Journal: Journal of Neuroinflammation

Article Title: Dual inhibition of IRAK1/TAK1 signaling in astrocytes reduces accelerated mortality in human APOE4 knock-in APPswe/PSEN1dE9/P301S-Tau triple transgenic mouse model

doi: 10.1186/s12974-025-03564-7

Figure Lengend Snippet: Inhibition of A1 astrocyte activation alleviated mitochondrial dysfunction in differentiating neurons. A Schematic overview of the in vitro study using differentiating MSP-1 newborn neurons. MSP-1 neurons were treated with cultured medium (CM) collected from MSP-1 astrocytes for 24 h. B Representative images of AT8 staining of MSP-1 neurons treated with TNF CM and TNF+Ans CM (scale bar, 10 μm). C Quantification of AT8 fluorescence intensity in each group, ( n = 3–5). D Representative electron microscopy images of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (scale bar, 2 μm (up), 1 μm (down)). N, nucleus, MT, normal mitochondria (blue), sMT, swollen mitochondria (yellow). E Quantification of mitochondrial length and the ratio of swollen mitochondria in each group (neuron number, 15–18; mitochondria number, 45–49). F Representative images of MT-1 staining of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (10 μM) (scale bar, 25 μm). G Quantification of MT-1 fluorescence intensity in each group ( n = 4–6). H Representative images of ROS staining of MSP-1 neurons treated with Sham, TNF CM, TNF+Ans CM and TNF CM-C3aRA (10 μM) (scale bar, 25 μm). I Quantification of ROS fluorescence intensity in each group ( n = 5–6). Data are presented as mean ± SEM ( C, G and I ) or presented as violin plots ( E ) (median: bold dashed line; quartiles (1/4 and 3/4: dashed lines)) and were analyzed by one-way ANOVA with Dunnett's multiple comparisons test ( C, G and I ) or Kruskal-Wallis test with Dunn's multiple comparisons test ( E ). All comparisons made were versus the TNF CM group. * p < 0.05, ** p < 0.01, *** p < 0.001. Some elements in panel A were created with BioRender.com

Article Snippet: For blocking experiments, necrostatin-1 (Nec-1) and the C3a receptor antagonist SB290157 (Selleck) were used.

Techniques: Inhibition, Activation Assay, In Vitro, Cell Culture, Staining, Fluorescence, Electron Microscopy

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: Glomerular and tubular C3 and C3aR staining in Stx2/LPS mice: ( A ) Representative images and quantification of intraglomerular and tubular C3 staining (green) in control and Stx2/LPS-injected mice at 48 h; ( B ) Representative images and quantification of intraglomerular and tubular C3aR expression (red) in control and Stx2/LPS-injected mice. Scale bars: 20 μm. Results are presented as mean ± SEM (control n = 4, Stx2/LPS n = 7), and unpaired Student’s t test was used. * p < 0.05, ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Staining, Control, Injection, Expressing

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: Treatment with the C3aR antagonist limits glomerular mitochondrial alterations in Stx2/LPS mice: ( A ) Representative transmission electron microscope images showing mitochondrial morphology in podocytes of control and Stx2/LPS mice, given vehicle or C3aR antagonist (C3aRa). Scale bars: 500 nm; ( B ) Quantification of mitochondrial length and area and ( C ) characterization of mitochondrial subpopulations in podocytes. Results are expressed as mean ± SEM (control: n = 516 mitochondria analyzed in n = 6 glomeruli of n = 2 mice; Stx2/LPS + vehicle: n = 411 mitochondria analyzed in n = 7 glomeruli of n = 3 mice; Stx2/LPS + C3aRa: n = 529 mitochondria analyzed in n = 7 glomeruli of n = 3 mice); ( D ) Representative images and quantification of glomerular VDAC (upper panels) and ATP5I (bottom panels) staining in controls and Stx2/LPS mice given vehicle or C3aRa. Podocytes are indicated by red arrowheads. Scale bars: 20 μm. Results are expressed as mean ± SEM (VDAC: control n = 4, Stx2/LPS + vehicle n = 5, Stx2/LPS + C3aRa n = 4; ATP5I: control n = 4, Stx2/LPS + vehicle n = 7, Stx2/LPS + C3aRa n = 7), and ANOVA with Tukey multiple-comparisons test was used. * p < 0.05, ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Transmission Assay, Microscopy, Control, Staining

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: The C3aR blockade reduced proximal tubular cell apoptosis and restored megalin expression in Stx2/LPS mice. ( A , B ) Representative images and quantification of ( A ) cleaved caspase-3 (red) and ( B ) megalin (red) in control and Stx2/LPS mice given vehicle or C3aRa. Arrowheads indicate loss of megalin expression on the proximal tubular cell brush border. Cell membranes and nuclei were stained with FITC-WGA-lectin (green) and DAPI (blue), respectively. Scale bars: 20 μm. Results are expressed as mean ± SEM ( n = 4 per each group), and ANOVA with Tukey multiple-comparisons test was used. * p < 0.05, ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Expressing, Control, Staining

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: The C3aR blockade restores mitochondrial morphology and function in proximal tubular cells in Stx2/LPS mice. ( A , B ) Representative transmission electron microscope images and morphometric analysis showing mitochondrial morphology in proximal tubular cells of control and Stx2/LPS mice, given vehicle or C3aRa. Scale bars: 500 nm. Results are expressed as mean ± SEM (control: n = 6768 mitochondria analyzed in n = 3 mice; Stx2/LPS + vehicle: n = 4238 mitochondria analyzed in n = 3 mice; Stx2/LPS + C3aRa: n = 7100 mitochondria analyzed in n = 3 mice). ( C ) Representative images and quantification of tubular VDAC (upper panels) and ATP5I (bottom panels) stainings in control and Stx2/LPS mice with or without C3aRa. Scale bars: 20 μm. Results are expressed as mean ± SEM (VDAC: control n = 4, Stx2/LPS + vehicle n = 5, Stx2/LPS + C3aRa n = 4; ATP5I: control n = 4, Stx2/LPS + vehicle n = 7, Stx2/LPS + C3aRa n = 7). ANOVA with Tukey multiple-comparisons test was used. * p < 0.05, ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Transmission Assay, Microscopy, Control

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: Treatment with the C3aR antagonist limits tubulin alterations in proximal tubules of Stx2/LPS mice. Representative images of tubulin expression (green) in control and Stx2/LPS mice receiving vehicle or C3aRa. Nuclei were stained with DAPI (blue). Scale bars: 20 μm.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Expressing, Control, Staining

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: Stx2 increases podocyte and tubular cell sensitivity to C3a through the upregulation of C3aR expression in vitro. ( A ) Representative images and quantification of C3aR staining (red) in podocytes and RPTECs exposed to control medium or Stx2 (50 pM) for 15 h. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. Results are presented as mean ± SEM (number of biological samples: n = 3 control and Stx2-treated podocytes; n = 4 control RPTECs and n = 6 Stx2-treated RPTECs), and unpaired Student’s t test was used. ( B ) Representative images of mitochondria labeled with MitoTracker in live podocytes and RPTECs incubated with control medium, C3a (1 μM, 6 h) alone, or with Stx2 (50 pM, 24 h) in the presence of C3a, added in the last 6 h. Nuclei were counterstained with Hoechst (blue). Arrowhead indicates mitochondrial swelling. The percentage of cells with an altered mitochondrial pattern, in terms of fragmentation and perinuclear redistribution, on total cells per field was quantified. Scale bars: 20 μm. Results are expressed as mean ± SEM (number of biological samples: n = 3 control, C3a and Stx2 + C3a-treated podocytes and RPTECs), and ANOVA with Tukey multiple-comparisons test was used. ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Expressing, In Vitro, Staining, Control, Labeling, Incubation

Journal: Cells

Article Title: Shiga Toxin 2 Triggers C3a-Dependent Glomerular and Tubular Injury through Mitochondrial Dysfunction in Hemolytic Uremic Syndrome

doi: 10.3390/cells11111755

Figure Lengend Snippet: C3a decreases tubulin expression and reduces mitochondrial antioxidant defense in cultured podocytes and RPTECs. Representative western blot and densitometric analysis of ( A ) tubulin and ( B ) acetylated SOD2 (SOD2 AcK68 ) and total SOD2 protein expression in protein extracts obtained from podocytes and RPTECs incubated with control medium, C3a (1 μM, 6h) alone, or with Stx2 (50 pM, 24 h) in the presence of C3a, added in the last 6 h. SOD2 acetylation was expressed as the ratio between SOD2 AcK68 and total SOD2. Results are presented as mean ± SEM (number of biological samples for tubulin: n = 3 control and n = 6 C3a-treated podocytes; n = 6 control and n = 12 C3a-treated RPTECs), (number of biological samples for SOD2: n = 3 control, C3a and Stx2+C3a-treated podocytes and RPTECs), and unpaired Student’s t test or ANOVA with Tukey multiple-comparisons test were used. * p < 0.05, ** p < 0.01.

Article Snippet: Animals were randomly allocated to the following groups: group 1 ( n = 7), C57BL/6 mice given purified Stx2 (120 ng per mouse) plus LPS (60 μg per mouse; L2630, Merck, Darmstadt, Germany) by intraperitoneal injection to induce HUS; group 2 ( n = 7), Stx2/LPS-treated mice given the C3a receptor antagonist SB290157 (Cayman Chemical, Ann Arbor, MI, USA) at a dose of 2.5 mg/kg [ ], by intraperitoneal injection, 1 h before Stx2/LPS injection and twice a day for two days.

Techniques: Expressing, Cell Culture, Western Blot, Incubation, Control

Journal: FEBS Open Bio

Article Title: Expression of a Crry/p65 is reduced in acute lung injury induced by extracellular histones

doi: 10.1002/2211-5463.13322

Figure Lengend Snippet: Reduced expression of Crry/p65 associated with lung oedema. C57BL/6J mice received a single tail vein injection of calf thymus histones (45 μg·g −1 body weight) or saline ( n = 9 per group). Lung weight per body weight (%) and Crry/p65 expression are shown in panels A and B, respectively. The correlation between Crry/p65 expression and lung weight per body weight is shown in panel E. The concentrations of C3a in plasma are shown in panel C. The correlation between Crry/p65 expression and C3a in plasma is shown in panel F. The concentrations of LDH in plasma are shown in panel D. The correlation between Crry/p65 expression and LDH in plasma is shown in panel G. Values are shown as experimental means ± SD. * P < 0.01 vs. vehicle (Student's t ‐test) † P < 0.01 vs. vehicle (Welch’s t ‐test). Correlation analysis was conducted by Pearson's correlation analysis.

Article Snippet: Briefly, mice were randomized prior to receiving an intraperitoneal injection of either C3a receptor antagonist (1 mg·kg −1 SB290157; Calbiochem, Darmstadt, Germany) diluted in PBS and dimethyl sulfoxide (DMSO; 1.16%, v/v), or an equal volume of this vehicle 45 min before being administered a single tail vein injection of calf thymus histones (45 μg·g −1 body weight).

Techniques: Expressing, Injection, Saline, Clinical Proteomics

Journal: FEBS Open Bio

Article Title: Expression of a Crry/p65 is reduced in acute lung injury induced by extracellular histones

doi: 10.1002/2211-5463.13322

Figure Lengend Snippet: Treatment with C3a receptor antagonist ameliorates lung injury induced by extracellular histones. C57BL/6J mice received a single intraperitoneal injection of C3a receptor antagonist (C3aRA) 45 min prior to histone injection (histone group; n = 9, C3aRA group; n = 8). Panel A shows the proportion of bleeding area in a tissue section. Lung weight per body weight (%) after histone injection is shown in panel B. Values are shown as experimental means ± SD. ** P < 0.05 vs. histone (Student’s t ‐test).

Article Snippet: Briefly, mice were randomized prior to receiving an intraperitoneal injection of either C3a receptor antagonist (1 mg·kg −1 SB290157; Calbiochem, Darmstadt, Germany) diluted in PBS and dimethyl sulfoxide (DMSO; 1.16%, v/v), or an equal volume of this vehicle 45 min before being administered a single tail vein injection of calf thymus histones (45 μg·g −1 body weight).

Techniques: Injection