Saeedeh Asgarbeik

• Background: Acute pancreatitis (AP) is a serious inflammatory condition marked by the premature activation of digestive proteases, resulting in pancreatic self-digestion and tissue injury. A pivotal event in the early stages of AP is the conversion of the zymogen trypsinogen into its active form, trypsin. Among the potential mediators of this process, the lysosomal protease cathepsin B has been identified as a key initiator. Thus, the regulation of cathepsin B activity is considered a critical factor influencing the onset and severity of pancreatitis. The activity of cathepsin B can be modulated in various cellular compartments, including the cytosol, where cystatin B acts as a regulator. In addition to its role in activating proteases, cytosolic cathepsin B has been linked to inducing apoptosis by activating the BID and caspase pathways. In addition to its role in protease activation, cytosolic cathepsin B has been associated with inducing apoptosis by activating BID and caspase pathways. In addition to its role in activating proteases, cytosolic cathepsin B has been linked to inducing apoptosis by activating the BID and caspase pathways. Furthermore, cystatin B inhibits cathepsin B, a key mediator involved in inflammasome formation in macrophages, thereby modulating the inflammatory response. This study aimed to investigate the regulatory effect of cystatin B on cathepsin B activity during pancreatitis. Method: Proteomic analysis using LC-MS/MS was conducted to evaluate the presence of protease inhibitors in various subcellular fractions. Acute pancreatitis (AP) was induced in C57Bl/6 and Cstb-/- mice through hourly repetitive injections of caerulein (50 μg/kg body weight). Disease severity was assessed through the measurement of serum lipase and amylase activities, in conjunction with histological evaluation of pancreatic tissue. Protease activity was analyzed using fluorogenic substrates in both whole pancreatic homogenates and isolated subcellular fractions. Apoptotic cell death was quantified via the TUNEL assay, while mitochondrial function was assessed by determining mitochondrial membrane potential. Furthermore, protease activation and cellular injury were further investigated in isolated pancreatic acinar cells. Inflammasome formation and IL-1β release were evaluated using an ELISA kit to quantify cytokine levels. Result: Our results demonstrated that the absence of cystatin B was associated with increased severity of pancreatitis, as evidenced by elevated serum amylase and lipase levels, along with greater pancreatic tissue damage. Additionally, trypsin activity was significantly higher in the cytosolic fraction of knock-out mice, accompanied by increased chymotrypsin activity. Both Cathepsin B and Cathepsin L exhibited elevated activity in the cytosolic compartment following one hour of pancreatitis induction. In vivo measurements further confirmed enhanced trypsin activity, which correlated with a greater extent of necrosis in knock-out mice. Moreover, higher levels of apoptosis, likely driven by increased cathepsin B activity, were observed in the absence of cystatin B. Analysis of mitochondrial function revealed depolarization of the mitochondrial membrane potential and the absence of TFAM as a transcription factor in the cytosol of knock-out mice. Finally, the cystatin B-deficient exhibited increased immune cell infiltration and enhanced inflammasome formation within macrophages residing in the pancreatic tissue. Conclusion: In the later stages of pancreatitis, cathepsin B translocates into the cytosol, where its activity is normally regulated and inhibited by cystatin B. In the absence of cystatin B, cathepsin B remains active in the cytosol, triggering multiple pathological events. Active cathepsin B facilitates the premature conversion of trypsinogen to trypsin, initiating a proteolytic cascade involving other serine proteases and thereby intensifying pancreatic injury. This aberrant protease activity contributes to increased apoptosis in acinar cells. It promotes necrotic cell death, as evidenced by elevated in vitro trypsin activity in living acinar cells from knock-out mice. Moreover, sustained cytosolic cathepsin B activity in the absence of cystatin B induces pronounced mitochondrial dysfunction, characterized by depolarization of the mitochondrial membrane potential and loss of mitochondrial integrity, as evidenced by the release of mitochondrial transcription factor A (TFAM) into the cytosol at later stages of pancreatitis. TFAM is a key regulator of mitochondrial maintenance, and its cytosolic localization likely reflects ongoing mitochondrial disruption. The resulting cellular stress further enhances immune cell infiltration and inflammatory responses within the pancreatic tissue. Importantly, the loss of cystatin B also leads to amplified inflammasome activation in macrophages, likely driven by increased release of damage-associated molecular patterns (DAMPs) and mitochondrial dysfunction. This contributes to the elevated secretion of proinflammatory cytokines such as IL-1β, which may, in turn, promote or precede pyroptotic cell death. In summary, cystatin B plays a critical protective role during the progression of pancreatitis by regulating cathepsin B activity, preserving mitochondrial function, and limiting inflammatory responses. Its deficiency leads to aggravated disease severity, characterized by elevated levels of acinar cell apoptosis and necrosis, as well as intensified pyroptotic cell death in macrophages.