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1.
Autoimmunity ; 57(1): 2350202, 2024 Apr 22.
Article in English | MEDLINE | ID: mdl-38721694

ABSTRACT

Yinchenhao Decoction (YCHD) is a classic prescription in traditional Chinese medicine (TCM). It appears to play an important role in anti-inflammation and autoimmunity protection. As one of the key active ingredients in YCHD, quercetin is a novel anti-inflammatory metabolite that exerts protective effects in many autoimmune diseases. However, its role in autoimmune hepatitis (AIH)-related hepatic injury has not been studied. The aim of this study was to reveal the hepatocyte protective mechanism of quercetin. In this study, we used Concanavalin A (Con A) to establish an in vitro hepatocyte injury-associated AIH model. Brl3a hepatocyte injury was induced by the supernatant of J774A.1 cells treated with Con A. We found that quercetin mitigated Con A-induced via macrophage-mediated Brl3a hepatocyte injury. Quercetin administration reduced the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in the supernatant of Con A-treated Brl3a cells and attenuated the infiltration of J774A.1 macrophages induced by Con A. Moreover, quercetin effectively inhibited the expression of proinflammatory cytokines including interleukin-1ß (IL-1ß) by Con A. Furthermore, quercetin decreased hepatocyte apoptosis and ferroptosis levels in the macrophage-induced hepatocyte injury model. In conclusion, our study indicates that quercetin alleviates macrophage-induced hepatocyte damage by reducing the inflammatory response, apoptosis and ferroptosis. Our work suggests that quercetin might be a potential therapeutic strategy for AIH.


Subject(s)
Anti-Inflammatory Agents , Apoptosis , Ferroptosis , Hepatocytes , Macrophages , Quercetin , Quercetin/pharmacology , Quercetin/therapeutic use , Animals , Hepatocytes/drug effects , Hepatocytes/metabolism , Macrophages/metabolism , Macrophages/drug effects , Macrophages/immunology , Ferroptosis/drug effects , Apoptosis/drug effects , Mice , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Cell Line , Hepatitis, Autoimmune/drug therapy , Hepatitis, Autoimmune/immunology , Hepatitis, Autoimmune/pathology , Hepatitis, Autoimmune/metabolism , Hepatitis, Autoimmune/etiology , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/immunology , Concanavalin A , Cytokines/metabolism
2.
BMC Gastroenterol ; 24(1): 163, 2024 May 14.
Article in English | MEDLINE | ID: mdl-38745150

ABSTRACT

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.


Subject(s)
CD11b Antigen , Liver Cirrhosis , Liver Regeneration , Mice, Inbred C57BL , Myeloid-Derived Suppressor Cells , Animals , Myeloid-Derived Suppressor Cells/metabolism , Myeloid-Derived Suppressor Cells/immunology , Mice , Liver Cirrhosis/pathology , Liver Cirrhosis/metabolism , Liver Regeneration/physiology , CD11b Antigen/metabolism , Male , Disease Models, Animal , Liver/pathology , Liver/metabolism , Vascular Endothelial Growth Factor A/metabolism , Carbon Tetrachloride , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/immunology , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/physiopathology , Concanavalin A , Ligation , Lipopolysaccharides , Interleukin-10/metabolism , Transforming Growth Factor beta/metabolism , Hepatic Stellate Cells/metabolism , Coculture Techniques , Hepatocytes/metabolism , Hepatocytes/pathology , Bile Ducts
3.
Phytomedicine ; 129: 155722, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38733905

ABSTRACT

BACKGROUND: Autoimmune hepatitis (AIH), primarily mediated by T cells, is characterized by liver inflammation. Despite the advancements in understanding its pathogenesis, effective therapeutic options are limited. Naringin, a flavonoid abundant in citrus fruits, is recognized for its anti-inflammatory properties and ability to protect against various inflammatory diseases, including drug-induced liver injury. However, the exact effects of naringin on AIH and the mechanisms involved remain poorly understood. PURPOSE: We aim to determine the role of naringin in AIH, exploring its targets and actions in this disease. METHODS: Network pharmacology, molecular docking, and molecular dynamics simulations were utilized to predict the HUB targets connecting naringin, T cell-mediated autoimmune disorders, and AIH. Cellular thermal shift assays were used to determine the binding abilities of naringin with the HUB targets. An in vivo experiment confirmed the impact of naringin treatment on AIH development and underlying mechanisms. RESULTS: Naringin demonstrated therapeutic effects on ConA-induced AIH. There were 455 shared targets between naringin, T cell-mediated autoimmune diseases, and AIH. Ten HUB genes (AKT1, ALB, IL-6, IL-1ß, CTNNB1, TNF, TP53, MAPK3, VEGFA, and JUN) were identified through the PPI network. Gene ontology analysis revealed involvement in gene expression regulation, lipopolysaccharide-mediated signaling, and I-kappa kinase/NFκB signaling. Pathway analysis suggested TNF, Th1/Th2 cell differentiation, and Toll-like receptor pathways, with favorable naringin-HUB gene binding. Molecular docking confirmed albumin (ALB), IL-1ß, IL-6, and TNF as primary targets for naringin. Molecular dynamics simulations showed stable binding in ALB-naringin, TNF-naringin, and IL-1ß-naringin complexes. Naringin's hepatoprotective effect on AIH was supported by increased serum ALB and decreased hepatic inflammatory cytokines including IL-1ß, IL-6, and TNF-α. CONCLUSION: Our data underscore the potential of naringin as a preventive or therapeutical agent in T cell-mediated autoimmune diseases including AIH.


Subject(s)
Flavanones , Hepatitis, Autoimmune , Molecular Docking Simulation , Flavanones/pharmacology , Flavanones/chemistry , Hepatitis, Autoimmune/drug therapy , Animals , Citrus/chemistry , Molecular Dynamics Simulation , Liver/drug effects , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/chemistry , Male , Network Pharmacology , Concanavalin A , Mice , Humans , T-Lymphocytes/drug effects
4.
ACS Biomater Sci Eng ; 10(5): 3017-3028, 2024 May 13.
Article in English | MEDLINE | ID: mdl-38655791

ABSTRACT

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.


Subject(s)
Cryogels , Oxidation-Reduction , Polyethylene Glycols , Cryogels/chemistry , Polyethylene Glycols/chemistry , Animals , Concanavalin A/chemistry , Concanavalin A/metabolism , Methacrylates/chemistry , Mice , Mannose/chemistry , Immobilized Proteins/chemistry , Immobilized Proteins/metabolism , Sulfhydryl Compounds/chemistry , Streptavidin/chemistry , Streptavidin/metabolism , Proteins/chemistry , Proteins/metabolism , Biotin/chemistry , Biotin/metabolism , Biotin/analogs & derivatives , Porosity
5.
Phytomedicine ; 129: 155652, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38663118

ABSTRACT

BACKGROUND: Autoimmune hepatitis (AIH) is a prevalent liver disease that can potentially lead to hepatic fibrosis and cirrhosis. The prolonged administration of immunosuppressive medications carries significant risks for patients. Purple sweet potato polysaccharide (PSPP), a macromolecule stored in root tubers, exhibits anti-inflammatory, antioxidant, immune-enhancing, and intestinal flora-regulating properties. Nevertheless, investigation into the role and potential mechanisms of PSPP in AIH remains notably scarce. PURPOSE: Our aim was to explore the possible protective impacts of PSPP against concanavalin A (Con A)-induced liver injury in mice. METHODS: Polysaccharide was isolated from purple sweet potato tubers using water extraction and alcohol precipitation, followed by purification through DEAE-52 cellulose column chromatography and Sephadex G-100 column chromatography. A highly purified component was obtained, and its monosaccharide composition was characterized by high performance liquid chromatography (HPLC). Mouse and cellular models induced by Con A were set up to investigate the impacts of PSPP on hepatic histopathology, apoptosis, as well as inflammation- and oxidative stress-related proteins in response to PSPP treatment. RESULTS: The administration of PSPP significantly reduced hepatic pathological damage, suppressed elevation of ALT and AST levels, and attenuated hepatic apoptosis in Con A-exposed mice. PSPP was found to mitigate Con A-induced inflammation by suppressing the TLR4-P2X7R/NLRP3 signaling pathway in mice. Furthermore, PSPP alleviated Con A-induced oxidative stress by activating the PI3K/AKT/mTOR signaling pathway in mice. Additionally, PSPP demonstrated the ability to reduce inflammation and oxidative stress in RAW264.7 cells induced by Con A in vitro. CONCLUSION: PSPP has the potential to ameliorate hepatic inflammation via the TLR4-P2X7R/NLRP3 pathway and inhibit hepatic oxidative stress through the PI3K/AKT/mTOR pathway during the progression of Con A-induced hepatic injury. The results of this study have unveiled the potential hepatoprotective properties of purple sweet potato and its medicinal value for humans. Moreover, this study serves as a valuable reference, highlighting the potential of PSPP-1 as a drug candidate for the treatment of immune liver injury.


Subject(s)
Concanavalin A , Ipomoea batatas , Oxidative Stress , Polysaccharides , Animals , Oxidative Stress/drug effects , Ipomoea batatas/chemistry , Polysaccharides/pharmacology , Polysaccharides/chemistry , Mice , Male , Chemical and Drug Induced Liver Injury/drug therapy , Liver/drug effects , RAW 264.7 Cells , Hepatitis, Autoimmune/drug therapy , Toll-Like Receptor 4/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Apoptosis/drug effects , Inflammation/drug therapy , Signal Transduction/drug effects , Anti-Inflammatory Agents/pharmacology , TOR Serine-Threonine Kinases/metabolism , Antioxidants/pharmacology , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Plant Tubers/chemistry , Proto-Oncogene Proteins c-akt/metabolism
6.
Viruses ; 16(4)2024 03 24.
Article in English | MEDLINE | ID: mdl-38675840

ABSTRACT

The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.


Subject(s)
COVID-19 , Interleukin-6 , Lectins, C-Type , Leukocytes, Mononuclear , Mannose Receptor , Mannose-Binding Lectins , Receptors, Cell Surface , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Humans , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/immunology , Spike Glycoprotein, Coronavirus/genetics , Lectins, C-Type/metabolism , Receptors, Cell Surface/metabolism , Leukocytes, Mononuclear/metabolism , Leukocytes, Mononuclear/immunology , COVID-19/immunology , COVID-19/virology , COVID-19/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/metabolism , Mannose-Binding Lectins/metabolism , Interleukin-6/metabolism , Cytokines/metabolism , Interferon-gamma/metabolism , Cells, Cultured , Polysaccharides/metabolism , Healthy Volunteers , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Lymphocyte Activation , Concanavalin A/metabolism
7.
Langmuir ; 40(15): 7974-7981, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38564230

ABSTRACT

An electrochemical impedimetric biosensing platform with lectin as a molecular recognition element has been established for the sensitive detection of glycoproteins, a class of important biomarkers in clinical diagnosis. One of the representative metal-organic framework materials, MIL-101(Cr)-NH2, was utilized as the supporting matrix, and its amino groups served as the anchors to immobilize the lectins of concanavalin A (Con A), constituting Con A@MIL-101(Cr)-NH2 for the determination of invertase (INV) as a model glycoprotein. The Con A concentration, immobilization time, and incubation time with INV were optimized. Under the optimal conditions, the degree of impedance increase was linearly proportional to the logarithm of INV concentration between 1.0 × 10-16 and 1.0 × 10-11 M, affording a limit of detection as low as 3.98 × 10-18 M. Good specificity, stability, reproducibility, and repeatability were demonstrated for the fabricated biosensing platform. Moreover, real mouse serum samples were spiked with different concentrations of INV. Excellent recoveries were obtained, which demonstrated the biosensing platform's capability of analyzing glycoproteins within a complex matrix.


Subject(s)
Biosensing Techniques , Metal-Organic Frameworks , Animals , Mice , Concanavalin A , Metal-Organic Frameworks/chemistry , Reproducibility of Results , Lectins/chemistry , Glycoproteins , Electrochemical Techniques , Limit of Detection
8.
J Vis Exp ; (205)2024 Mar 01.
Article in English | MEDLINE | ID: mdl-38497648

ABSTRACT

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.


Subject(s)
Chromatin , Satellite Cells, Skeletal Muscle , Animals , Mice , Chromatin Immunoprecipitation , Biological Assay , Concanavalin A
9.
Hepatol Commun ; 8(4)2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38466881

ABSTRACT

BACKGROUND: Autoimmune hepatitis (AIH) is an immune-mediated liver disease of unknown etiology accompanied by intestinal dysbiosis and a damaged intestinal barrier. Berberine (BBR) is a traditional antibacterial medicine that has a variety of pharmacological properties. It has been reported that BBR alleviates AIH, but relevant mechanisms remain to be fully explored. METHODS: BBR was orally administered at doses of 100 mg⋅kg-1⋅d-1 for 7 days to mice before concanavalin A-induced AIH model establishment. Histopathological, immunohistochemical, immunofluorescence, western blotting, ELISA, 16S rRNA analysis, flow cytometry, real-time quantitative PCR, and fecal microbiota transplantation studies were performed to ascertain BBR effects and mechanisms in AIH mice. RESULTS: We found that liver necrosis and apoptosis were decreased upon BBR administration; the levels of serum transaminase, serum lipopolysaccharide, liver proinflammatory factors TNF-α, interferon-γ, IL-1ß, and IL-17A, and the proportion of Th17 cells in spleen cells were all reduced, while the anti-inflammatory factor IL-10 and regulatory T cell proportions were increased. Moreover, BBR treatment increased beneficial and reduced harmful bacteria in the gut. BBR also strengthened ileal barrier function by increasing the expression of the tight junction proteins zonula occludens-1 and occludin, thereby blocking lipopolysaccharide translocation, preventing lipopolysaccharide/toll-like receptor 4 (TLR4)/ NF-κB pathway activation, and inhibiting inflammatory factor production in the liver. Fecal microbiota transplantation from BBR to model mice also showed that BBR potentially alleviated AIH by altering the gut microbiota. CONCLUSIONS: BBR alleviated concanavalin A-induced AIH by modulating the gut microbiota and related immune regulation. These results shed more light on potential BBR therapeutic strategies for AIH.


Subject(s)
Berberine , Gastrointestinal Microbiome , Hepatitis A , Hepatitis, Autoimmune , Mice , Animals , Hepatitis, Autoimmune/drug therapy , Hepatitis, Autoimmune/etiology , Berberine/pharmacology , Berberine/therapeutic use , Concanavalin A/pharmacology , Lipopolysaccharides/pharmacology , RNA, Ribosomal, 16S
10.
Chem Biodivers ; 21(5): e202400283, 2024 May.
Article in English | MEDLINE | ID: mdl-38485665

ABSTRACT

Fifteen bibenyls and four fluorenones, including five new bibenzyl-phenylpropane hybrids, were isolated from the aerial part of Dendrobium nobile Lindl. Their structures were determined by spectroscopic methods. Bioassay on the LPS-induced proliferations of mouse splenic B lymphocytes, and Con A-induced T lymphocytes showed that compounds 1, 2, and 14 showed excellent immunosuppressive activities with IC50 values of 1.23, 1.01, and 3.87 µM, respectively, while compounds 3-4, 7, 10, 13, and 15 exhibited moderate immunosuppressive activities with IC50 values ranging from 6.89 to 14.2 µM.


Subject(s)
Bibenzyls , Cell Proliferation , Dendrobium , Immunosuppressive Agents , Dendrobium/chemistry , Animals , Mice , Immunosuppressive Agents/pharmacology , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/isolation & purification , Bibenzyls/chemistry , Bibenzyls/pharmacology , Bibenzyls/isolation & purification , Cell Proliferation/drug effects , T-Lymphocytes/drug effects , B-Lymphocytes/drug effects , Molecular Structure , Structure-Activity Relationship , Lipopolysaccharides/pharmacology , Lipopolysaccharides/antagonists & inhibitors , Dose-Response Relationship, Drug , Concanavalin A/antagonists & inhibitors , Concanavalin A/pharmacology
11.
J Hepatol ; 80(2): 194-208, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38438948

ABSTRACT

BACKGROUND & AIMS: Hepatocyte apoptosis, a well-defined form of cell death in non-alcoholic steatohepatitis (NASH), is considered the primary cause of liver inflammation and fibrosis. However, the mechanisms underlying the regulation of hepatocyte apoptosis in NASH remain largely unclear. We explored the anti-apoptotic effect of hepatocyte CD1d in NASH. METHODS: Hepatocyte CD1d expression was analyzed in patients with NASH and mouse models. Hepatocyte-specific gene overexpression or knockdown and anti-CD1d crosslinking were used to investigate the anti-apoptotic effect of hepatocyte CD1d on lipotoxicity-, Fas-, and concanavalin (ConA)-mediated liver injuries. A high-fat diet, a methionine-choline-deficient diet, a Fas agonist, and ConA were used to induce lipotoxic and/or apoptotic liver injuries. Palmitic acid was used to mimic lipotoxicity-induced apoptosis in vitro. RESULTS: We identified a dramatic decrease in CD1d expression in hepatocytes of patients with NASH and mouse models. Hepatocyte-specific CD1d overexpression and knockdown experiments collectively demonstrated that hepatocyte CD1d protected against hepatocyte apoptosis and alleviated hepatic inflammation and injuries in NASH mice. Furthermore, decreased JAK2-STAT3 signaling was observed in NASH patient livers. Mechanistically, anti-CD1d crosslinking on hepatocytes induced tyrosine phosphorylation of the CD1d cytoplasmic tail, leading to the recruitment and phosphorylation of JAK2. Phosphorylated JAK2 activated STAT3 and subsequently reduced apoptosis in hepatocytes, which was associated with an increase in anti-apoptotic effectors (Bcl-xL and Mcl-1) and a decrease in pro-apoptotic effectors (cleaved-caspase 3/7). Moreover, anti-CD1d crosslinking effectively protected against Fas- or ConA-mediated hepatocyte apoptosis and liver injury in mice. CONCLUSIONS: Our study uncovered a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 axis in hepatocytes that conferred hepatoprotection and highlighted the potential of hepatocyte CD1d-directed therapy for liver injury and fibrosis in NASH, as well as in other liver diseases associated with hepatocyte apoptosis. IMPACT AND IMPLICATIONS: Excessive and/or sustained hepatocyte apoptosis is critical in driving liver inflammation and injury. The mechanisms underlying the regulation of hepatocyte apoptosis in non-alcoholic steatohepatitis (NASH) remain largely unclear. Here, we found that CD1d expression in hepatocytes substantially decreases and negatively correlates with the severity of liver injury in patients with NASH. We further revealed a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 signaling axis in hepatocytes, which confers significant protection against liver injury in NASH and acute liver diseases. Thus, hepatocyte CD1d-targeted therapy could be a promising strategy to manipulate liver injury in both NASH and other hepatocyte apoptosis-related liver diseases.


Subject(s)
Non-alcoholic Fatty Liver Disease , Animals , Humans , Mice , Apoptosis , Concanavalin A , Disease Models, Animal , Hepatocytes , Inflammation
12.
Antiviral Res ; 225: 105856, 2024 May.
Article in English | MEDLINE | ID: mdl-38447646

ABSTRACT

Four years after its outbreak, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global challenge for human health. At its surface, SARS-CoV-2 features numerous extensively glycosylated spike proteins. This glycan coat supports virion docking and entry into host cells and at the same time renders the virus less susceptible to neutralizing antibodies. Given the high genetic plasticity of SARS-CoV-2 and the rapid emergence of immune escape variants, targeting the glycan shield by carbohydrate-binding agents emerges as a promising strategy. However, the potential of carbohydrate-targeting reagents as viral inhibitors remains underexplored. Here, we tested seven plant-derived carbohydrate-binding proteins, called lectins, and one crude plant extract for their antiviral activity against SARS-CoV-2 in two types of human lung cells: A549 cells ectopically expressing the ACE2 receptor and Calu-3 cells. We identified three lectins and an Allium porrum (leek) extract inhibiting SARS-CoV-2 infection in both cell systems with selectivity indices (SI) ranging between >2 and >299. Amongst these, the lectin Concanavalin A (Con A) exerted the most potent and broad activity against a panel of SARS-CoV-2 variants. We used multiplex super-resolution microscopy to address lectin interactions with SARS-CoV-2 and its host cells. Notably, we discovered that Con A not only binds to SARS-CoV-2 virions and their host cells, but also causes SARS-CoV-2 aggregation. Thus, Con A exerts a dual mode-of-action comprising both, antiviral and virucidal, mechanisms. These results establish Con A and other plant lectins as candidates for COVID-19 prevention and basis for further drug development.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Onions/metabolism , Concanavalin A/metabolism , Lectins/metabolism , Polysaccharides , Antiviral Agents/pharmacology , Plant Extracts , Spike Glycoprotein, Coronavirus
13.
J Pharm Biomed Anal ; 242: 116022, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38354538

ABSTRACT

Human chorionic gonadotropin (hCG) is constituted of the hCGα and hCGß subunits and is a highly glycosylated protein. Affinity supports based on immobilized Concanavalin A (Con A) lectin were used in solid phase extraction (SPE) to fractionate the hCG glycoforms according to their glycosylation state. For the first time, the lectin SPE fractions were off-line analysed by a nano liquid chromatography - high-resolution mass spectrometry (nanoLC-HRMS) method keeping the glycoforms intact. For this, home-made Con A sorbents were prepared by immobilizing lectin on Sepharose with a mean grafting yield of 98.2% (relative standard deviation (RSD) of 3.5%, n = 15). A capacity of about 100 µg of purified urinary hCG (uhCG) per ml of sorbent, grafted with a density of 10 mg of Con A per ml, was estimated. Average extraction yields of around 60% for both hCGα and hCGß glycoforms were obtained after optimization of the extraction protocol. Intra- and inter-assay evaluation led to average RSD values of around 10%, indicating a repeatable extraction procedure. Similar results were obtained with commercial Con A-based sorbents but only after their 3rd use or after an extensive pre-conditioning step. Finally, the Con A SPE led to the fractionation of some glycoforms of uhCG, allowing the detection of an hCGα glycoform with two tetra-antennary N-glycans that couldn't be detected by direct analysis in nanoLC-HRMS without Con A SPE. Regarding a recombinant hCG, a fractionation was also observed leading to the detection of unretained hCGα glycoforms with tri-antennary N-glycans. Therefore, the combination of lectin SPE with intact protein analysis by nanoLC-HRMS can contribute to a more detailed glycosylation characterization of the hCG protein.


Subject(s)
Chorionic Gonadotropin , Lectins , Humans , Chorionic Gonadotropin/analysis , Concanavalin A , Chorionic Gonadotropin, beta Subunit, Human/chemistry , Mass Spectrometry , Polysaccharides/analysis , Chromatography
14.
Carbohydr Res ; 536: 109050, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38335804

ABSTRACT

Lectin Con A, with specificity to interact with α-d-mannopyranoside, achieves tight binding affinity with the aid of optimal multivalent ligand valencies, distances and orientations between the ligands. A series of synthetic arabinomannans, possessing arabinan core and mannan at the non-reducing ends, is studied to assess the above constraints involved with lectin binding in this report. Trisaccharides, with (1 â†’ 2)(1 â†’ 3), (1 â†’ 2)(1 â†’ 5) and (1 â†’ 3)(1 â†’ 5) glycosidic bond connectivities, and a pentasaccharide with mannopyranosides at the non-reducing ends are synthesized. The binding affinities of the mannose bivalent ligands are studied with tetrameric Con A lectin by isothermal titration calorimetry (ITC). Among the derivatives, trisaccharide with (1 â†’ 2)(1 â†’ 3) glycosidic bond connectivity and the pentasaccharide undergo lectin interaction, clearly fulfilling the bivalent structural and functional valencies. Remaining oligosaccharides exhibit only a functional monovalency, defying the bivalent structural valency. The trisaccharide fulfilling the structural and functional valencies represent the smallest bivalent ligand, undergoing the lectin interaction in a trans-mode.


Subject(s)
Lectins , Mannans , Lectins/chemistry , Ligands , Concanavalin A/chemistry , Mannose/chemistry , Glycosides/chemistry , Oligosaccharides , Trisaccharides , Protein Binding
15.
Int Immunopharmacol ; 129: 111618, 2024 Mar 10.
Article in English | MEDLINE | ID: mdl-38354508

ABSTRACT

BACKGROUND: Acute hepatitis is a progressive inflammatory disorder that can lead to liver failure. Endothelial permeability is the vital pathophysiological change involved in infiltrating inflammatory factors. DDX24 has been implicated in immune signaling. However, the precise role of DDX24 in immune-mediated hepatitis remains unclear. Here, we investigate the phenotype of endothelium-targeted Ddx24 conditional knockout mice with Concanavalin A (ConA)-induced hepatitis. METHODS: Mice with homozygous endothelium-targeted Ddx24 conditional knockout (Ddx24flox/flox; Cdh5-Cre+) were established using the CRISPR/Cas9 mediated Cre-loxP system. We investigated the biological functions of endothelial cells derived from transgenic mice and explored the effects of Ddx24 in mice with ConA-induced hepatitis in vivo. The mass spectrometry was performed to identify the differentially expressed proteins in liver tissues of transgenic mice. RESULT: We successfully established mice with endothelium-targeted Ddx24 conditional knockout. The results showed migration and tube formation potentials of murine aortic endothelial cells with DDX24 silencing were significantly promoted. No differences were observed between Ddx24flox/flox; Cdh5-Cre+ and control regarding body weight and length, pathological tissue change and embryogenesis. We demonstrated Ddx24flox/flox; Cdh5-Cre+ exhibited exacerbation of ConA-induced hepatitis by up-regulating TNF-α and IFN-γ. Furthermore, endothelium-targeted Ddx24 conditional knockout caused vascular hyper-permeability in ConA-injected mice by down-regulating vascular integrity-associated proteins. Mechanistically, we identified Ddx24 might regulate immune-mediated hepatitis by inflammation-related permeable barrier pathways. CONCLUSION: These findings prove that endothelium-targeted Ddx24 conditional knockout exacerbates ConA-induced hepatitis in mice because of vascular hyper-permeability. The findings indicate a crucial role of DDX24 in regulating immune-mediated hepatitis, suggesting DDX24 as a potential therapeutic target in the disorder.


Subject(s)
Endothelial Cells , Hepatitis , Animals , Mice , Concanavalin A/toxicity , Endothelial Cells/metabolism , Endothelium/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic
16.
J Leukoc Biol ; 115(6): 1070-1083, 2024 May 29.
Article in English | MEDLINE | ID: mdl-38366630

ABSTRACT

FICZ (6-formylindolo[3,2-b]carbazole) is a potent aryl hydrocarbon receptor agonist that has a poorly understood function in the regulation of inflammation. In this study, we investigated the effect of aryl hydrocarbon receptor activation by FICZ in a murine model of autoimmune hepatitis induced by concanavalin A. High-throughput sequencing techniques such as single-cell RNA sequencing and assay for transposase accessible chromatin sequencing were used to explore the mechanisms through which FICZ induces its effects. FICZ treatment attenuated concanavalin A-induced hepatitis, evidenced by decreased T-cell infiltration, decreased circulating alanine transaminase levels, and suppression of proinflammatory cytokines. Concanavalin A revealed an increase in natural killer T cells, T cells, and mature B cells upon concanavalin A injection while FICZ treatment reversed the presence of these subsets. Surprisingly, concanavalin A depleted a subset of CD55+ B cells, while FICZ partially protected this subset. The immune cells showed significant dysregulation in the gene expression profiles, including diverse expression of migratory markers such as CCL4, CCL5, and CXCL2 and critical regulatory markers such as Junb. Assay for transposase accessible chromatin sequencing showed more accessible chromatin in the CD3e promoter in the concanavalin A-only group as compared to the naive and concanavalin A-exposed, FICZ-treated group. While there was overall more accessible chromatin of the Adgre1 (F4/80) promoter in the FICZ-treated group, we observed less open chromatin in the Itgam (CD11b) promoter in Kupffer cells, supporting the ability of FICZ to reduce the infiltration of proinflammatory cytokine producing CD11b+ Kupffer cells. Taken together, these data demonstrate that aryl hydrocarbon receptor activation by FICZ suppresses liver injury through the limitation of CD3+ T-cell activation and CD11b+ Kupffer cell infiltration.


Subject(s)
CD11b Antigen , Carbazoles , Concanavalin A , Kupffer Cells , Lymphocyte Activation , Receptors, Aryl Hydrocarbon , T-Lymphocytes , Animals , Concanavalin A/pharmacology , Carbazoles/pharmacology , Kupffer Cells/metabolism , Kupffer Cells/drug effects , Kupffer Cells/pathology , Lymphocyte Activation/drug effects , Receptors, Aryl Hydrocarbon/metabolism , Mice , CD11b Antigen/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Hepatitis, Autoimmune/pathology , Hepatitis, Autoimmune/drug therapy , Hepatitis, Autoimmune/immunology , Hepatitis, Autoimmune/metabolism , Hepatitis, Autoimmune/etiology , Mice, Inbred C57BL , Ligands , Male , Cytokines/metabolism
17.
Arch Microbiol ; 206(1): 54, 2024 Jan 05.
Article in English | MEDLINE | ID: mdl-38180520

ABSTRACT

Pseudomonas aeruginosa is an opportunistic bacterium that can form a biofilm with the ability to colonize different surfaces and for increasing resistance to antibiotics. An alternative to solve this problem may be the use of non-glucose/mannose glycosylated proteins from Melipona beecheii honey, which are capable of inhibiting the growth of this pathogen. In this work, the antibiofilm activity of the conA-unbound protein fraction (F1) from M. beecheii was evaluated. The crude protein extract (CPE) and the F1 fraction inhibited the P. aeruginosa biofilm growth above 80% at 4 and 1.3 µg/mL, respectively. These proteins affected the structure of the biofilm, as well as fleQ and fleR gene expressions involved in the formation and regulation of the P. aeruginosa biofilm. The results demonstrated that the F1 fraction proteins of M. beecheii honey inhibit and affect the formation of the P. aeruginosa biofilm.


Subject(s)
Honey , Pseudomonas Infections , Bees , Animals , Pseudomonas aeruginosa/genetics , Anti-Bacterial Agents/pharmacology , Biofilms , Concanavalin A
18.
Article in English | MEDLINE | ID: mdl-38199060

ABSTRACT

In this study, a precursor carboxy-silica support was demonstrated in the immobilization of two different lectins, namely concanavalin A (Con A) and wheat germ agglutinin (WGA) for use in high performance lectin affinity chromatography (LAC) for the selective capturing and enrichment of glycoproteins from healthy/disease free and cancer human sera. The lectin columns thus obtained (i.e., Con A- and WGA-columns) showed no nonspecific interactions toward some chosen standard glycoproteins and non-glycoproteins. Both columns were shown in sub-glycoproteomics enrichment from human sera including disease free and adenocarcinoma cancer sera. The collected fractions were subjected to LC-MS/MS for identification of the captured glycoproteins, whereby the total number of identified proteins using Con A column from disease-free and cancer sera were 164 and 188, respectively while 133 and 103 proteins were identified in the fractions captured by the WGA column from disease-free and cancer sera samples, respectively. Differentially expressed proteins (DEPs) between the disease free and cancer sera in both the Con A and WGA column fractions were identified via the plot of the abundance vs. the protein ratio whereby the binary logarithm of average intensities of cancer and disease free sera were plotted against the binary logarithm of cancer/disease free sera ratios. The proteins that exhibit log 2 (cancer/healthy) ratio values greater than +2 and less than -2 in both categories are considered as DEPs. Furthermore, for visualization of the data arrangement, Q-Q scatterplot were also used whereby the binary logarithm of cancer serum was plotted against the binary logarithm of disease-free serum for both Con A and WGA. For Con A column, 28 up-regulated and 10 down regulated proteins were identified with a total of 38 DEPs while only two being non-glycoproteins. Furthermore, the up-regulated, and down regulated proteins recorded for WGA column are 14 and 6, respectively, totaling 20 proteins including 3 non-glycoproteins. Some of the non-specific binding to lectin are most likely due to protein-protein interactions.


Subject(s)
Lectins , Neoplasms , Humans , Lectins/chemistry , Chromatography, Liquid/methods , Silicon Dioxide/chemistry , Tandem Mass Spectrometry , Glycoproteins/chemistry , Concanavalin A , Chromatography, Affinity/methods , Wheat Germ Agglutinins/chemistry
19.
Bioorg Chem ; 144: 107145, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38278050

ABSTRACT

Hyperadamans A-G (1-7), seven new adamantane type polycyclic polyprenylated acylphloroglucinols (PPAPs), were isolated from Hypericum wilsonii N. Robson. Structurally, 1-4 were the first adamantanes bearing an unusual 2,7-dioxabicyclo-[2.2.1]-heptane fragment, and compound 5 was the first adamantane with a rare 1,6-dioxaspiro[4.4]nonane section. Importantly, 1-7 exhibited significant immunosuppressive activity on Con A-induced T-lymphocyte proliferation in vitro, with IC50 values ranging from 3.97 ± 0.10 to 18.12 ± 1.07 µM. Pretreatment with 1 in Con A-challenged autoimmune hepatitis mice could dramatically ameliorate the levels of hepatic injury indexes (ALT and AST) and reduce the product of proinflammatory cytokines (COX-2, IL-6, IL-1ß, IL-18, IL-23A and TNF-α). Furthermore, the protective effect of 1 on the Con A-induced liver injury was corroborated by the histological analysis and the immunohistochemistry.


Subject(s)
Adamantane , Hepatitis, Autoimmune , Mice , Animals , Concanavalin A , Hepatitis, Autoimmune/drug therapy , Hepatitis, Autoimmune/prevention & control , Adamantane/pharmacology , Adamantane/chemistry , Cytokines , Tumor Necrosis Factor-alpha , Molecular Structure
20.
ACS Appl Mater Interfaces ; 16(5): 5536-5547, 2024 Feb 07.
Article in English | MEDLINE | ID: mdl-38267397

ABSTRACT

Autoimmune hepatitis (AIH) is a severe immune-mediated inflammatory liver disease whose standard of care is immunosuppressive treatment with inevitable undesired outcomes. Macrophage is acknowledged to aggravate liver damage, providing a promising AIH therapeutic target. Accordingly, in this study, a kind of curdlan-decorated fullerene nanoparticle (Cur-F) is fabricated to alleviate immune-mediated hepatic injury for treating AIH via reducing macrophage infiltration in a concanavalin A (Con A)-induced AIH mouse model. After intravenous administration, Cur-F primarily distributes in liver tissues, efficiently eliminates the excessive reactive oxygen species, significantly attenuates oxidative stress, and subsequently suppresses the nuclear factor kappa-B-gene binding (NF-κB) signal pathway, resulting in the lowered production of pro-inflammatory cytokines and the balancing of the immune homeostasis with the prevention of macrophage infiltration in the liver. The regulation of hepatic inflammation contributes to inhibiting inflammatory cytokines-induced hepatocyte apoptosis, decreasing the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) contents and thus ameliorating immune-mediated hepatic injury. Notably, there is no detectable toxicity to the body. Our findings may open up novel avenues for AIH based on curdlan and fullerene materials.


Subject(s)
Fullerenes , Hepatitis, Autoimmune , beta-Glucans , Animals , Mice , Hepatitis, Autoimmune/drug therapy , Hepatitis, Autoimmune/metabolism , Fullerenes/pharmacology , Fullerenes/therapeutic use , Fullerenes/metabolism , Liver/metabolism , Cytokines/metabolism , NF-kappa B/metabolism , Concanavalin A , Macrophages/metabolism
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