30 ne inhibitor sivelestat  (MedChemExpress)

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of GPR4, GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Disruption, Quantitative RT-PCR, Expressing, Transfection, Activity Assay, Control, Permeability, Cell Stimulation, Mutagenesis, Incubation

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA‐induced GPR68 activation and endothelial barrier disruption. (A) HPAECs were transfected with GPR68 PRESTO‐Tango plasmids for 24 h followed by the addition of HKSA for indicated time periods with or without OGM‐8345 pretreatment (3 μM, 30 min). GPR68 activity was determined by measuring luminescence signal. The data are presented after normalizing to non‐treated controls. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (B) HPAEC were pre‐incubated with OGM‐8345 (3 μM, 30 min), and HKSA (5 × 10 8 particles/mL) was added for 3 h. qRT‐PCR was carried out to determine the mRNA expression levels of GPR68, GPR65, and GPR4. * p < .05, vs. GPR68 controls; ** p < .05, vs. GPR68 HKSA; ND p > .05 vs. GPR65 and GPR4 controls, n = 3. (C) HPAEC monolayers were treated with HKSA with or without pre‐treatment OGM‐8345 as above. Endothelial macromolecular permeability was determined by the XPerT assay. Increased FITC fluorescence reflects an increase in endothelial permeability. Bar = 20 μm. (D) HPAEC were pre‐incubated for 30 min with OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) followed by the addition of HKSA. Endothelial permeability was monitored by TER measurements. The bar graph represents normalized pooled data at 15 h of EC stimulation. * p < .05, vs. HKSA, n = 6.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Activation Assay, Disruption, Transfection, Activity Assay, Control, Incubation, Quantitative RT-PCR, Expressing, Permeability, Fluorescence

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA and acidosis‐induced endothelial dysfunction. (A) HPAEC were pretreated with 3 μM OGM‐8345 at both normal and acidic pH (30 min) and stimulated with HKSA for 6 h. XPerT assay was performed to evaluate EC monolayer permeability for macromolecules. FITC fluorescence signal reflects an increase in endothelial permeability. Bar = 20 μm. (B) HPAEC were exposed to HKSA at indicated pH media with or without OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) pretreatment followed by qRT‐PCR analysis of mRNA expression of selected pro‐inflammatory marker genes. * p < .05, vs. corresponding controls, ** p < .05, vs. pH 6.5 + HKSA, n = 3. (C) Cells were challenged with HKSA at pH 6.5 or pH 7.4 in the presence or absence of OGM‐8345 for 6 h, followed by western blot analysis of ICAM‐1 protein levels; β‐tubulin was used as an internal loading control. Bar graph: Normalized densitometry data; * p < .05, vs. HKSA pH 7.4; ** p < .05, vs. pH 6.5 + HKSA, n = 3.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Permeability, Fluorescence, Quantitative RT-PCR, Expressing, Marker, Western Blot, Control

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 rescues HKSA‐induced lung injury in vivo. C57BL/6 mice were exposed to HKSA (2 × 10 8 bacterial particles/mouse, intranasal) for 18 h. (A, B) Total RNA was extracted from various tissues of control or HKSA‐challenged mice, and qRT‐PCR was used to measure mRNA expression levels of GPR4 and GPR68. * p < .05, vs. control, n = 3. (C–F) C57BL/6 mice were injected with OGM‐8345 (20 mg/kg, i.p.) immediately followed by intranasal administration of HKSA. BAL fluid was collected to analyze total PMNs (C) and protein content (D). * p < .05, vs. HKSA alone, n = 8. (E) Total RNA was extracted from lung tissues, and qRT‐PCR was carried out to determine mRNA expression of indicated pro‐inflammatory genes. p < .05, vs. HKSA alone, n = 6. (F) HKSA‐induced Evans blue accumulation in the lung reflects lung vascular hyperpermeability and the protective effect of OGM‐8345. Shown are representative images of lungs from five independent experiments.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: In Vivo, Control, Quantitative RT-PCR, Expressing, Injection