Journal: Acta Physiologica (Oxford, England)

Article Title: Sex Differences in the Effects of Etonogestrel on Respiratory Recovery in an In Vivo Rat Model of Central Chemoreflex Impairment

doi: 10.1111/apha.70194

Figure Lengend Snippet: Molecular changes following ETO administration in the retrotrapezoid nucleus (RTN), the solitary tract nucleus (NTS), and the medial hypothalamus (MH) of female and male rats. (A–C) Quantitative PCR analysis of Nmb , transcription factor Phox2b , and proton‐sensing channels, Gpr4 and Task2 , in the RTN (A), NTS (B), and MH (C) of females and males. Naive males showed higher expression levels of Nmb and Gpr4 in the RTN (A) and Phox2b , Gpr4 , and Pgr in the NTS (B) compared with females. Following lesion or ETO treatment, Nmb expression was significantly reduced in both SHAM‐ and ETO‐treated animals compared with naive controls, in males and females (A). ETO‐treated females showed upregulation of Task2 and Gpr4 mRNA levels in the RTN (A), and downregulation of Phox2b and Task2 mRNA levels in the NTS (B), consistent with the observed recovery of the CO 2 chemoreflex. No significant changes were observed in the MH (C). In males, no significant transcriptional changes were detected in the RTN, NTS, or MH, except for the reduction of Nmb in the RTN. Graphs show mean ± SD with individual values represented as 2 −ΔCt with all statistical analyses performed on ΔCt values. Comparisons between naive males and females were analyzed using unpaired t ‐tests or Mann–Whitney U tests, whereas differences among experimental groups within each sex were analyzed using one‐way ANOVA (Tukey post hoc) or Kruskal–Wallis ANOVA (Dunn's post hoc). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The assays used were: rat Phox2b (ID: Rn01413076_mH), rat Pgr (ID: Rn01448227_m1), rat Gpr4 (ID: Rn02585915), rat Task2 (ID: Rn01755927), rat Nmb (ID: Rn01478123_m1), and the endogenous control rat Actb (ID: Rn00667869_m1).

Techniques: Real-time Polymerase Chain Reaction, Expressing, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Microscopy, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Microscopy, Two Tailed Test

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

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