
Fig. 6 C23 administration improved mitochondrial function and reduced ROS accumulation in the L-arginine-induced AP model.A Ultrastructural alterations in the pancreas (Transmission electron microscopy).Scale bars:2 µm and 5 µm.B Representative images of pancreatic sections subjected to dihydroethidium (DHE) immunofluorescence analysis.Scale bar: 20 µm.C DHE relative fluorescence intensity analysis.D−F Western blotting analysis of Tfam, PINK1, Mfn2, LAMP2A, LC3-II in the pancreas; (G) Superoxide dismutase (SOD) levels in the pancreatic tissue.H Glutathione (GSH) levels in the pancreatic tissue.n = 6/group.*p < 0.05 versus sham group; #p < 0.05 versus vehicle group.Data are expressed as means ± SEM.