GBP2 upregulated in LPS-stimulated macrophages-derived exosomes accelerates septic lung injury by activating epithelial cell NLRP3 signaling.

Macrophages infiltration is a crucial factor causing Sepsis-associated acute lung injury (ALI). Accumulating evidence suggests macrophages-alveolar epithelial cells communication is proven to be critical in ALI. However, little is known regarding how activated macrophages regulated sepsis-associated ALI. To explore the role of macrophages-alveolar epithelial cells communication in the ALI process, our data revealed that Lipopolysaccharides-induced macrophages-derived exosomes (L-Exo) induced sepsis-associated ALI and caused alveolar epithelial cells damage. Moreover, Guanylate-binding protein 2 (GBP2) was significantly upregulated in L-Exo, and NLRP3 inflammasomes was the direct target of GBP2. Further experimentation showed that GBP2 inhibition in vitro and in vivo reserves L-Exo effects, while GBP2 overexpression in vitro and in vivo promotes L-Exo effects. These results demonstrated that L-Exo contains excessive GBP2 and promotes inflammation through targeting NLRP3 inflammasomes, which induced alveolar epithelial cells dysfunction and pyroptosis. These findings demonstrate that L-Exo exerted a deleterious effect on ALI by regulating the GBP2/NLRP3 axis, which might provide new insight on ALI prevention and treatment.

LPS-induced macrophage HMGB1-loaded extracellular vesicles trigger hepatocyte pyroptosis by activating the NLRP3 inflammasome.

Extracellular vesicles (EVs) have emerged as important vectors of intercellular dialogue. High mobility group box protein 1 (HMGB1) is a typical damage-associated molecular pattern (DAMP) molecule, which is cytotoxic and leads to cell death and tissue injury. Whether EVs are involved in the release of HMGB1 in lipopolysaccharide (LPS)-induced acute liver injuries need more investigation. EVs were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. The co-localization of HMGB1, RAGE (receptor for advanced glycation end-products), EEA1, Rab5, Rab7, Lamp1 and transferrin were detected by confocal microscopy. The interaction of HMGB1 and RAGE were investigated by co-immunoprecipitation. EVs were labeled with the PKH67 and used for uptake experiments. The pyroptotic cell death was determined by FLICA 660-YVAD-FMK. The expression of NLRP3 (NOD-like receptor family pyrin domain containing 3) inflammasomes were analyzed by western-blot or immunohistochemistry. Serum HMGB1, ALT (alanine aminotransferase), AST (aspartate aminotransferase), LDH (lactate dehydrogenase) and MPO (myeloperoxidase) were measured using a commercial kit. The extracellular vesicle HMGB1 was detected in the serums of sepsis patients. Macrophages were found to contribute to HMGB1 release through the EVs. HMGB1-RAGE interactions participated in the loading of HMGB1 into the EVs. These EVs shuttled HMGB1 to target cells by transferrin-mediated endocytosis leading to hepatocyte pyroptosis by the activation of NLRP3 inflammasomes. Moreover, a positive correlation was verified between the sepsis serum EVs-HMGB1 level and clinical liver damage. This finding provides insights for the development of novel diagnostic and therapeutic strategies for acute liver injuries.

Proteomic Profiling of LPS-Induced Macrophage-Derived Exosomes Indicates Their Involvement in Acute Liver Injury.

Exosomes are typically involved in cellular communication and signaling. Macrophages play a key role in lipopolysaccharide (LPS)-induced sepsis. However, the molecular comparison of exosomes derived from LPS-induced macrophage has not been well analyzed. The macrophage-exosomes are validated and the protein composition of those exosomes are investigated by isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. A total of 5056 proteins are identified in macrophage-exosomes. We discovered 341 increased proteins and 363 reduced proteins in LPS-treated macrophage-exosomes compared with control exosomes. In addition, gene ontology analysis demonstrates that macrophage-exosomes proteins are mostly linked to cell, organelle, extracellular region, and membrane. The bioinformatics analysis also indicates that these proteins are mainly involved in cellular process, single-organism process, metabolic process, and biological regulation. Among these 341 upregulated proteins, Kyoto Encyclopedia of Genes and Genomes analysis reveals that 22 proteins are involved in the NOD-like receptor signaling pathway. Finally, hepatocytes can uptake macrophage-exosomes and subsequently NLRP3 inflammasome is activated in vitro and in vivo. These data emphasize the fundamental importance of macrophage-exosomes in sepsis-induced liver injury. Therefore, the iTRAQ proteomic strategy brings new insights into macrophage-derived exosomes. It may improve our understanding of macrophage-exosomes' functions and their possible use as therapeutic targets for sepsis.

Down-regulation of microRNA-224 -inhibites growth and epithelial-to-mesenchymal transition phenotype -via modulating SUFU expression in bladder cancer cells.

Aberrant expression of miR-224 is usually found in cancer studies; however, the role of miR-224 has seldom been reported in bladder cancer (BC). We explored miR-224's function and the underlying mechanism in BC. It was found that miR-224 expression was significantly up-regulated in BC tissues and cell lines. Knockdown of miR-224 decreased BC cell growth and invasion both in vitro and in vivo. We identified the SUFU protein as a downstream target of miR-224 by using luciferase and western blot assays. We proposed that miR-224 promoted BC cell growth and invasion via sustaining the activity of Hedgehog pathway, which was negatively regulated by SUFU. Taken together, our study demonstrated that miR-224 may function as an onco-miR in BC and suggested that miR-224 may be a potential therapeutic target for BC patients.

MicroRNA-21 Mediates Angiotensin II-Induced Liver Fibrosis by Activating NLRP3 Inflammasome/IL-1ß Axis via Targeting Smad7 and Spry1.

AIMS: Angiotensin II (AngII), a vasoconstrictive peptide of the renin-angiotensin system (RAS), promotes hepatic fibrogenesis and induces microRNA-21(mir-21) expression. Angiotensin-(1-7) [Ang-(1-7)] is a peptide of the RAS, which attenuates liver fibrosis. Recently, it was reported that the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome participated in liver fibrosis. However, it remains unclear how mir-21 mediates AngII-induced NLRP3 inflammasome activation. We investigate the role of AngII-induced mir-21 in the regulation of NLRP3 inflammasome/IL-1ß axis in liver fibrosis. RESULTS: In vivo, circulating mir-21 was upregulated in patients with liver fibrosis and was positively correlated with liver fibrosis and oxidation. Treatment with Ang-(1-7) inhibited mir-21, NLRP3 inflammasome, and liver fibrosis after bile duct ligation (BDL) or AngII infusion. Inhibition of mir-21 suppressed the Smad7/Smad2/3/NOX4, Spry1/ERK/NF-κB pathway, NLRP3 inflammasome, and liver fibrosis induced by AngII infusion. In vitro, AngII upregulated mir-21 expression via targeting Smad7 and Spry1 in primary hepatic stellate cells (HSCs). In contrast, Ang-(1-7) suppressed mir-21 expression and oxidation induced by AngII. Overexpression of mir-21 promoted oxidation, and collagen production enhanced the effect of AngII on NLRP3 inflammasome activation via the Spry1/ERK/NF-κB, Smad7/Smad2/3/NOX4 pathways. However, downregulation of mir-21 exerted the opposite effects. Innovation and Conclusions: Mir-21 mediates AngII-activated NLRP3 inflammasome and resultant HSC activation via targeting Spry1 and Smad7. Ang-(1-7) protected against BDL or AngII infusion-induced hepatic fibrosis and inhibited mir-21 expression. Antioxid. Redox Signal. 27, 1-20.

[Losartan regulates oxidative stress via caveolin-1 and NOX4 in mice with ventilator- induced lung injury].

OBJECTIVE: To investigate the effect of losartan in regulating oxidative stress and the underlying mechanism in mice with ventilator-induced lung injury. METHODS: Thirty-six male C57 mice were randomly divided into control group, losartan treatment group, mechanical ventilation model group, and ventilation plus losartan treatment group. After the corresponding treatments, the lung injuries in each group were examined and the expressions of caveolin-1 and NOX4 in the lung tissues were detected. RESULTS: The mean Smith score of lung injury was significantly higher in mechanical ventilation model group (3.3) than in the control group (0.4), and losartan treatment group (0.3); the mean score was significantly lowered in ventilation plus losartan treatment group (2.3) compared with that in the model group (P<0.05). The expressions of caveolin-1 and NOX4 were significantly higher in the model group than in the control and losartan treatment groups (P<0.05) but was obviously lowered after losartan treatment (P<0.05). Co-expression of caveolin-1 and NOX4 in the lungs was observed in the model group, and was significantly decreased after losartan treatment. CONCLUSION: Losartan can alleviate ventilator-induced lung injury in mice and inhibit the expression of caveolin-1 and NOX4 and their interaction in the lungs.