ca-074-methyl-ester and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Delayed secondary degeneration in the non-ischemic sites such as ipsilateral thalamus would occur after cortical infarction. Hence, alleviating secondary damage is considered to be a promising novel target for acute stroke therapy. In the current study, the neuroprotective effects of bis(propyl)-cognitin (B3C), a multifunctional dimer, against secondary damage in the VPN of ipsilateral thalamus were investigated in a distal middle cerebral artery occlusion (dMCAO) stroke model in adult rats. It was found that B3C (0.5 and 1 mg/kg, ip) effectively improved neurological function of rats at day 7 and day 14 after dMCAO. Additionally, the treatment with B3C alleviated neuronal loss and gliosis in ipsilateral VPN after dMCAO, as evidenced by the higher immunoreactivity of neuron-specific nuclear-binding protein (NeuN) as well as lower immunostaining intensity of glial fibrillary acidic protein (GFAP) and cluster of differentiation 68 (CD68). Most encouragingly, immunohistochemistry and western blotting further revealed that B3C treatment greatly reduced Aβ deposits and cathepsin B expression in the VPN of ipsilateral thalamus at day 7 and day 14 after dMCAO. In parallel, we demonstrated herein that the neuroprotective effects of B3C in dMCAO model were similar to L-3-trans-(Propyl-carbamoyloxirane-2-carbonyl)- L-isoleucyl-l-proline methyl ester (CA-074Me), a specific inhibitor of cathepsin B, suggesting that B3C attenuated secondary damage and Aβ deposits in the VPN of ipsilateral thalamus after dMCAO possibly through the reduction of cathepsin B. These findings taken together provide novel molecular sights into the potential application of B3C for the treatment of secondary degeneration after cortical infarction.

Topics: Amyloid beta-Peptides; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Antigens, Nuclear; Cathepsin B; Dipeptides; Disease Models, Animal; GABA-A Receptor Antagonists; Glial Fibrillary Acidic Protein; Gliosis; Infarction, Middle Cerebral Artery; Nerve Tissue Proteins; Neuroglia; Neurons; Neuroprotective Agents; Rats; Tacrine; Thalamus; Ventral Thalamic Nuclei

Autism spectrum disorder (ASD) is a wide range of neurodevelopmental disorders involving deficits in social interaction and communication. Unfortunately, autism remains a scientific and clinical challenge owing to the lack of understanding the cellular and molecular mechanisms underlying it. This study aimed to investigate the pathophysiological mechanism underlying leukocyte-endothelial adhesion in autism-related neurovascular inflammation.. Male BTBR T+tf/J mice were used as an autism model. The dynamic pattern of leukocyte-endothelial adhesion in mouse cerebral vessels was detected by two-photon laser scanning microscopy (TPLSM). Using FACS, RT-PCR, and Western blotting, we explored the expression of cell adhesion molecules, the mRNA expression of endothelial chemokine, the protein levels of cathepsin B, and inflammatory mediators.. We found a significant increase in leukocyte-endothelial adhesion in BTBR mice, accompanied by elevated expression of the adhesion molecule neutrophils CD11b and endothelial ICAM-1. Our data further indicate that elevated neutrophil cathepsin B levels contribute to elevated endothelial chemokine CXCL7 levels in BTBR mice. The pharmacological inhibition of cathepsin B reverses the enhanced leukocyte-endothelial adhesion in the cerebral vessels of autistic mice.. Our results revealed the prominent role of cathepsin B in modulating leukocyte-endothelial adhesion during autism-related neurovascular inflammation and identified a promising novel approach for autism treatment.

Topics: Animals; Autistic Disorder; Cathepsin B; Cell Adhesion; Dipeptides; Disease Models, Animal; Endothelium, Vascular; Leukocytes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic

Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after

Topics: Animals; Antigen-Presenting Cells; Cathepsin B; Cathepsins; Cell Proliferation; Cells, Cultured; Cytokines; Dipeptides; Disease Models, Animal; Galactosylceramides; Humans; Lipopolysaccharides; Liver; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Natural Killer T-Cells

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most common chronic liver disease. NLRP3 inflammasome activation has been widely studied in the pathogenesis of NAFLD. Cathepsin B (CTSB) is a ubiquitous cysteine cathepsin, and the role of CTSB in the progression and development of NAFLD has received extensive concern. However, the exact roles of CTSB in the NAFLD development and NLRP3 inflammasome activation are yet to be evaluated. In the present study, we used methionine choline-deficient (MCD) diet to establish mice NASH model. CTSB inhibitor (CA-074) was used to suppress the expression of CSTB. Expressions of CTSB and caspase-1 were evaluated by immunohistochemical staining. Serum IL-1β and IL-18 levels were also determined. Palmitic acid was used to stimulate Kupffer cells (KCs), and protein expressions of CTSB, NLRP3, ASC (apoptosis-associated speck-like protein containing CARD), and caspase-1 in KCs were detected. The levels of IL-1β and IL-18 in the supernatant of KCs were evaluated by enzyme-linked immunosorbent assay (ELISA). Our results showed that CTSB inhibition improved the liver function and reduced hepatic inflammation and ballooning, and the levels of pro-inflammatory cytokines IL-1β and IL-18 were decreased. The expressions of CTSB and caspase-1 in liver tissues were increased in the NASH group. In in vitro experiments, PA stimulation could increase the expressions of CTSB and NLRP3 inflammasome in KCs, and CTSB inhibition downregulated the expression of NLRP3 inflammasome in KCs, when challenged by PA. Moreover, CTSB inhibition effectively suppressed the expression and activity of caspase-1 and subsequently secretions of IL-1β and IL-18. Collectively, these results suggest that CTSB inhibition limits NLRP3 inflammasome-dependent NASH formation through regulating the expression and activity of caspase-1, thus providing a novel anti-inflammatory signal pathway for the therapy of NAFLD.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Caspase 1; Cathepsin B; Cells, Cultured; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Enzyme Activation; Fluorescent Antibody Technique, Indirect; Immunohistochemistry; Inflammasomes; Injections, Intraperitoneal; Interleukin-18; Interleukin-1beta; Kupffer Cells; Liver; Male; Mice, Inbred C57BL; NLR Family, Pyrin Domain-Containing 3 Protein; Non-alcoholic Fatty Liver Disease; Random Allocation

Many studies have demonstrated the key role of lysosomes in ischemic cell death in the brain and have led to the "lysosomocentric" hypothesis. In this hypothesis, the release of cathepsin-B due to a change of lysosomal membrane permeabilization (LMP) or rupture is critical, and this can be prevented by its inhibitors CA074 and CA074-me. However, the role of CA074-me in neuronal death and its effect on the change of lysosomal membrane integrity after global cerebral ischemia/reperfusion (I/R) injury is not clear, so we investigated this here. Rat hippocampal CA1 neuronal death was evaluated after 20-min global cerebral I/R injury. CA074-me (1 μg, 10 μg) were given intracerebroventricularly 1h before ischemia or 1h post reperfusion. The changes of heat shock protein 70 (Hsp70), cathepsin-B, lysosomal-associated membrane protein 1 (LAMP-1), receptor-interacting protein 3 (RIP3), and the change of lysosomal pH were evaluated respectively. Hippocampal CA1 neuronal programmed necrosis induced by global cerebral I/R injury was prevented by CA074-me both pre-treatment and post-treatment. Diffuse cytoplasmic cathepsin-B and LAMP-1 immunostaining synchronized with the pyknotic nuclear changes 2 days post reperfusion, and a rise of lysosomal pH with the leakage of DND-153, a dye of lysosomes, after oxygen-glucose deprivation (OGD) was detected. Both of these changes demonstrated the rupture of lysosomal membrane and the leakage of cathepsin-B, and this was strongly inhibited by CA074-me pre-treatment. The overexpression and nuclear translocation of RIP3 and the reduction of NAD(+) level after I/R injury were also inhibited, while the upregulation of Hsp70 was strengthened by CA074-me pre-treatment. Delayed fulminant leakage of cathepsin-B due to lysosomal rupture is a critical harmful factor in neuronal programmed necrosis induced by 20-min global I/R injury. In addition to being an inhibitor of cathepsin-B, CA074-me may have an indirect neuroprotective effect by maintaining lysosomal membrane integrity and protecting against lysosomal rupture.

Topics: Active Transport, Cell Nucleus; Animals; Brain Ischemia; CA1 Region, Hippocampal; Cathepsin B; Cell Hypoxia; Cells, Cultured; Dipeptides; Disease Models, Animal; Glucose; HSP70 Heat-Shock Proteins; Male; NAD; Necrosis; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases; Reperfusion Injury

During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD.

Topics: Animals; Cathepsin D; Chloroquine; Collagen; Dipeptides; Disease Models, Animal; Extracellular Matrix; Female; Fibrosis; Humans; Lysosomes; Mice; Myofibroblasts; Pepstatins; Renal Insufficiency, Chronic

Cathepsin B (CB) is involved in the turnover of proteins and has various roles in maintaining the normal metabolism of cells. In our recent study, CB is increased in the muscles of polymyositis/dermatomyositis (PM/DM). However, the role of CB in interstitial lung disease (ILD) has not been reported. ILD is a frequent complication of PM/DM, which is the leading cause of death in PM/DM. It carries high morbidity and mortality in connective tissue diseases, characterized by an overproduction of inflammatory cytokines and induced fibrosis, resulting in respiratory failure. The etiology and pathogenesis of ILD remain incompletely understood. This study investigated whether treatment with CA-074Me, a specific inhibitor of CB, attenuates ILD in PM. CB expression, inflammation, and fibrosis were analyzed in the lung tissues from patients with PM/DM. The animal model of PM was induced in guinea pigs with Coxsackie virus B1 (CVB1). CA-074Me was given 24 h after CVB1 injection for 7 consecutive days. At the end of the experiment, the animals were killed and lung tissues were collected for the following analysis. Inflammation, fibrosis and apoptosis cells, and cytokines were assessed by histological examinations and immunohistochemical analyses, western blot analysis and transferase-mediated dUTP nick-end labeling assay. In patients with PM/DM, the protein levels of CB were significantly elevated in lung tissues compared with healthy controls, which correlated with increases in inflammation and fibrosis. Similarly, the expression of CB, inflammation and fibrosis, CD8(+) T cell, CD68(+) cell, tumor necrosis factor-alpha, transforming growth factor-beta1 infiltrations, and apoptotic cell death were significantly increased in lung tissues of the guinea-pig model of CVB1-induced PM. These changes were attenuated by the administration of CA-074Me. In conclusion, this study demonstrates that PM/DM increases CB expression in lung tissues and inhibition of CB reduces ILD in a guinea-pig model of CVB1-induced PM. This finding suggests that CB may be a potential therapeutic target for ILD.

Topics: Animals; Apoptosis; Cathepsin B; Dermatomyositis; Dipeptides; Disease Models, Animal; Female; Guinea Pigs; Lung Diseases, Interstitial; Pulmonary Fibrosis; Rats; Up-Regulation

Recently, cathepsin B has been demonstrated to be involved in myocardial infarction (MI). This study aimed to elucidate the effects of a specific cathepsin B inhibitor, CA-074Me, on cardiac dysfunction, remodeling and fibrosis following MI in a rat model. Furthermore, the potential mechanisms of action of this inhibitor were investigated. In the present study, Sprague-Dawley rats were anesthetized and subjected to a sham operation or left anterior descending coronary artery ligation, followed by intraperitoneal injection of CA-074Me (10 mg/kg/day) or an equal volume of vehicle for 4 weeks. Activation of the cathepsin B and NLRP3 pathway was detected. Cardiac function was assessed by echocardiography, while hypertrophy and fibrosis were determined by Masson's trichrome, immunofluorescence and morphometry. The results demonstrated that cathepsin B-NLRP3 activation was inhibited by CA-074Me treatment. Following such treatment for 4 weeks, the rats demonstrated smaller decreases in cardiac function, and a decrease in cardiomyocyte hypertrophy and the level of fibrosis. Cathepsin B inhibition significantly attenuated cardiac dysfunction, and reduced cardiomyocyte size and cardiac fibrosis in the experimental MI model, by inhibiting NLRP3 activation. This suggested that targeting the cathepsin B-NLRP3 pathway may represent a novel therapeutic strategy to prevent heart failure and remodeling following MI.

Topics: Animals; Carrier Proteins; Cathepsin B; Dipeptides; Disease Models, Animal; Fibrosis; Interleukin-1beta; Male; Myocardial Infarction; Myocardium; Myocytes, Cardiac; NLR Family, Pyrin Domain-Containing 3 Protein; Rats; Signal Transduction; Ventricular Remodeling

Polymyositis (PM) cause pain and weakness of muscle, even threatens patient's lives, but the etiology and pathogenesis of it remains partially understood. Previous studies have proved Cathepsin B (CB) was strongly stained in muscle tissues of PM patients. But no further studies were performed to investigate the role of CB in PM.. To investigate the protective effects of CB inhibitor CA-074Me in PM.. CB expression, inflammation and apoptosis were analyzed in muscle tissues from patients with PM. Guinea pigs were inoculated intraperitoneally with Coxsackie virus B1 (CVB1) and were then immunized with completely emulsified 0.6ml rabbit muscle homogenates in Freund's Complete Adjuvant (FCA) once a week for consecutive three weeks. The effects of CB inhibitor CA-074Me on CB expression, inflammation and apoptosis were then investigated. Inflammation was assessed by histological examination. Both immunohistochemistry and western blot were used to determine the protein expression. The mRNA levels of CB were measured by Real-Time RT-PCR. The apoptosis was determined by TUNEL assay.. In patients with PM, the protein levels of CB were significantly up-regulated in muscle tissues compared with healthy controls, which correlated with increases in inflammation score and apoptotic rate in PM patients. Consistently, the expression of CB, inflammation score, CD8(+) T-cell, CD68(+) cell, tumor necrosis factor-alpha (TNF-α) infiltration and apoptotic rate were significantly increased in the guinea-pig model of CVB1-induced polymyositis. Administration of CA-074Me reduced CB expression, decreased inflammation score and attenuated apoptosis in muscle tissues of the guinea-pig model of CVB1-induced polymyositis. The inhibitory effect of CA-074Me on apoptosis was associated with down-regulation of Bax expression and consequent increase in the ratio of Bcl-2/Bax. However, CA-074Me had effect not on CD8(+) T-cells infiltrations but on CD68(+) cells and TNF-α(+) cells infiltrations in the guinea-pig model of CVB1-induced polymyositis.. This study confirms up-regulation of CB in PM patients and demonstrates that inhibition of CB provides protective effects in a guinea pig model of CVB1-induced PM. Thus, CB will be an important therapeutic target for PM.

Topics: Adult; Aged; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apoptosis; Cathepsin B; Dipeptides; Disease Models, Animal; Female; Guinea Pigs; Humans; Inflammation; Macrophages; Male; Middle Aged; Muscles; Polymyositis; Proto-Oncogene Proteins c-bcl-2; Tumor Necrosis Factor-alpha

Interleukin (IL)-1β and IL-18 play critical roles in the induction of chronic pain hypersensitivity. Their inactive forms are activated by caspase-1. However, little is known about the mechanism underlying the activation of pro-caspase-1. There is increasing evidence that cathepsin B (CatB), a typical lysosomal cysteine protease, is involved in the pro-caspase-1 activation and the subsequent maturation of IL-1β and IL-18. In this context, CatB is considered to be an important molecular target to control chronic pain. However, no information is currently available about the role of CatB in chronic pain hypersensitivity. We herein show that CatB deficiency or the intrathecal administration of CA-074Me, a specific CatB inhibitor, significantly inhibited the induction of complete Freund's adjuvant-induced tactile allodynia in mice without affecting peripheral inflammation. In contrast, CatB deficiency did not affect the nerve injury-induced tactile allodynia. Furthermore, CatB deficiency or CA-074Me treatment significantly inhibited the maturation and secretion of IL-1β and IL-18 by cultured microglia following treatment with the neuroactive glycoprotein chromogranin A (CGA), but not with ATP. Moreover, the IL-1β expression in spinal microglia and the induction of tactile allodynia following the intrathecal administration of CGA depended on CatB, whereas those induced by the intrathecal administration of ATP or lysophosphatidic acid were CatB independent. These results strongly suggest that CatB is an essential enzyme for the induction of chronic inflammatory pain through its activation of pro-caspase-1, which subsequently induces the maturation and secretion of IL-1β and IL-18 by spinal microglia. Therefore, CatB-specific inhibitors may represent a useful new strategy for treating inflammation-associated pain.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Analysis of Variance; Animals; Calcium-Binding Proteins; Carrier Proteins; Cathepsin B; CD11b Antigen; CD4 Antigens; Cells, Cultured; Chromogranin A; Chronic Pain; Cyclooxygenase 2; Dipeptides; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Gene Expression Regulation; Hyperalgesia; Inflammation; Interleukin-18; Interleukin-1beta; Lysophospholipids; Lysosomes; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Microglia; Motor Activity; Nerve Tissue Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Pain Threshold; RNA, Small Interfering; Spinal Cord; Transfection

Endostatin (ES) has been recognized as a potent anti-angiogenic factor. We here investigated the expression of ES in ischemic brain and the consequence of cells expressing ES after stroke in adult stroke-prone renovascular hypertensive rats. A single dose of Ca-074ME, a membrane-permeable cathepsin B (CB) specific inhibitor, or vehicle was given by intraperitoneal injection immediately after distal middle cerebral artery occlusion (dMCAO), ES expression was evaluated using fluorescent immunohistochemistry staining, and CB enzyme activity was tested by measuring the free 7-amino-4-methylcoumarin (AMC) released by CB from its' specific substrate, the Z-Arg-Arg-7-amido-4-methylcoumarin. ES immunoreactivity (IR) was significantly up-regulated as early as 6 h and returned to baseline level at 3 days in peri-infarct area following dMCAO. Double-staining experiment revealed that the majority of ischemia-induced ES positive cells were neurons. Furthermore, ES was co-labeled with CB and Cleaved Caspase-3(Asp175) whereas treatment with Ca-074ME reduced up-regulation of ES expression and attenuated apoptosis in peri-infarct neurons. Collectively, our data suggest that peri-infarct neurons express ES during the early stage of cerebral ischemia and treatment with Ca-074ME attenuates ES expression and apoptosis in peri-infarct neurons.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Caspase 3; Cathepsin B; Coumarins; Dipeptides; Disease Models, Animal; Endostatins; Enzyme Inhibitors; Hypertension; Infarction, Middle Cerebral Artery; Male; Neurons; Random Allocation; Rats; Rats, Sprague-Dawley; Stroke; Time Factors

Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Cathepsin B; Cattle; Cysteine Proteinase Inhibitors; Dipeptides; Disease Models, Animal; Drug Evaluation, Preclinical; Gene Expression; Humans; Leucine; Maze Learning; Memory; Mice; Mice, Transgenic

We have hypothesized that the colocalization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the colocalization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pretreated with the cathepsin B inhibitor CA-074 me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pretreatment with CA-074 me prevents intrapancreatic zymogen activation and reduces organellar fragility, but it does not alter the caerulein-induced colocalization phenomenon or subcellular F-actin redistribution or prevent caerulein-induced activation of NF-kappaB, ERK1/2, and JNK or upregulated expression of cytochemokines. We conclude 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity.

Topics: Actins; Acute Disease; Amylases; Animals; Arylsulfatases; Cathepsin B; Ceruletide; Chemokine CCL2; Dipeptides; Disease Models, Animal; Enzyme Activation; Enzyme Inhibitors; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lysosomes; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; NF-kappa B; Pancreas; Pancreatitis; Protein Transport; Secretory Vesicles; Severity of Illness Index; Time Factors; Trypsin; Trypsinogen

We previously reported that CA074, a specific inhibitor of cathepsin B, significantly deviated immune responses from the disease-promoting Th2 type to the protective Th1 type in BALB/c mice infected with Leishmania major. Herein, we found that pepstatin A-sensitive aspartic proteases (PSAP) in lysosomes seem to play a different role from that of cathepsin B in antigen-processing and Ii-degradation. That is, cathepsin B appears to digest 16-, 28-, and 31-kDa peptides of soluble leishmania antigen (SLA), whereas PSAP seems to process mainly 28-kDa peptides. Furthermore, the latter protease contributed to the degradation of Ii but cathepsin B did not. Following treatment with pepstatin A, both Th1 and Th2 responses were profoundly suppressed in resistant DBA/2 mice (H-2(d)) and in susceptible BALB/c mice (H-2(d)), and both strains of mice became markedly susceptible compared with the untreated groups, probably owing to failure in degradation of Ii and partly to failure in digestion of 28-kDa peptide.

Topics: Animals; Antibody Formation; Antigen Presentation; Antigen-Presenting Cells; Antigens, Differentiation, B-Lymphocyte; Antigens, Protozoan; Aspartic Acid Endopeptidases; Cathepsin B; CD4-Positive T-Lymphocytes; Cell Division; Cysteine Proteinase Inhibitors; Cytokines; Dipeptides; Disease Models, Animal; Female; Histocompatibility Antigens Class II; Leishmania major; Leishmaniasis, Cutaneous; Lymphocytes; Lysosomes; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Pepstatins; Th1 Cells; Th2 Cells