scholarly journals The METTL3/MALAT1/PTBP1/USP8/TAK1 axis promotes pyroptosis and M1 polarization of macrophages and contributes to liver fibrosis

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Bo Shu ◽  
Ying-Xia Zhou ◽  
Hao Li ◽  
Rui-Zhi Zhang ◽  
Chao He ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Western Blot ◽  
Rna Stability ◽  
Interferon Γ ◽  
In Vivo Model ◽  
Loss Of Function ◽  
M1 Polarization ◽  
The Impact

AbstractPro-inflammatory M1 macrophages, via activating hepatic stellate cells, contribute to liver fibrosis. In this study, we examined the mechanism and the significance of a signaling axis, METTL3/MALAT1/PTBP1/USP8/TAK1, in regulating pyroptosis and M1 polarization of hepatic macrophages. Liver fibrosis model was established in vivo by CCl4 treatment; M1 polarization was induced in vitro by treating macrophages with lipopolysaccharide or interferon γ. Expressions of METTL3, MALAT1, PTBP1, USP8, and TAK1 were measured by RT-PCR and/or Western blot in Kupffer cells (KCs) isolated from in vivo model or in vitro activated macrophages. Macrophage phenotypes including inflammation (RT-qPCR analysis of a panel of proinflammatory cytokines and ELISA on productions of interleukin (IL)−1β and IL-18) and pyroptosis (Western blot of NLRP3, Caspase-1, and GSDMD) were investigated. The impact of METTL3 on m6A methylation of MALAT1 was examined by methylated RNA immunoprecipitation (RIP), the interaction between PTBP1 and MALAT1 or USP8 mRNA by combining RNA pull-down, RIP, and RNA stability assays, and the crosstalk between USP8 and TAK1 by co-immunoprecipitation and protein degradation assays. Functional significance of individual component of METTL3/MALAT1/PTBP1/USP8/TAK1 axis was assessed by combining gain-of-function and loss-of-function approaches. In KCs isolated from in vivo liver fibrosis model or in vitro M1-polarized macrophages, METTL3 was up-regulated, and sequentially, it increased MALAT1 level via m6A methylation, which promoted USP8 mRNA degradation through the interaction with PTBP1. Reduced USP8 expression regulated the ubiquitination and protein stability of TAK1, which promoted pyroptosis and inflammation of macrophages. The signaling cascade METTL3/MALAT1/PTBP1/USP8/TAK1, by essentially stimulating pyroptosis and inflammation of macrophages, aggravates liver fibrosis. Therefore, targeting individual components of this axis may benefit the treatment of liver fibrosis.

scholarly journals Impact of the acidic environment on gene expression and functional parameters of tumors in vitro and in vivo

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Mandy Rauschner ◽  
Luisa Lange ◽  
Thea Hüsing ◽  
Sarah Reime ◽  
Alexander Nolze ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Death ◽  
Western Blot ◽  
Tumor Cell ◽  
Low Ph ◽  
Strong Increase ◽  
Acidic Ph ◽  
The Impact

Abstract Background The low extracellular pH (pHe) of tumors resulting from glycolytic metabolism is a stress factor for the cells independent from concomitant hypoxia. The aim of the study was to analyze the impact of acidic pHe on gene expression on mRNA and protein level in two experimental tumor lines in vitro and in vivo and were compared to hypoxic conditions as well as combined acidosis+hypoxia. Methods Gene expression was analyzed in AT1 prostate and Walker-256 mammary carcinoma of the rat by Next Generation Sequencing (NGS), qPCR and Western blot. In addition, the impact of acidosis on tumor cell migration, adhesion, proliferation, cell death and mitochondrial activity was analyzed. Results NGS analyses revealed that 147 genes were uniformly regulated in both cell lines (in vitro) and 79 genes in both experimental tumors after 24 h at low pH. A subset of 25 genes was re-evaluated by qPCR and Western blot. Low pH consistently upregulated Aox1, Gls2, Gstp1, Ikbke, Per3, Pink1, Tlr5, Txnip, Ypel3 or downregulated Acat2, Brip1, Clspn, Dnajc25, Ercc6l, Mmd, Rif1, Zmpste24 whereas hypoxia alone led to a downregulation of most of the genes. Direct incubation at low pH reduced tumor cell adhesion whereas acidic pre-incubation increased the adhesive potential. In both tumor lines acidosis induced a G1-arrest (in vivo) of the cell cycle and a strong increase in necrotic cell death (but not in apoptosis). The mitochondrial O2 consumption increased gradually with decreasing pH. Conclusions These data show that acidic pHe in tumors plays an important role for gene expression independently from hypoxia. In parallel, acidosis modulates functional properties of tumors relevant for their malignant potential and which might be the result of pH-dependent gene expression.

scholarly journals Liver X Receptor Nuclear Receptors Are Transcriptional Regulators of Dendritic Cell Chemotaxis

2018 ◽  
Vol 38 (10) ◽  
Author(s):  
Susana Beceiro ◽  
Attila Pap ◽  
Zsolt Czimmerer ◽  
Tamer Sallam ◽  
Jose A. Guillén ◽  
...  
Keyword(s):  
Nuclear Receptors ◽  
Low Density Lipoprotein ◽  
Myeloid Cells ◽  
Density Lipoprotein ◽  
Loss Of Function ◽  
Transcriptional Responses ◽  
Cd38 Expression ◽  
The Impact

ABSTRACTThe liver X receptors (LXRs) are ligand-activated nuclear receptors with established roles in the maintenance of lipid homeostasis in multiple tissues. LXRs exert additional biological functions as negative regulators of inflammation, particularly in macrophages. However, the transcriptional responses controlled by LXRs in other myeloid cells, such as dendritic cells (DCs), are still poorly understood. Here we used gain- and loss-of-function models to characterize the impact of LXR deficiency on DC activation programs. Our results identified an LXR-dependent pathway that is important for DC chemotaxis. LXR-deficient mature DCs are defective in stimulus-induced migrationin vitroandin vivo. Mechanistically, we show that LXRs facilitate DC chemotactic signaling by regulating the expression of CD38, an ectoenzyme important for leukocyte trafficking. Pharmacological or genetic inactivation of CD38 activity abolished the LXR-dependent induction of DC chemotaxis. Using the low-density lipoprotein receptor-deficient (LDLR−/−) LDLR−/−mouse model of atherosclerosis, we also demonstrated that hematopoietic CD38 expression is important for the accumulation of lipid-laden myeloid cells in lesions, suggesting that CD38 is a key factor in leukocyte migration during atherogenesis. Collectively, our results demonstrate that LXRs are required for the efficient emigration of DCs in response to chemotactic signals during inflammation.

Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits fibrogenesis in an in vivo model of liver fibrosis

1999 ◽  
Vol 117 (5) ◽  
pp. 1198-1204 ◽  
Author(s):  
Jianliang Zhu ◽  
Jian Wu ◽  
Edward Frizell ◽  
Shu-Ling Liu ◽  
Reza Bashey ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Liver Fibrosis ◽  
Hepatic Stellate Cell ◽  
Stellate Cell ◽  
In Vivo Model ◽  
Proliferation In Vitro

scholarly journals The Effect of Lecithins Coupled Decorin Nanoliposomes on Treatment of Carbon Tetrachloride-Induced Liver Fibrosis

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Guojun Chen ◽  
Yiping Zhu ◽  
Xiao Liang ◽  
Xianfa Wang ◽  
Weihua Yu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Carbon Tetrachloride ◽  
Bile Duct ◽  
Liver Fibrosis ◽  
Western Blot ◽  
Immunofluorescence Staining ◽  
Type I ◽  
Masson Trichrome Staining

This study aimed to investigate the effect of bile duct-targeting lecithins- (PC-) coupled decorin (DCN) (PC-DCN) nanoliposomes against liver fibrosis in vitro and in vivo. We prepared PC-DCN nanoliposomes by using rat astrocytes, HSC-T6, to verify the antifibrosis effect of PC-DCN in vitro. First, we established a rat model of carbon tetrachloride-induced fibrosis. PC-DCN nanoliposomes were then injected into fibrotic rats via the portal vein or bile duct. The EdU assay was performed to analyze cell proliferation. Immunofluorescence staining was used to detect α-smooth muscle actin (α-SMA) expression. Western blot was performed to examine the expression of α-SMA, collagen type I alpha 1 (COL1A1), and transforming growth factor-β (TGF-β) protein. The levels of aspartate transaminase (AST), alanine transaminase (ALT), and total bilirubin (TBIL) were examined by enzyme-linked immunosorbent assay (ELISA) analysis. Hematoxylin and eosin (H&E) staining and Masson trichrome staining were used to determine liver tissue lesions and liver fibrosis. Compared with TGF-β group, PC-DCN treatment could significantly reduce cell proliferation. Western blot analysis indicated that the expression of α-SMA, COL1A1, and TGF-β was downregulated after treatment with PC-DCN in vitro and in vivo. Immunofluorescence staining confirmed that α-SMA expression was reduced by PC-DCN. Furthermore, H&E staining and Masson trichrome staining showed that the administration of PC-DCN nanoliposomes via the bile duct could reduce the extent of liver fibrosis. PCR analysis showed that PC-DCN administration could reduce proinflammatory cytokines IL-6, TNF-α, and IL-1β expression via the bile duct. The administration of PC-DCN nanoliposomes also significantly downregulated liver function indicators ALT, AST, and TBIL. The results of our study indicated that PC-DCN could effectively reduce the extent of liver fibrosis.

scholarly journals Degradation of Mitochondria and Oxidative Stress as the Main Mechanism of Toxicity of Pristine Graphene on U87 Glioblastoma Cells and Tumors and HS-5 Cells

2019 ◽  
Vol 20 (3) ◽  
pp. 650 ◽  
Author(s):  
Sławomir Jaworski ◽  
Barbara Strojny ◽  
Ewa Sawosz ◽  
Mateusz Wierzbicki ◽  
Marta Grodzik ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Chorioallantoic Membrane ◽  
In Vivo Model ◽  
Pristine Graphene ◽  
Infrared Measurements ◽  
The Impact

Due to the development of nanotechnologies, graphene and graphene-based nanomaterials have attracted immense scientific interest owing to their extraordinary properties. Graphene can be used in many fields, including biomedicine. To date, little is known about the impact graphene may have on human health in the case of intentional exposure. The present study was carried out on U87 glioma cells and non-cancer HS-5 cell lines as in vitro model and U87 tumors cultured on chicken embryo chorioallantoic membrane as in vivo model, on which the effects of pristine graphene platelets (GPs) were evaluated. The investigation consisted of structural analysis of GPs using transmission electron microscopy, Fourier transmission infrared measurements, zeta potential measurements, evaluation of cell morphology, assessment of cell viability, investigation of reactive oxygen species production, and investigation of mitochondrial membrane potential. The toxicity of U87 glioma tumors was evaluated by calculating the weight and volume of tumors and performing analyses of the ultrastructure, histology, and protein expression. The in vitro results indicate that GPs have dose-dependent cytotoxicity via ROS overproduction and depletion of the mitochondrial membrane potential. The mass and volume of tumors were reduced in vivo after injection of GPs. Additionally, the level of apoptotic and necrotic markers increased in GPs-treated tumors.

scholarly journals Alpha-Synuclein Aggregation Induced by Brief Ischemia Negatively Impacts Neuronal Survival in vivo: A Study in [A30P]alpha-Synuclein Transgenic Mouse

2010 ◽  
Vol 31 (3) ◽  
pp. 913-923 ◽  
Author(s):  
Isin Unal-Cevik ◽  
Yasemin Gursoy-Ozdemir ◽  
Muge Yemisci ◽  
Sevda Lule ◽  
Gunfer Gurer ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Neuronal Survival ◽  
Alpha Synuclein ◽  
In Vivo Model ◽  
Neuronal Necrosis ◽  
Mca Occlusion ◽  
Nitrative Stress ◽  
The Impact

Alpha-synuclein oligomerization and aggregation are considered to have a role in the pathogenesis of neurodegenerative diseases. However, despite numerous in vitro studies, the impact of aggregates in the intact brain is unclear. In vitro, oxidative/nitrative stress and acidity induce α-synuclein oligomerization. These conditions favoring α-synuclein fibrillization are present in the ischemic brain, which may serve as an in vivo model to study α-synuclein aggregation. In this study, we show that 30-minute proximal middle cerebral artery (MCA) occlusion and 72 hours reperfusion induce oligomerization of wild-type α-synuclein in the ischemic mouse brain. The nonamyloidogenic isoform β-synuclein did not form oligomers. Alpha-synuclein aggregates were confined to neurons and colocalized with ubiquitin immunoreactivity. We also found that 30 minutes proximal MCA occlusion and 24 hours reperfusion induced larger infarcts in C57BL/6(Thy1)-h[A30P]alphaSYN transgenic mice, which have an increased tendency to form synuclein fibrils. Trangenics also developed more selective neuronal necrosis when subjected to 20 minutes distal MCA occlusion and 72 hours reperfusion. Enhanced 3-nitrotyrosine immunoreactivity in transgenic mice suggests that oxidative/nitrative stress may be one of the mechanisms mediating aggregate toxicity. Thus, the increased vulnerability of transgenic mice to ischemia suggests that α-synuclein aggregates not only form during ischemia but also negatively impact neuronal survival, supporting the idea that α-synuclein misfolding may be neurotoxic.

scholarly journals The In Vitro and In Vivo Effects of Hypoxis hemerocallidea on Indinavir Pharmacokinetics: Modulation of Efflux

Planta Medica ◽  
2018 ◽  
Vol 84 (12/13) ◽  
pp. 895-901
Author(s):  
Kaylee Havenga ◽  
Efrem Abay ◽  
Lubbe Wiesner ◽  
Alvaro Viljoen ◽  
Dewald Steyn ◽  
...  
Keyword(s):  
Aqueous Extract ◽  
Plant Material ◽  
Commercial Product ◽  
In Vivo Model ◽  
Cell Monolayers ◽  
Reference Plant ◽  
The Impact ◽  
Hypoxis Hemerocallidea

Abstract Hypoxis hemerocallidea (African potato) is a popular medicinal plant that has been used traditionally for the treatment of various disorders. Some HIV/AIDS patients use this traditional medicine together with their antiretroviral therapy. This study aimed to determine the impact of selected H. hemerocallidea materials (i.e., a commercial product, an aqueous extract, and biomass reference plant material) on the bidirectional permeability of indinavir across Caco-2 cell monolayers as well as the bioavailability of indinavir during an acute, single administration study in Sprague-Dawley rats. All of the selected H. hemerocallidea test materials demonstrated inhibition effects on indinavir efflux across Caco-2 cell monolayers, albeit to different extents. An increase in the bioavailability of indinavir was obtained in vivo when administered concomitantly with the H. hemerocallidea materials, albeit not statistically significantly. The change in bioavailability directly correlated with the in vitro permeability results. It can therefore be concluded that the change in permeability and bioavailability of indinavir was caused by efflux inhibition and this effect was dependent on the type of H. hemerocallidea material investigated, which was found to be in the following order: commercial product > aqueous extract > reference plant material. The clinical significance of the combined effect of efflux and metabolism inhibition by H. hemerocallidea should be determined in another in vivo model that expresses the cytochrome P450 3A4 enzyme.

scholarly journals A Novel Role of Bergamottin in Attenuating Cancer Associated Cachexia by Diverse Molecular Mechanisms

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1347
Author(s):  
Young Yun Jung ◽  
Jeong-Hyeon Ko ◽  
Jae-Young Um ◽  
Gautam Sethi ◽  
Kwang Seok Ahn
Keyword(s):  
Western Blot ◽  
Cancer Cachexia ◽  
Molecular Mechanisms ◽  
Suppressive Effect ◽  
In Vivo Experiment ◽  
Hematoxylin And Eosin ◽  
Loss Method ◽  
The Impact

Purpose: The potential effects of bergamotiin (BGM) on the suppression of cancer cachexia was evaluated under in vitro and in vivo conditions to investigate its possible inhibitory effects on the muscle and fat loss. Method: The differentiated C2C12 and 3T3L1 cells were treated with BGM after the induction of cancer-cachexia with pancreatic cancer conditioned media (CM). The expression levels of the various molecules involved in the differentiation and loss of muscle and fat (MuRF-1, Atrogin-1, C/EBPα, and PPARγ) were analyzed by Western blot and oil red O staining. For in vivo experiment, MIA PaCa-2 cells were injected into the mice (n = 6), and then BGM (1 mg/kg) was intraperitoneally administered to analyze muscle and adipose tissue by Hematoxylin and Eosin staining and Western blot. Result: BGM displayed a significant effect on the inhibition of muscle and fat catabolism under both in vitro and in vivo conditions. The results of the in vivo experiment revealed a remarkable suppressive effect of BGM on the weight loss in mice. Conclusions: The potential effects of BGM on the inhibition of muscle and fat catabolism in vitro and in vivo were thus confirmed. Based on the results, the impact of BGM on cancer cachexia could be possibly analyzed in the future clinical studies.

scholarly journals Endothelial lipase mediates efficient lipolysis of triglyceride-rich lipoproteins

PLoS Genetics ◽  
2021 ◽  
Vol 17 (9) ◽  
pp. e1009802
Author(s):  
Sumeet A. Khetarpal ◽  
Cecilia Vitali ◽  
Michael G. Levin ◽  
Derek Klarin ◽  
Joseph Park ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Endothelial Lipase ◽  
Primary Role ◽  
Loss Of Function ◽  
Extracellular Lipase ◽  
High Fat ◽  
Peripheral Tissues ◽  
The Impact

Triglyceride-rich lipoproteins (TRLs) are circulating reservoirs of fatty acids used as vital energy sources for peripheral tissues. Lipoprotein lipase (LPL) is a predominant enzyme mediating triglyceride (TG) lipolysis and TRL clearance to provide fatty acids to tissues in animals. Physiological and human genetic evidence support a primary role for LPL in hydrolyzing TRL TGs. We hypothesized that endothelial lipase (EL), another extracellular lipase that primarily hydrolyzes lipoprotein phospholipids may also contribute to TRL metabolism. To explore this, we studied the impact of genetic EL loss-of-function on TRL metabolism in humans and mice. Humans carrying a loss-of-function missense variant in LIPG, p.Asn396Ser (rs77960347), demonstrated elevated plasma TGs and elevated phospholipids in TRLs, among other lipoprotein classes. Mice with germline EL deficiency challenged with excess dietary TG through refeeding or a high-fat diet exhibited elevated TGs, delayed dietary TRL clearance, and impaired TRL TG lipolysis in vivo that was rescued by EL reconstitution in the liver. Lipidomic analyses of postprandial plasma from high-fat fed Lipg-/- mice demonstrated accumulation of phospholipids and TGs harboring long-chain polyunsaturated fatty acids (PUFAs), known substrates for EL lipolysis. In vitro and in vivo, EL and LPL together promoted greater TG lipolysis than either extracellular lipase alone. Our data positions EL as a key collaborator of LPL to mediate efficient lipolysis of TRLs in humans and mice.

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