Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Dual Strategy to Design New Agents Targeting Schistosoma mansoni: Advancing Phenotypic and SmCB1 Inhibitors for Improved Efficacy.

In this study, we have identified and optimized two lead structures from an in-house screening, with promising results against the parasitic flatworm Schistosoma mansoni and its target protease S. mansoni cathepsin B1 (SmCB1). Our correlation analysis highlighted the significance of physicochemical properties for the compounds' in vitro activities, resulting in a dual approach to optimize the lead structures, regarding both phenotypic effects in S. mansoni newly transformed schistosomula (NTS), adult worms, and SmCB1 inhibition. The optimized compounds from both approaches ("phenotypic" vs "SmCB1" approach) demonstrated improved efficacy against S. mansoni NTS and adult worms, with 2h from the "SmCB1" approach emerging as the most potent compound. 2h displayed nanomolar inhibition of SmCB1 (Ki = 0.050 µM) while maintaining selectivity toward human off-target cathepsins. Additionally, the greatly improved efficacy of compound 2h toward S. mansoni adults (86% dead worms at 10 µM, 68% at 1 µM, 35% at 0.1 µM) demonstrates its potential as a new therapeutic agent for schistosomiasis, underlined by its improved permeability.

Specific photodamage on HT-29 cancer cells leads to endolysosomal failure and autophagy blockage by cathepsin depletion.

Endolysosomes perform a wide range of cellular functions, including nutrient sensing, macromolecule digestion and recycling, as well as plasma membrane repair. Because of their high activity in cancerous cells, endolysosomes are attractive targets for the development of novel cancer treatments. Light-activated compounds termed photosensitizers (PS) can catalyze the oxidation of specific biomolecules and intracellular organelles. To selectively damage endosomes and lysosomes, HT-29 colorectal cancer cells were incubated with nanomolar concentrations of meso-tetraphenylporphine disulfonate (TPPS2a), an amphiphilic PS taken up via endocytosis and activated by green light (522 nm, 2.1 J.cm-1). Several cellular responses were characterized by a combination of immunofluorescence and immunoblotting assays. We showed that TPPS2a photosensitization blocked autophagic flux without extensive endolysosomal membrane rupture. Nevertheless, there was a severe functional failure of endolysosomes due to a decrease in CTSD (cathepsin D, 55%) and CTSB (cathepsin B, 52%) maturation. PSAP (prosaposin) processing (into saposins) was also considerably impaired, a fact that could be detrimental to glycosphingolipid homeostasis. Therefore, photosensitization of HT-29 cells previously incubated with a low concentration of TPPS2a promotes endolysosomal dysfunction, an effect that can be used to improve cancer therapies.

Design, synthesis, and biological evaluation of cathepsin B cleavage albumin-binding SN38 prodrug in breast cancer.

Here, we introduce a novel and effective approach utilizing a cathepsin B cleavage albumin-binding SN38 prodrug specifically designed for the treatment of metastatic breast cancer. Termed Mal-va-mac-SN38, our prodrug exhibits a unique ability to rapidly and covalently bind with endogenous albumin, resulting in the formation of HSA-va-mac-SN38. This prodrug demonstrates exceptional stability in human plasma. Importantly, HSA-va-mac-SN38 showcases an impressive enhancement in cellular uptake by 4T1 breast cancer cells, primarily facilitated through caveolin-mediated endocytosis. Intriguingly, the release of the active SN38, is triggered by the enzymatic activity of cathepsin B within the lysosomal environment. In vivo studies employing a lung metastasis 4T1 breast cancer model underscore the potency of HSA-va-mac-SN38. Histological immunohistochemical analyses further illuminate the multifaceted impact of our prodrug, showcasing elevated levels of apoptosis, downregulated expression of matrix metalloproteinases, and inhibition of angiogenesis, all critical factors contributing to the anti-metastatic effect observed. Biodistribution studies elucidate the capacity of Mal-va-mac-SN38 to augment tumor accumulation through covalent binding to serum albumin, presenting a potential avenue for targeted therapeutic interventions. Collectively, our findings propose a promising therapeutic avenue for metastatic breast cancer, through the utilization of a cathepsin B-cleavable albumin-binding prodrug.

Cathepsin B as a key regulator of ferroptosis in microglia following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a subtype of stroke marked by elevated mortality and disability rates. Recently, mounting evidence suggests a significant role of ferroptosis in the pathogenesis of ICH. Through a combination of bioinformatics analysis and basic experiments, our goal is to identify the primary cell types and key molecules implicated in ferroptosis post-ICH. This aims to propel the advancement of ferroptosis research, offering potential therapeutic targets for ICH treatment. Our study reveals pronounced ferroptosis in microglia and identifies the target gene, cathepsin B (Ctsb), by analyzing differentially expressed genes following ICH. Ctsb, a cysteine protease primarily located in lysosomes, becomes a focal point in our investigation. Utilizing in vitro and in vivo models, we explore the correlation between Ctsb and ferroptosis in microglia post-ICH. Results demonstrate that ICH and hemin-induced ferroptosis in microglia coincide with elevated levels and activity of Ctsb protein. Effective alleviation of ferroptosis in microglia after ICH is achieved through the inhibition of Ctsb protease activity and protein levels using inhibitors and shRNA. Additionally, a notable increase in m6A methylation levels of Ctsb mRNA post-ICH is observed, suggesting a pivotal role of m6A methylation in regulating Ctsb translation. These research insights deepen our comprehension of the molecular pathways involved in ferroptosis after ICH, underscoring the potential of Ctsb as a promising target for mitigating brain damage resulting from ICH.

Cathepsin B: The dawn of tumor therapy.

Cathepsin B (CTSB) is a key lysosomal protease that plays a crucial role in the development of cancer. This article elucidates the relationship between CTSB and cancer from the perspectives of its structure, function, and role in tumor growth, migration, invasion, metastasis, angiogenesis and autophagy. Further, we summarized the research progress of cancer treatment related drugs targeting CTSB, as well as the potential and advantages of Traditional Chinese medicine in treating tumors by regulating the expression of CTSB.

In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Human ß-Defensin 3 Inhibition of P. gingivalis LPS-Induced IL-1ß Production by BV-2 Microglia through Suppression of Cathepsins B and L.

Cathepsin B (CatB) is thought to be essential for the induction of Porphyromonas gingivalis lipopolysaccharide (Pg LPS)-induced Alzheimer's disease-like pathologies in mice, including interleukin-1ß (IL-1ß) production and cognitive decline. However, little is known about the role of CatB in Pg virulence factor-induced IL-1ß production by microglia. We first subjected IL-1ß-luciferase reporter BV-2 microglia to inhibitors of Toll-like receptors (TLRs), IκB kinase, and the NLRP3 inflammasome following stimulation with Pg LPS and outer membrane vesicles (OMVs). To clarify the involvement of CatB, we used several known CatB inhibitors, including CA-074Me, ZRLR, and human ß-defensin 3 (hBD3). IL-1ß production in BV-2 microglia induced by Pg LPS and OMVs was significantly inhibited by the TLR2 inhibitor C29 and the IκB kinase inhibitor wedelolactonne, but not by the NLRPs inhibitor MCC950. Both hBD3 and CA-074Me significantly inhibited Pg LPS-induced IL-1ß production in BV-2 microglia. Although CA-074Me also suppressed OMV-induced IL-1ß production, hBD3 did not inhibit it. Furthermore, both hBD3 and CA-074Me significantly blocked Pg LPS-induced nuclear NF-κB p65 translocation and IκBα degradation. In contrast, hBD3 and CA-074Me did not block OMV-induced nuclear NF-κB p65 translocation or IκBα degradation. Furthermore, neither ZRLR, a specific CatB inhibitor, nor shRNA-mediated knockdown of CatB expression had any effect on Pg virulence factor-induced IL-1ß production. Interestingly, phagocytosis of OMVs by BV-2 microglia induced IL-1ß production. Finally, the structural models generated by AlphaFold indicated that hBD3 can bind to the substrate-binding pocket of CatB, and possibly CatL as well. These results suggest that Pg LPS induces CatB/CatL-dependent synthesis and processing of pro-IL-1ß without activation of the NLRP3 inflammasome. In contrast, OMVs promote the synthesis and processing of pro-IL-1ß through CatB/CatL-independent phagocytic mechanisms. Thus, hBD3 can improve the IL-1ß-associated vicious inflammatory cycle induced by microglia through inhibition of CatB/CatL.

Allulose mitigates chronic enteritis by reducing mitochondria dysfunction via regulating cathepsin B production.

Metabolic changes have been linked to the development of inflammatory bowel disease (IBD), which includes colitis. Allulose, an endogenous bioactive monosaccharide, is vital to the synthesis of numerous compounds and metabolic processes within living organisms. Nevertheless, the precise biochemical mechanism by which allulose inhibits colitis remains unknown. Allulose is an essential and intrinsic protector of the intestinal mucosal barrier, as it maintains the integrity of tight junctions in the intestines, according to the current research. It is also important to know that there is a link between the severity of inflammatory bowel disease (IBD) and colorectal cancer (CRC), chemically-induced colitis in rodents, and lower levels of allulose in the blood. Mice with colitis, either caused by dextran sodium sulphate (DSS) or naturally occurring colitis in IL-10-/- mice, had less damage to their intestinal mucosa after being given allulose. Giving allulose to a colitis model starts a chain of reactions because it stops cathepsin B from ejecting and helps lysosomes stick together. This system effectively stops the activity of myosin light chain kinase (MLCK) when intestinal epithelial damage happens. This stops the breakdown of tight junction integrity and the start of mitochondrial dysfunction. To summarise, the study's findings have presented data that supports the advantageous impact of allulose in reducing the advancement of colitis. Its ability to stop the disruption of the intestinal barrier enables this. Therefore, allulose has potential as a medicinal supplement for treating colitis.

Cathepsin B degrades RbcL during freezing-induced programmed cell death in Arabidopsis.

KEY MESSAGE: Cathepsin B plays an important role that degrades the Rubisco large subunit RbcL in freezing stress. Programmed cell death (PCD) has been well documented in both development and in response to environmental stresses in plants, however, PCD induced by freezing stress and its molecular mechanisms remain poorly understood. In the present study, we characterized freezing-induced PCD and explored its mechanisms in Arabidopsis. PCD induced by freezing stress was similar to that induced by other stresses and senescence in Arabidopsis plants with cold acclimation. Inhibitor treatment assays and immunoblotting indicated that cathepsin B mainly contributed to increased caspase-3-like activity during freezing-induced PCD. Cathepsin B was involved in freezing-induced PCD and degraded the large subunit, RbcL, of Rubisco. Our results demonstrate an essential regulatory mechanism of cathepsin B for Rubisco degradation in freezing-induced PCD, improving our understanding of freezing-induced cell death and nitrogen and carbohydrate remobilisation in plants.

Cathepsin B Processing Is Required for the In Vivo Efficacy of Albumin-Drug Conjugates.

Targeted drug delivery approaches that selectively and preferentially deliver therapeutic agents to specific tissues are of great interest for safer and more effective pharmaceutical treatments. We investigated whether cathepsin B cleavage of a valine-citrulline [VC(S)]-containing linker is required for the release of monomethyl auristatin E (MMAE) from albumin-drug conjugates. In this study, we used an engineered version of human serum albumin, Veltis High Binder II (HBII), which has enhanced binding to the neonatal Fc (fragment crystallizable) receptor (FcRn) to improve drug release upon binding and FcRn-mediated recycling. The linker-payload was conjugated to cysteine 34 of albumin using a carbonylacrylic (caa) reagent which produced homogeneous and plasma stable conjugates that retained FcRn binding. Two caa-linker-MMAE reagents were synthesized─one with a cleavable [VC(S)] linker and one with a noncleavable [VC(R)] linker─to question whether protease-mediated cleavage is needed for MMAE release. Our findings demonstrate that cathepsin B is required to achieve efficient and selective antitumor activity. The conjugates equipped with the cleavable [VC(S)] linker had potent antitumor activity in vivo facilitated by the release of free MMAE upon FcRn binding and internalization. In addition to the pronounced antitumor activity of the albumin conjugates in vivo, we also demonstrated their preferable tumor biodistribution and biocompatibility with no associated toxicity or side effects. These results suggest that the use of engineered albumins with high FcRn binding combined with protease cleavable linkers is an efficient strategy to target delivery of drugs to solid tumors.

Cathepsin B-Responsive Programmed Brain Targeted Delivery System for Chemo-Immunotherapy Combination Therapy of Glioblastoma.

Tumor-associated macrophages (TAMs) are closely related to the progression of glioblastoma multiform (GBM) and its development of therapeutic resistance to conventional chemotherapy. TAM-targeted therapy combined with conventional chemotherapy has emerged as a promising strategy to combat GBM. However, the presence of the blood-brain barrier (BBB) severely limits the therapeutic efficacy. Meanwhile, the lack of ability to distinguish different targeted cells also poses a challenge for precise therapy. Herein, we propose a cathepsin B (CTSB)-responsive programmed brain-targeted delivery system (D&R-HM-MCA) for simultaneous TAM-targeted and GBM-targeted delivery. D&R-HM-MCA could cross the BBB via low density lipoprotein receptor-associated protein 1 (LRP1)-mediated transcytosis. Upon reaching the GBM site, the outer angiopep-2 modification could be detached from D&R-HM-MCA via cleavage of the CTSB-responsive peptide, which could circumvent abluminal LRP1-mediated efflux. The exposed p-aminophenyl-α-d-mannopyranoside (MAN) modification could further recognize glucose transporter-1 (GLUT1) on GBM and macrophage mannose receptor (MMR) on TAMs. D&R-HM-MCA could achieve chemotherapeutic killing of GBM and simultaneously induce TAM polarization from anti-inflammatory M2 phenotype to pro-inflammatory M1 phenotype, thus resensitizing the chemotherapeutic response and improving anti-GBM immune response. This CTSB-responsive brain-targeted delivery system not only can improve brain delivery efficiency, but also can enable the combination of chemo-immunotherapy against GBM. The effectiveness of this strategy may provide thinking for designing more functional brain-targeted delivery systems and more effective therapeutic regimens.

In vivo imaging of cathepsin B in activated glia in the brain after orofacial formalin test.

PURPOSE Cathepsin B (Cat B) is a cysteine lysosomal protease that is upregulated in many inflammatory diseases and widely expressed in the brain. Here, we used a Cat B activatable near-infrared (NIR) imaging probe to measure glial activation in vivo in the formalin test, a standard orofacial inflammatory pain model. The probe's efficacy was quantified with immunohistochemical analysis of the somatosensory cortex. PROCEDURES Three different concentrations of Cat B imaging probe (30, 50, 100 pmol/200 g bodyweight) were injected intracisternally into the foramen magnum of rats under anesthesia. Four hours later formalin (1.5%, 50 µl) was injected into the upper lip and the animal's behaviors recorded for 45 min. Subsequently, animals were repeatedly scanned using the IVIS Spectrum (8, 10, and 28 h post imaging probe injection) to measure extracellular Cat B activity. Aldehyde fixed brain sections were immunostained with antibodies against microglial marker Iba1 or astrocytic GFAP and detected with fluorescently labeled secondary antibodies to quantify co-localization with the fluorescent probe. RESULTS The Cat B imaging probe only slightly altered the formalin test results. Nocifensive behavior was only reduced in phase 1 in the 100 pmol group. In vivo measured fluorescence efficiency was highest in the 100 pmol group 28 h post imaging probe injection. Post-mortem immunohistochemical analysis of the somatosensory cortex detected the greatest amount of NIR fluorescence localized on microglia and astrocytes in the 100 pmol imaging probe group. Sensory neuron neuropeptide and cell injury marker expression in ipsilateral trigeminal ganglia was not altered by the presence of fluorescent probe. CONCLUSIONS These data demonstrate a concentration- and time-dependent visualization of extracellular Cat B in activated glia in the formalin test using a NIR imaging probe. Intracisternal injections are well suited for extracellular CNS proteinase detection in conditions when the blood-brain barrier is intact.

Gasdermin D regulates soluble fms-like tyrosine kinase 1 release in macrophages.

Preeclampsia (PE) is a serious complication, and soluble fms-like tyrosine kinase (sFLT1) released from the placenta is one of the causes of PE pathology. Trophoblasts are the primary source of sFLT1; however, monocytes/macrophages exist enough in the placenta can also secrete sFLT1. Sterile inflammatory responses, especially NLRP3 inflammasome and its downstream gasdermin D (GSDMD)-regulated pyroptosis, may be involved in the development of PE pathology. In this study, we investigated whether human monocyte/macrophage cell line THP-1 cells secrete sFLT1 depending on the NLRP3 inflammasome and GSDMD. To differentiate THP-1 monocytes into macrophages, treatment with phorbol 12-myristate 13-acetate (PMA) induced sFLT1 with interleukin (IL)- 1ß, but did not induce cell lytic death. IL-1ß secretion induced by PMA inhibited by deletion of NLRP3 and inhibitors of NLRP3 and caspase-1, but deletion of NLRP3 and these inhibitors did not affect sFLT1 secretion in THP-1 cells. Both gene deletion and inhibition of GSDMD dramatically decreased IL-1ß and sFLT1 secretion from THP-1 cells. Treatment with CA074-ME (a cathepsin B inhibitor) also reduced the secretion of both sFLT1 and IL-1ß in THP-1 cells. In conclusion, THP-1 macrophages release sFLT1 in a GSDMD-dependent manner, but not in the NLRP3 inflammasome-dependent manner, and this sFLT1 release may be associated with the non-lytic role of GSDMD. In addition, sFLT1 levels induced by PMA are associated with lysosomal cathepsin B in THP-1 macrophages. We suggest that sFLT1 synthesis regulated by GSDMD are involved in the pathology of PE.

Cathepsin B serves as a potential prognostic biomarker and correlates with ferroptosis in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a long-term, systemic, and progressive autoimmune disorder. It has been established that ferroptosis, a type of iron-dependent lipid peroxidation cell death, is closely associated with RA. Fibroblast-like synoviocytes (FLS) are the main drivers of RA joint destruction, and they possess a high concentration of endoplasmic reticulum structure. Therefore, targeting ferroptosis and RA-FLS may be a potential treatment for RA. METHODS: Four machine learning algorithms were utilized to detect the essential genes linked to RA, and an XGBoost model was created based on the identified genes. SHAP values were then used to visualize the factors that affect the development and progression of RA, and to analyze the importance of individual features in predicting the outcomes. Moreover, WGCNA and PPI were employed to identify the key genes related to RA, and CIBERSORT was used to analyze the correlation between the chosen genes and immune cells. Finally, the findings were validated through in vitro cell experiments, such as CCK-8 assay, lipid peroxidation assay, iron assay, GSH assay, and Western blot. RESULTS: Bioinformatics and machine learning were employed to identify cathepsin B (CTSB) as a potential biomarker for RA. CTSB is highly expressed in RA patients and has been found to have a positive correlation with macrophages M2, neutrophils, and T cell follicular helper cells, and a negative correlation with CD8 T cells, monocytes, Tregs, and CD4 memory T cells. To investigate the effect of CTSB on RA-FLS from RA patients, the CTSB inhibitor CA-074Me was used and it was observed to reduce the proliferation and migration of RA-FLS, as indicated by the accumulation of lipid ROS and ferrous ions, and induce ferroptosis in RA-FLS. CONCLUSIONS: This study identified CTSB, a gene associated with ferroptosis, as a potential biomarker for diagnosing and managing RA. Moreover, CA-074Me, a CTSB inhibitor, was observed to cause ferroptosis and reduce the migratory capacity of RA-FLS.

Cathepsin B aggravates atherosclerosis in ApoE-deficient mice by modulating vascular smooth muscle cell pyroptosis through NF-κB / NLRP3 signaling pathway.

Atherosclerosis (AS) is a chronic inflammatory disease involving cell death and inflammatory responses. Pyroptosis, a newly discovered pro-inflammatory programmed cell death process, exacerbates inflammatory responses. However, the roles of cathepsin B (CTSB) in pyroptosis and AS remain unclear. To gain further insight, we fed ApoE-/- mice a high-fat diet to investigate the effects and mechanisms of CTSB overexpression and silencing on AS. We also explored the specific role of CTSB in vascular smooth muscle cells (VSMCs) in vitro. The study revealed that high-fat diet led to the formation of AS plaques, and CTSB was found to increase the AS plaque lesion area. Immunohistochemical and TUNEL/caspase-1 staining revealed the existence of pyroptosis in atherosclerotic plaques, particularly in VSMCs. In vitro studies, including Hoechst 33342/propidium iodide staining, a lactate dehydrogenase (LDH) release assay, detection of protein indicators of pyroptosis, and detection of interleukin-1ß (IL-1ß) in cell culture medium, demonstrated that oxidized low-density lipoprotein (ox-LDL) induced VSMC pyroptosis. Additionally, CTSB promoted VSMC pyroptosis. Ox-LDL increased the expression of CTSB, which in turn activated the NOD-like receptor protein 3 (NLRP3) inflammasome and promoted NLRP3 expression by facilitating nuclear factor kappa B (NF-κB) p65 nuclear translocation. This effect could be attenuated by the NF-κB inhibitor SN50. Our research found that CTSB not only promotes VSMC pyroptosis by activating the NLRP3 inflammasome, but also increases the expression of NLRP3.

Extracellular vesicle proteome unveils cathepsin B connection to Alzheimer's disease pathogenesis.

Extracellular vesicles (EVs) are membrane vesicles that are released extracellularly and considered to be implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's disease. Here, CSF EVs of 16 ATN-classified cases were subjected to quantitative proteome analysis. In these CSF EVs, levels of 11 proteins were significantly altered during the ATN stage transitions (P < 0.05 and fold-change > 2.0). These proteins were thought to be associated with Alzheimer's disease pathogenesis and represent candidate biomarkers for pathogenic stage classification. Enzyme-linked immunosorbent assay analysis of CSF and plasma EVs revealed altered levels of cathepsin B (CatB) during the ATN transition (seven ATN groups in validation set, n = 136). The CSF and plasma EV CatB levels showed a negative correlation with CSF amyloid-ß42 concentrations. This proteomic landscape of CSF EVs in ATN classifications can depict the molecular framework of Alzheimer's disease progression, and CatB may be considered a promising candidate biomarker and therapeutic target in Alzheimer's disease amyloid pathology.

Chitosan-coated nanostructured lipid carriers for effective brain delivery of Tanshinone IIA in Parkinson's disease: interplay between nuclear factor-kappa ß and cathepsin B.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder associated with increased oxidative stress, the underlying vital process contributing to cell death. Tanshinone IIA (TAN) is a phytomedicine with a documented activity in treating many CNS disorders, particularly PD owing to its unique anti-inflammatory and antioxidant effect. However, its clinical utility is limited by its poor aqueous solubility, short half-life, and hence low concentration reaching targeted cells. This work aimed to develop a biocompatible chitosan-coated nanostructured lipid carriers (CS-NLCs) for effective brain delivery of TAN for PD management. The proposed nanosystem was successfully prepared using a simple melt-emulsification ultra-sonication method, optimized and characterized both in vitro and in vivo in a rotenone-induced PD rat model. The developed TAN-loaded CS-NLCs (CS-TAN-NLCs) showed good colloidal properties (size ≤ 200 nm, PDI ≤ 0.2, and ζ-potential + 20 mV) and high drug entrapment efficiency (> 97%) with sustained release profile for 24 h. Following intranasal administration, CS-TAN-NLCs succeeded to achieve a remarkable antiparkinsonian and antidepressant effect in diseased animals compared to both the uncoated TAN-NLCs and free TAN suspension as evidenced by the conducted behavioral tests and improved histopathological findings. Furthermore, biochemical evaluation of oxidative stress along with inflammatory markers, nuclear factor-kabba ß (NF-Kß) and cathepsin B further confirmed the potential of the CS-TAN-NLCs in enhancing brain delivery and hence the therapeutic effect of TAN of treatment of PD. Accordingly, CS-TAN-NLCs could be addressed as a promising nano-platform for the effective management of PD.

Lycopene Alleviates Chronic Stress-Induced Hippocampal Microglial Pyroptosis by Inhibiting the Cathepsin B/NLRP3 Signaling Pathway.

Lycopene (LYC) exerts a strong neuroprotective and antipyroptotic effects. This study explored the effects and mechanisms of LYC on chronic stress-induced hippocampal microglial damage and depression-like behaviors. The caspase-1 inhibitor VX-765 attenuated chronic restrain stress (CRS)-induced hippocampal microglial pyroptosis and depression-like behaviors. Moreover, the alleviation of CRS-induced hippocampal microglial pyroptosis and depression-like behaviors by LYC was associated with the cathepsin B/NLRP3 pathway. In vitro, the caspase-1 inhibitor Z-YVAD-FMK alleviated pyroptosis in highly aggressively proliferating immortalized (HAPI) cells. Additionally, the alleviation of corticosterone-induced HAPI cell damage and pyroptosis by LYC was associated with the cathepsin B/NLRP3 pathway. Furthermore, the cathepsin B agonist pazopanib promoted HAPI cell pyroptosis, whereas LYC inhibited pazopanib-induced pyroptosis via the cathepsin B/NLRP3 pathway. Similarly, Z-YVAD-FMK inhibited pazopanib-induced HAPI cell pyroptosis. These results suggest that LYC alleviates chronic stress-induced hippocampal microglial pyroptosis via the cathepsin B/NLRP3 pathway inhibition. This study provides a new strategy for treating chronic stress encephalopathy.

Particulate matter 10 induces oxidative stress and apoptosis in rhesus macaques skin fibroblast.

Background: Particulate matter (PM) is a major air pollutant that affects human health worldwide. PM can pass through the skin barrier, thus causing skin diseases such as heat rash, allergic reaction, infection, or inflammation. However, only a few studies have been conducted on the cytotoxic effects of PM exposure on large-scale animals. Therefore, herein, we investigated whether and how PM affects rhesus macaque skin fibroblasts. Methods: Rhesus macaque skin fibroblasts were treated with various concentrations of PM10 (1, 5, 10, 50, and 100 µg/mL) and incubated for 24, 48, and 72 h. Then, cell viability assay, TUNEL assay, and qRT-PCR were performed on the treated cells. Further, the reactive oxygen species, glutathione, and cathepsin B levels were determined. The MTT assay revealed that PM10 (>50 µg/mL) proportionately reduced the cell proliferation rate. Results: PM10 treatment increased TUNEL-positive cell numbers, following the pro-apoptosis-associated genes (CASP3 and BAX) and tumor suppressor gene TP53 were significantly upregulated. PM10 treatment induced reactive oxidative stress. Cathepsin B intensity was increased, whereas GSH intensity was decreased. The mRNA expression levels of antioxidant enzyme-related genes (CAT, GPX1 and GPX3) were significantly upregulated. Furthermore, PM10 reduced the mitochondrial membrane potential. The mRNA expression of mitochondrial complex genes, such as NDUFA1, NDUFA2, NDUFAC2, NDUFS4, and ATP5H were also significantly upregulated. In conclusion, these results showed that PM10 triggers apoptosis and mitochondrial damage, thus inducing ROS accumulation. These findings provide potential information on the cytotoxic effects of PM10 treatment and help to understand the mechanism of air pollution-induced skin diseases.