Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Evaluation of cytotoxicity of PF8380 and AS2717638 in BV-2 microglia . Cells were treated with increasing concentrations of ( A ) the ATX inhibitor PF8380 (‘PF’) and ( B ) the LPA5 inhibitor AS2717638 (‘AS’) for 24 h. Dimethylsulfoxide (DMSO) was used as vehicle control. Dose-dependent effect of the inhibitors on MTT reduction in cells was compared to control. Results are shown as mean ± SEM of three independent experiments. (**** p < 0.0001, compared to control; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Effect of LPS on ATX protein expression, PLD activity and LPA receptor expression in BV-2 microglia . ( A ) Cells were treated in the absence (‘-‘) or presence (‘+’) of LPS (20 ng/mL) for the indicated times. Cell protein lysates were collected and ATX was detected by immunoblotting. β-actin was used as loading control. One representative blot of ATX is shown. Densitometric evaluation of immunoreactive bands is shown in the bar graphs. Values are expressed as mean ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01 compared to control; unpaired Student’s t test). ( B ) Cells were treated with LPS (20 ng/mL) in the absence or presence of PF8380 (‘PF’; 10 and 1 µM) for the indicated times. DMSO was used as vehicle control. PLD activity in the supernatant was measured by phospholipase D kit (Sigma) and compared with their appropriate control. Results are presented as mean ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01; # p < 0.05, ## p < 0.01 compared to LPS-treated cells; one-way ANOVA with Bonferroni correction). ( C ) Cells were incubated with LPS (20 ng/mL) for 24 h. Expression of LPA2, -3, -5 and -6 was detected by immunoblotting. β-actin was used as loading control. One representative blot for each protein is shown. Densitometric evaluation of immunoreactive bands is shown in the bar graphs. Values are expressed as mean ± SEM of three independent experiments. (** p < 0.01 compared to control; unpaired Student’s t test).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Expressing, Activity Assay, Western Blot, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX and LPA5 regulates LPS-induced transcription factor phosphorylation . Cells were treated with DMSO (vehicle control), LPS (20 ng/mL) plus DMSO, and ( A ) LPS plus PF8380 (‘PF’; 10 and 1 µM) or ( B ) LPS plus AS2717638 (‘AS’; 1 and 0.1 µM) for the indicated times. Cell lysate were collected and protein expression of phosphorylated STAT1, p65, and c-Jun along with their total protein was monitored using immunoblotting. β-actin was used as loading control. One representative blot for each protein is shown. Densitometric evaluation of immunoreactive bands is shown in the bar graphs. Values are expressed as mean ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to DMSO control; # p < 0.05, ### p < 0.001, #### p < 0.0001 compared to LPS-treated cells; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX and LPA5 down-regulates LPS-induced TLR4, COX-2 expression, nitric oxide production in BV-2 cells and reduces neurotoxicity of BV-2-conditioned medium. Cells were treated with DMSO (vehicle control), LPS (20 ng/mL) plus DMSO, ( A ) LPS plus PF8380 (‘PF’; 10 and 1 µM) or ( B ) LPS plus AS2717638 (‘AS’; 1 and 0.1 µM) for the indicated times. Cell lysates were collected and protein expression of TLR4 and COX2 was monitored by immunoblotting. β-actin was used as loading control. One representative blot for each protein is shown. Densitometric evaluation of immunoreactive bands is shown in the bar graphs. ( C ) Cells were treated with DMSO, LPS plus DMSO, and LPS plus PF8380 (‘PF’; 10 µM) or LPS plus AS2717638 (‘AS’; 1 µM) for 24 h. The production of NO was determined by measuring the total nitrate concentration in the supernatants. ( D ) CATH.a neurons were incubated for 24 h with conditioned media collected from LPS-treated (in the absence or presence of PF8380 (‘PF’; 10 µM) or AS2717638 (‘AS’; 1 µM)) BV-2 cells for 24 h. LDH levels were detected and neurotoxixity was calculated according to manufacturer’s protocol. Values are expressed as mean ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to DMSO control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to LPS-treated cells; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Expressing, Western Blot, Concentration Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX by PF8380 attenuates LPS-induced secretion of cyto-/chemokines in BV-2 microglia. Cells were treated with DMSO control, LPS (20 ng/mL) plus DMSO in the absence or presence of PF8380 (‘PF’; 10 and 1 µM) for the indicated times. Supernatants were collected. ( A – F ) Concentration of cytokines TNFα, IL6, and IL-1β and chemokines CXCL10, CXCL2, and CCL5 were quantified by ELISA. Values are expressed as mean ± SEM of three independent experiments. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to DMSO control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to LPS-treated cells; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: PF8380 crosses the blood–brain barrier and attenuates LPA levels in mouse brain. Animals received PF8380 (‘PF’) dissolved in oral formulation vehicle at a dose of 30 mg/kg by gavage to get an indication about oral bioavailability. At the indicated times, mice were sacrificed, perfused, and brains were dissected and snap frozen. The tissue homogenates were extracted using a modified Bligh & Dyer HCl method. Quantification of ( A ) PF8380 (‘PF’) and ( B ) LPA in brain was performed by LC-MS/MS. Results are presented as mean values ± SEM of three mice per group (* p < 0.05 compared to control; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Modification, Liquid Chromatography with Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX and LPA5 regulates expression of pro-inflammatory genes in LPS-injected C57Bl/6 mice. Mice (n = 6–8 per group) were injected intraperitoneally (i.p.) with DMSO, LPS (5 mg/kg) plus DMSO, with or without PF8380 (‘PF’; 30 mg/kg) or AS2717638 (‘AS’; 10 mg/kg). After 24 h, the animals were sacrificed and the brains were perfused. The right hemisphere from each mouse was processed for RNA isolation and gene expression of ( A ) iNOS, ( B ) TNFα, ( C ) IL6, ( D ) IL-1β, ( E ) CXCL10, ( F ) CXCL2, and ( G ) CCL5 was evaluated by qPCR. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as housekeeping gene. Expression was calculated using the 2−ddCt method. Results are presented as mean values ± SEM of 6–8 mice per group (** p < 0.01, **** p < 0.0001 compared to DMSO control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to LPS-treated mice; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Expressing, Injection, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX and LPA5 regulates proteins involved in neuroinflammation in LPS-injected C57Bl/6 mice. Mice (n = 6–8 per group) were injected intraperitoneally (i.p.) with DMSO, LPS (5 mg/kg) plus DMSO, with or without ( A ) PF8380 (‘PF’; 30 mg/kg) or ( B ) AS2717638 (‘AS’; 10 mg/kg). After 24 h, the animals were sacrificed and the brains were perfused. The left hemisphere from each mouse was processed for protein analyses by immunoblotting. Protein expression of TLR4, Iba1, GFAP, COX2, synaptophysin, Bax, and Bcl2 were monitored by Western blot analyses. β-actin was used as loading control. One representative blot for each protein is shown. Densitometric evaluation of immunoreactive bands is shown in the bar graphs. Values are expressed as mean ± SEM of 6–8 mice per group (* p < 0.05, ** p < 0.01, *** p < 0.001 compared to DMSO control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared to LPS-treated mice; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Injection, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Autotaxin and Lysophosphatidic Acid Receptor 5 Attenuates Neuroinflammation in LPS-Activated BV-2 Microglia and a Mouse Endotoxemia Model

doi: 10.3390/ijms22168519

Figure Lengend Snippet: Inhibition of ATX and LPA5 downregulates peripheral cytokine concentrations in LPS-injected C57Bl/6 mice. Mice (n = 6–8 per group) were injected i.p. with DMSO, LPS (5 mg/kg) plus DMSO, with or without PF8380 (‘PF’; 30 mg/kg) or AS2717638 (‘AS’; 10 mg/kg). After 24 h, the animals were sacrificed, blood was collected and processed to obtain serum. Serum was diluted at 1:10 for further analysis. ( A–C ) The concentrations of TNFα, IL6 and IL-1β were quantified using ELISA. Values are expressed as mean ± SEM of 6–8 mice per group ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to DMSO control; # p < 0.05 compared to LPS-treated mice; one-way ANOVA with Bonferroni correction).

Article Snippet: Briefly, C57BL/6 mice were administered PF8380 (900 µg) dissolved in 450 µL oral formulation vehicle (Echelon), resulting in a dose of 30 mg/kg body weight.

Techniques: Inhibition, Injection, Enzyme-linked Immunosorbent Assay