Danggui-Shaoyao-San protects against non-alcoholic steatohepatitis via modulation of hepatic APP protein, Lysosomal CTSB release, and NF-κB activation.

Background: Non-alcoholic steatohepatitis (NASH), an escalating global health concern, is a primary factor behind cirrhosis, liver transplantation, and hepatocellular carcinoma. Effective treatments remain elusive. Danggui-Shaoyao-San (DGSY), a classic famous prescription employed in treating NASH, could hold promise, although its molecular underpinnings are still under investigation. This study undertakes an exploration of the impacts of DGSY on NASH and seeks to illuminate the mechanisms at play. Methods: UHPLC-Q-Orbitrap HRMS was employed to identify compounds within DGSY. Mice underwent a 25-week regimen of HFHC diet and high-sugar water, with 4 weeks of DGSY treatment for efficacy and pathogenic mechanism exploration in vivo. L02 cells were cultured with 0.2 mM FFA for 24 h, exposed to DGSY at 1 mg/ml and 2 mg/ml for efficacy and pathogenic mechanism exploration in vitro. Using online databases, we sought potential targets for NASH treatment, and through PPI networks, identified key targets. Expression levels of genes and proteins were examined by western blotting, RT-PCR, and immunofluorescence staining. Results: Thirty-four compounds were identified within DGSY. DGSY brought about marked reductions in biochemical indicators and yielded significant improvements in NASH mice histological features. Additionally, it mitigated hepatic steatosis and inflammation both in vivo and in vitro. The top 10 targets from two network pharmacology analyses, one focusing on structural prediction and the other on literature mining, identified APOE and APP as potential therapeutic targets for DGSY in NASH treatment. PCR validation confirmed that DGSY reduced APP expression after treatment, and further investigation revealed that DGSY significantly suppressed hepatic APP and Aß expression, indicating its effectiveness in treating NASH. Furthermore, it inhibited Aß-induced Cathepsin B lysosomal release, reducing hepatic inflammation. Conclusion: Danggui-Shaoyao-San has anti-steatohepatitis effects in ameliorating hepatic APP protein expression, reducing hepatic lysosomal CTSB release, and suppressing hepatic NF-κB activation. The study provided a more theoretical basis for the future clinical application of DGSY.

Genetic Heterogeneity in Cellular Angiofibromas.

BACKGROUND: Cellular angiofibroma, a rare benign mesenchymal neoplasm, is classified within the 13q/RB1 family of tumors due to morphological, immunohistochemical, and genetic similarities with spindle cell lipoma. Here, genetic data reveal pathogenetic heterogeneity in cellular angiofibroma. METHODS: Three cellular angiofibromas were studied using G-banding/Karyotyping, array comparative genomic hybridization, RNA sequencing, and direct cycling sequencing. RESULTS: The first tumor carried a del(13)(q12) together with heterozygous loss and minimal expression of the RB1 gene. Tumors two and three displayed chromosome 8 abnormalities associated with chimeras of the pleomorphic adenoma gene 1 (PLAG1). In tumor 2, the cathepsin B (CTSB) fused to PLAG1 (CTSB::PLAG1) while in tumor 3, the mir-99a-let-7c cluster host gene (MIR99AHG) fused to PLAG1 (MIR99AHG::PLAG1), both leading to elevated expression of PLAG1 and insulin growth factor 2. CONCLUSION: This study uncovers two genetic pathways contributing to the pathogenetic heterogeneity within cellular angiofibromas. The first aligns with the 13q/RB1 family of tumors and the second involves PLAG1-chimeras. These findings highlight the diverse genetic landscape of cellular angiofibromas, providing insights into potential diagnostic strategies.

miR-25-5p in exosomes derived from UVB-induced fibroblasts regulates melanogenesis via TSC2-dominated cellular organelle dysfunction.

BACKGROUND: Few reports have confirmed whether exosomes derived from fibroblasts can regulate the process of melanogenesis. We wondered whether exosomes derived from fibroblasts could have a potent regulatory effect on melanogenesis and explored the underlying mechanisms. OBJECTIVE: This study aimed to find the role of fibroblasts in melanocytes and revealed the related mechanisms. METHODS: RT-qPCR, Western blot analysis were conducted to measure the RNA and protein expression level of various related genes. miRNA sequencing, mass spectrum analysis and subsequent bioinformatics analysis were employed to find the underlying targets. Zebrafish were employed to measure the melanin synthesis related process in vivo. Furthermore, electron microscopy, ROS measurement and dual-luciferase reporter assay were adopted to investigate the relationship between these processes. RESULTS: We found that exosomes derived from human primary dermal fibroblasts were internalized by human primary melanocytes and MNT1 cells and that the melanin content and the expression of melanin synthesis-related proteins TYR and MITF was inhibited by exosomes derived from UVB-induced human primary dermal fibroblasts. The miRNA expression profile in secreted exosomes changed significantly, with miR-25-5p identified as capable of regulating TSC2 expression via the CDS region. The miR-25-5p-TSC2 axis could affect the melanin content through subsequent cellular organelle dysfunction, such as mitochondrial dysfunction, endoplasmic reticulum stress and dysregulation of lysosomal cysteine proteases. CONCLUSION: We unveiled a novel regulatory role of fibroblasts in melanocytes, facilitated by the secretion of exosomes. miR-25-5p within exosomes plays a pivotal role in regulating melanogenesis via TSC2-induced cellular organelle dysfunction.

MiR-223 enhances lipophagy by suppressing CTSB in microglia following lysolecithin-induced demyelination in mice.

BACKGROUND: Lipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability. Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved. METHODS: C57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM. RESULTS: Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1ß. CONCLUSION: These findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of lipophagy and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.

Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

hCeO2@ Cu5.4O nanoparticle alleviates inflammatory responses by regulating the CTSB-NLRP3 signaling pathway.

Inflammatory responses, especially chronic inflammation, are closely associated with many systemic diseases. There are many ways to treat and alleviate inflammation, but how to solve this problem at the molecular level has always been a hot topic in research. The use of nanoparticles (NPs) as anti-inflammatory agents is a potential treatment method. We synthesized new hollow cerium oxide nanomaterials (hCeO2 NPs) doped with different concentrations of Cu5.4O NPs [the molar ratio of Cu/(Ce + Cu) was 50%, 67%, and 83%, respectively], characterized their surface morphology and physicochemical properties, and screened the safe concentration of hCeO2@Cu5.4O using the CCK8 method. Macrophages were cultured, and P.g-lipopolysaccharide-stimulated was used as a model of inflammation and co-cultured with hCeO2@Cu5.4O NPs. We then observe the effect of the transcription levels of CTSB, NLRP3, caspase-1, ASC, IL-18, and IL-1ß by PCR and detect its effect on the expression level of CTSB protein by Western blot. The levels of IL-18 and IL-1ß in the cell supernatant were measured by enzyme-linked immunosorbent assay. Our results indicated that hCeO2@Cu5.4O NPs could reduce the production of reactive oxygen species and inhibit CTSB and NLRP3 to alleviate the damage caused by the inflammatory response to cells. More importantly, hCeO2@Cu5.4O NPs showed stronger anti-inflammatory effects as Cu5.4O NP doping increased. Therefore, the development of the novel nanomaterial hCeO2@Cu5.4O NPs provides a possible new approach for the treatment of inflammatory diseases.

Gastric cancer prognosis: unveiling autophagy-related signatures and immune infiltrates.

Background: Autophagy played a crucial regulatory role in tumor initiation and progression. Therefore, we aimed to comprehensively analyze autophagy-related genes (ARGs) in gastric cancer, focusing on their expression, prognostic value, and potential functions. Methods: The gastric cancer gene chip datasets (GSE79973 and GSE54129) were collected from the Gene Expression Omnibus (GEO) database. Subsequently, the Limma package was employed to identify differentially expressed genes (DEGs) between the normal and disease groups. The selected ARGs were further authenticated using the Human Protein Atlas (HPA) database, The Cancer Genome Atlas (TCGA) database, and GSE19826 database. Results: A total of 15 autophagy-related DEGs, eight of which were upregulated [FKBP1A, IL24, PEA15, HSP90AB1, cathepsin B (CTSB), ITGB1, SPHK1, HIF1A], while seven were downregulated (DAPK2, EIF2AK3, FKBP1B, PTK6, NKX2-3, NFE2L2, PRKCD). Analysis revealed that CTSB was specifically associated with the prognosis of gastric cancer patients. Gene set enrichment analysis (GSEA) showcased a significant enrichment of CTSB-related genes within immune-related pathways. Moreover, correlation analysis demonstrated a clear association between the expression of CTSB and immune infiltration. The upregulation of CTSB in gastric cancer was linked to poor survival and increased immune infiltration. Conclusions: We conjectured that CTSB likely played a critical role in regulating immunity and autophagy in gastric cancer.

Exploring the Role of Non-synonymous and Deleterious Variants Identified in Colorectal Cancer: A Multi-dimensional Computational Scrutiny of Exomes.

Introduction: Colorectal cancers are the world's third most commonly diagnosed type of cancer. Currently, there are several diagnostic and treatment options to combat it. However, a delay in detection of the disease is life-threatening. Additionally, a thorough analysis of the exomes of cancers reveals potential variation data that can be used for early disease prognosis. Methods: By utilizing a comprehensive computational investigation, the present study aimed to reveal mutations that could potentially predispose to colorectal cancer. Ten colorectal cancer exomes were retrieved. Quality control assessments were performed using FastQC and MultiQC, gapped alignment to the human reference genome (hg19) using Bowtie2 and calling the germline variants using Haplotype caller in the GATK pipeline. The variants were filtered and annotated using SIFT and PolyPhen2 successfully categorized the mutations into synonymous, non-synonymous, start loss and stop gain mutations as well as marked them as possibly damaging, probably damaging and benign. This mutational profile helped in shortlisting frequently occurring mutations and associated genes, for which the downstream multi-dimensional expression analyses were carried out. Results: Our work involved prioritizing the non-synonymous, deleterious SNPs since these polymorphisms bring about a functional alteration to the phenotype. The top variations associated with their genes with the highest frequency of occurrence included LGALS8, CTSB, RAD17, CPNE1, OPRM1, SEMA4D, MUC4, PDE4DIP, ELN and ADRA1A. An in-depth multi-dimensional downstream analysis of all these genes in terms of gene expression profiling and analysis and differential gene expression with regard to various cancer types revealed CTSB and CPNE1 as highly expressed and overregulated genes in colorectal cancer. Conclusion: Our work provides insights into the various alterations that might possibly lead to colorectal cancer and suggests the possibility of utilizing the most important genes identified for wet-lab experimentation.

Plant cathepsin B, a versatile protease.

Plant proteases are essential enzymes that play key roles during crucial phases of plant life. Some proteases are mainly involved in general protein turnover and recycle amino acids for protein synthesis. Other proteases are involved in cell signalling, cleave specific substrates and are key players during important genetically controlled molecular processes. Cathepsin B is a cysteine protease that can do both because of its exopeptidase and endopeptidase activities. Animal cathepsin B has been investigated for many years, and much is known about its mode of action and substrate preferences, but much remains to be discovered about this potent protease in plants. Cathepsin B is involved in plant development, germination, senescence, microspore embryogenesis, pathogen defence and responses to abiotic stress, including programmed cell death. This review discusses the structural features, the activity of the enzyme and the differences between the plant and animal forms. We discuss its maturation and subcellular localisation and provide a detailed overview of the involvement of cathepsin B in important plant life processes. A greater understanding of the cell signalling processes involving cathepsin B is needed for applied discoveries in plant biotechnology.

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.

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.

3-Monochloropropane-1,2-diol esters induce HepG2 cells necroptosis via CTSB/TFAM/ROS pathway.

3-monochloropropane-1,2-diol esters (3-MCPDE) are toxic substances that form in food thermal processing and have a diverse range of toxicities. In this study, we found that 3-MCPDE triggered necroptosis by RIPK1/RIPK3/MLKL pathway in HepG2 cells. Previous studies have shown that ROS is an important activator of RIPK1 and RIPK3. The data showed that 3-MCPDE induced excessive ROS production through mitochondrial damage. After treatment with ROS inhibitor N-acetylcysteine (NAC), 3-MCPDE-induced necroptosis was relieved. Further, we explored how 3-MCPDE destroys mitochondria. The data suggested that 3-MCPDE induced mitochondrial dysfunction through the CTSB/TFAM pathway. Overall, the results indicated that 3-MCPDE induced necroptosis through CTSB/TFAM/ROS pathway in HepG2 cells. Our study provided a new mechanism for 3-MCPDE hepatotoxicity.

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.

CTSB Nuclear Translocation Facilitates DNA Damage and Lysosomal Stress to Promote Retinoblastoma Cell Death.

Retinoblastoma (RB) is a pernicious tumor originating from photoreceptor precursor cells that often endangers the lives of children. The purpose of our study was to further investigate the influence of cathepsin B (CTSB) nuclear translocation on RB cell death. Y79 cells were injected into the vitreous cavity of nude mice at a dose of 4 µL/mouse to establish an animal model of RB. Real-time quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, a comet assay, a Cell Counting Kit-8 (CCK-8) assay and flow cytometry were used to measure the levels of the interrelated genes and proteins and to evaluate alterations in autophagy, apoptosis, proliferation, DNA damage and cell cycle arrest. CTSB was found to be expressed at low levels in RB animal model samples and RB cell lines. Functionally, CTSB nuclear translocation promoted DNA damage, cell cycle arrest, ferroptosis and autophagy in Y79 cells and inhibited their proliferation. Downstream mechanistic studies showed that nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting breast cancer 1 protein (BRCA1) expression and also activates the signal transducer and activator of transcription 3/stimulator of interferon response cGAMP interactor 1 (STAT3/STING1) pathway to induce lysosomal stress, leading to ferroptosis and autophagy in Y79 cells and alleviating RB. Nuclear translocation of CTSB facilitates DNA damage and cell cycle arrest in RB cells by inhibiting BRCA1 expression and activating the STAT3/STING1 pathway and induces lysosomal stress, which eventually leads to ferroptosis and autophagy and mitigates RB.

Circ_0020014 mediates CTSB expression and participates in IL-1ß-prompted chondrocyte injury via interacting with miR-24-3p.

BACKGROUND: Recent studies have shown that circRNAs are involved in the pathogenesis of osteoarthritis (OA) by affecting various fundamental cellular characteristics of chondrocytes. The purpose of this paper is to investigate the role and regulatory mechanism of hsa_circ_0020014 (circ_0020014) in chondrocytes of OA. METHODS: The inflammatory cytokine interleukin 1 beta (IL-1ß) was used to stimulate human chondrocytes. Cell viability, proliferation, and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. Several protein levels were determined by western blotting (WB). Levels of inflammatory cytokines and malondialdehyde (MDA) were determined by enzyme-linked immunosorbent assay (ELISA). Relative expression of circ_0020014 was estimated by real-time polymerase quantitative chain reaction (RT-qPCR). Bioinformatics prediction combined with dual-luciferase reporter, RIP and RNA pull-down assays were done to probe into the regulatory mechanism of circ_0020014. RESULTS: Circ_0020014 was overexpressed in OA patient-derived articular cartilages and IL-1ß-stimulated chondrocytes. Silencing of circ_0020014 lighted IL-1ß-prompted chondrocyte proliferation repression, apoptosis, inflammation, and oxidative stress. Mechanically, circ_0020014 functioned as a miR-24-3p molecular sponge to regulate cathepsin B (CTSB) expression. Furthermore, miR-24-3p inhibition alleviated circ_0020014 knockdown-mediation repression of IL-1ß-urged chondrocyte injury. In addition, CTSB overexpression whittled miR-24-3p upregulation-mediated suppression of IL-1ß-urged chondrocyte injury. CONCLUSION: Our findings demonstrated that the circ_0020014/miR-24-3p/CTSB axis was associated with IL-1ß-prompted chondrocyte injury, supporting the involvement of circ_0020014 in the OA pathogenesis.

Elaidic acid induced hepatocyte pyroptosis via autophagy-CTSB-NLRP3 pathway.

Elaidic acid (EA, C18:1 trans) is a kind of principal Trans fatty acid (TFA) and is widely found in processed food. Pyroptosis is a form of programmed cell death, distinct from apoptosis and traditional necrosis. Excessive pyroptosis could induce body injury and serious inflammation. However, the effect of EA on pyroptosis has not been reported. In the study, we found that EA exposure caused liver damage and hepatocyte pyroptosis by testing GSDMD-N, Caspase 1, IL-18, and IL-1ß in mice and HepG2 cells. Further exploring the mechanisms, we found that EA-induced pyroptosis depended on Cathepsin B (CTSB)-mediated NLRP3 inflammasome activation. Cell autophagy was closely related to lysosomes. Our study revealed that EA promoted hepatocyte autophagy, and activated autophagy induced lysosomal membrane permeabilization (LMP) and CTSB leakage. Inhibition of autophagy by 3-MA mitigated the CTSB leak, reduced the activation of the NLRP3 inflammasome, and then attenuated the EA-induced pyroptosis. In summary, these results indicated that EA induced hepatocyte pyroptosis via autophagy-CTSB-NLRP3 inflammasome pathway. The study revealed new insights into the toxicity mechanism of EA.

Hederagenin inhibits high glucose-induced fibrosis in human renal cells by suppression of NLRP3 inflammasome activation through reducing cathepsin B expression.

Diabetic nephropathy is a major complication of diabetes mellitus and is related to dysfunction of renal cells. Hederagenin is a triterpenoid saponin from some Chinese herbs with anti-inflammatory and anti-diabetic activities. However, its role in diabetic nephropathy progression is still obscure. This study aimed to explore the effects of hederagenin on renal cell dysfunction in vitro. Human renal mesangial cells (HRMCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) were cultured under high glucose (HG) conditions to mimic diabetic nephropathy-like injury. Cell proliferation was evaluated by CCK-8. mRNA and protein levels were determined by qRT-PCR and western blotting, respectively. The secretion levels of fibrosis-related biomarkers were analyzed by ELISA. Results showed that hederagenin reduced HG-induced proliferation increase in HRMCs and HRPTEpiCs. Hederagenin attenuated HG-induced increase in mRNA and protein expression of NLRP3, ASC, and IL-1ß. Hederagenin also suppressed HG-induced increase in mRNA and secretion levels of FN, Col. IV, PAI-1, and TGF-ß1. NLRP3 inhibitor MCC950 attenuated HG-induced fibrosis of renal cells, and its activator nigericin reversed the suppressive effect of hederagenin on HG-induced fibrosis. Bioinformatics analysis predicted cathepsin B (CTSB) as a target of hederagenin to modulate NOD-like receptor (NLR) pathway. Hederagenin decreased CTSB level, and CTSB overexpression reversed the suppressive effect of hederagenin on HG-induced NLRP3 inflammasome activation and fibrosis in HRMCs and HRPTEpiCs. In conclusion, hederagenin attenuates HG-induced fibrosis of renal cells by inhibiting NLRP3 inflammasome activation via reducing CTSB expression, indicating a therapeutic potential of hederagenin in diabetic nephropathy.

The MMP-2 histone H3 N-terminal tail protease is selectively targeted to the transcription start sites of active genes.

BACKGROUND: Proteolysis of the histone H3 N-terminal tail (H3NT) is an evolutionarily conserved epigenomic feature of nearly all eukaryotes, generating a cleaved H3 product that is retained in ~ 5-10% of the genome. Although H3NT proteolysis within chromatin was first reported over 60 years ago, the genomic sites targeted for H3NT proteolysis and the impact of this histone modification on chromatin structure and function remain largely unknown. The goal of this study was to identify the specific regions targeted for H3NT proteolysis and investigate the consequence of H3NT "clipping" on local histone post-translational modification (PTM) dynamics. RESULTS: Leveraging recent findings that matrix metalloproteinase 2 (MMP-2) functions as the principal nuclear H3NT protease in the human U2OS osteosarcoma cell line, a ChIP-Seq approach was used to map MMP-2 localization genome wide. The results indicate that MMP-2 is selectively targeted to the transcription start sites (TSSs) of protein coding genes, primarily at the + 1 nucleosome. MMP-2 localization was exclusive to highly expressed genes, further supporting a functional role for H3NT proteolysis in transcriptional regulation. MMP-2 dependent H3NT proteolysis at the TSSs of these genes resulted in a > twofold reduction of activation-associated histone H3 PTMs, including H3K4me3, H3K9ac and H3K18ac. One of genes requiring MMP-2 mediated H3NT proteolysis for proficient expression was the lysosomal cathepsin B protease (CTSB), which we discovered functions as a secondary nuclear H3NT protease in U2OS cells. CONCLUSIONS: This study revealed that the MMP-2 H3NT protease is selectively targeted to the TSSs of active protein coding genes in U2OS cells. The resulting H3NT proteolysis directly alters local histone H3 PTM patterns at TSSs, which likely functions to regulate transcription. MMP-2 mediated H3NT proteolysis directly activates CTSB, a secondary H3NT protease that generates additional cleaved H3 products within chromatin.

Apigenin Alleviated High-Fat-Diet-Induced Hepatic Pyroptosis by Mitophagy-ROS-CTSB-NLRP3 Pathway in Mice and AML12 Cells.

Apigenin is considered the most-known natural flavonoid and is abundant in a wide variety of fruits and vegetables. A high fat diet (HFD) can induce liver injury and hepatocyte death in multiple ways. Pyroptosis is an innovative type of programmed cell death. Moreover, excessive pyroptosis of hepatocytes leads to liver injury. We used HFD to induce liver cell pyroptosis in C57BL/6J mice in this work. After gavage of apigenin, apigenin can significantly reduce the level of lactate dehydrogenase (LDH) in liver tissue ignited by HFD and reduce the levels of NLRP3 (NOD-like receptor family pyrin domain containing 3), the N-terminal domain of GSDMD (GSDMD-N), cleaved-caspase 1, cathepsin B (CTSB), interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) protein expression and the colocalization of NLRP3 and CTSB and increase the level of lysosomal associated membrane protein-1 (LAMP-1) protein expression, thus alleviating cell pyroptosis. In a further in vitro mechanism study, we find that palmitic acid (PA) can induce pyroptosis in AML12 cells. After adding apigenin, apigenin can clear the damaged mitochondria through mitophagy and reduce the generation of intracellular reactive oxygen species (ROS), thus alleviating CTSB release caused by lysosomal membrane permeabilization (LMP), reducing the LDH release caused by PA and reducing the levels of NLRP3, GSDMD-N, cleaved-caspase 1, CTSB, IL-1ß, and IL-18 protein expression. By adding the mitophagy inhibitor cyclosporin A (CsA), LC3-siRNA, the CTSB inhibitor CA-074 methyl ester (CA-074 Me), and the NLRP3 inhibitor MCC950, the aforementioned results were further confirmed. Therefore, our results show that HFD-fed and PA can damage mitochondria, promote the production of intracellular ROS, enhance the lysosomal membrane permeabilization (LMP), and cause the leakage of CTSB, thus activating the NLRP3 inflammatory body and inducing pyroptosis in C57BL/6J mice and AML12 cells, while apigenin alleviates this phenomenon through the mitophagy-ROS-CTSB-NLRP3 pathway.

Low-dose of zeolitic imidazolate framework-8 nanoparticle cause energy metabolism disorder through lysosome-mitochondria dysfunction.

Understanding the underlying interaction between nanoparticle and organelles is conclusive to the nanotoxicology. According to existing literatures, lysosome is a crucial target of the nanoparticle carrier. Meanwhile, mitochondria could provide the essential energy for nanopaticles entering/exiting the cell. Based on the investigation of lysosome-mitochondria connection, we decoded the effects of low-dose ZIF-8 on energy metabolism, which are still largely obscure beforehand. In this research, low-dose ZIF-8 NPs were utilized to explore the effects on vascular endothelial cells, the first cells exposed to NPs during intravenous injection. Consequently, ZIF-8 could damage the energy metabolism, mainly manifested as mitochondrial fission, the decreased ATP production, and lysosomal dysfuction, which would subsequently affect the cell survival, proliferation and protein expression. This study highlights the fundamental understanding for exploring the regulation of nanoscale ZIF-8 in biological processes and its further application in biomedical field.