Journal: Pharmaceutics

Article Title: Anti-Inflammatory Activity of a Cyclic Tetrapeptide in Mouse and Human Experimental Models

doi: 10.3390/pharmaceutics12111030

Figure Lengend Snippet: Antagonists of EP4 and EP3 receptors for PGE2 abolish the suppressive action of the peptide in carrageenan-induced foot pad edema in mice. The mice were injected with 4B8M (250 µg/mouse) i.p. 1 h before s.c. carrageenan administration. PGE receptor antagonists AH23848 (EP4 receptor antagonist) and L-798106 (EP3 receptor antagonist) were given at doses of 40 µg/mouse intramuscularly (i.m.) 45 min following 4B8M. The footpad thickness was measured after 3 h with a spring caliper, and the results are presented as the antigen-specific increase of the edema (by subtracting BG values). Mean values ± SE for respective groups are presented. * Control vs. 4B8M p < 0.05; # 4B8M vs. 4B8M/AH23848; 4B8M vs. 4B8M/KL-798106 p < 0.05 (test of K-W).

Article Snippet: The following antibodies were used in this study: EP1 goat polyclonal IgG (L-15, Sc-22646, 2 μg/mL), EP2 goat polyclonal IgG (E-13, Sc-22196, 1 μg/mL), EP3 rabbit polyclonal IgG (H-200, Sc-20676, 2 μg/mL), EP4 rabbit polyclonal IgG (H-160 Sc-20677, 2 μg/mL) (all Santa Cruz), and secondary polyclonal goat anti-rabbit immunoglobulins/HRP (Dako, Brookings, SD, USA, P0448, 1:2000) and polyclonal rabbit anti-goat immunoglobulins/HRP (Dako, P0449, 1:1000).

Techniques: Injection, Control

Journal: bioRxiv

Article Title: Targeting the Schwann Cell EP2/cAMP Nanodomain to Block Pain but not Inflammation

doi: 10.1101/2024.09.10.612200

Figure Lengend Snippet: a, d, f, Dose-dependent acute nociception and b, e, g, dose- and time-dependent allodynia after intraplantar (i.pl.) injection of PGE 2 , L-902,688 (L-902), butaprost (Buta) or vehicle (Veh) in C57BL/6J mice (B6). c, Schematic representation of agonists/antagonists targeting EP2 and EP4 receptors. h, Acute nociception and i, allodynia after i.pl. PGE 2 (1.5 nmol) or Veh in B6 pretreated with PF-04448948 (PF, 5nmol), BGC 20-1531 (BGC, 5 nmol) or Veh (n=8 mice per group). j, RT-qPCR for Ptger2, Ptger4 Avil and S100 mRNA in mouse dorsal root ganglia (DRG) sciatic and cutaneous Schwann cells (SCs) (DRG and sciatic SCs n=4, cutaneous n=3 independent experiments). k, l, Representative images of EP2, EP4, NeuN and S100B expression in mouse DRG and sciatic nerve tissue (scale bar: 20 μm) (n=3 subjects). m, Representative images of EP2, EP4 and SOX10 expression in mouse sciatic and cutaneous SCs (n=3 independent experiments). n-q, Acute nociception (left panel) and allodynia (right panel) after i.pl. PGE 2 or Veh in Plp-Cre, Adv-Cre or Control mice infected with AAV for selective silencing of EP4 (-Ptger4 ) ( P1p-Ptger4 or Adv-Ptger4 ) ( n,o ) or EP2 (-Ptger2 ) ( P1p-Ptger2 or Adv-Ptger2 ) ( p,q ) (n=8 mice per group). Data are mean ± s.e.m. a,d, f, h, n, o, p, q 1-way or b, e, g, i, n, o, p , q 2-way ANOVA, Bonferroni correction. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. Veh † P<0.05, †† P<0.01, ††† P<0.001, †††† P<0.0001 vs. PGE 2 /Veh, Control/ PGE 2 .

Article Snippet: Human DRG (#0062-HP-240, Gentaur), mouse DRG, human sciatic nerve (#0062-HP-261, Gentaur) and mouse sciatic nerve formalin-fixed paraffin-embedded (FFPE) sections (5 µm) were incubated with the following primary antibodies EP2 (#ab167171, rabbit monoclonal, Abcam, 1:250), EP4 (#BS-8538R, rabbit polyclonal, Bioss, 1:200), S100B (#MA1-26621, mouse monoclonal, Invitrogen, 1:50), NeuN (#MAB377, mouse monoclonal, Merck, 1:250), diluted in fresh blocking solution (PBS, pH 7.4, 5% normal goat serum (NGS)), after antibody retrieval preformed with tri-sodium citrate buffer (pH 6) for EP2 and tris-EDTA buffer (pH 9) for EP4.

Techniques: Injection, Quantitative RT-PCR, Expressing, Control, Infection

Journal: bioRxiv

Article Title: Targeting the Schwann Cell EP2/cAMP Nanodomain to Block Pain but not Inflammation

doi: 10.1101/2024.09.10.612200

Figure Lengend Snippet: a , Global cAMP formation in human Schwann cells (hSCs), after blue light stimulation (450 nm, 10 min) (n=121 cells, n=3 independent experiments) or forskolin (FSK 100 μM) (n=78 cells, n=3 independent experiments). b , Schematic representation of Cre recombinase dependent expression of bPAC and mCherry separated by T2A self-cleaving peptide sequence. c , Representative images of mCherry expression in S100B+ cells in mouse paw tissue after intraplantar (i.pl.) infection with AAV for selective expression of bPAC in SCs (scale bar: 20 μm) (n=4 subjects). d , Illustration of bPAC activation after blue light stimulation and allodynia induced by blue light stimulation (pulsed 1 s/5 s for 10 min) in Plp-Cre or Control mice infected with AAV- bPAC ( Plp-bPAC ) (n=8 mice per group). e , Representative images and cumulative data of membrane and intracellular localization of EP2 and EP4 in hSCs cells, with or without pre-incubation with unlabeled PGE 2 (10 µM, 30 min). Scale bar, 20 µm. (n=4 independent experiments). f , Concentration-dependent global cAMP formation in hSCs induced by PGE 2 (n=8 replicates). g , Membrane confined cAMP formation induced by PGE 2 (10 nM) in hSCs in the presence of PF-04418948 (PF,1 μM), BGC 20-1531 (BGC, 1 μM) or vehicle (Veh) (cells number: PGE 2 =80, PF=90, BGC=88, n=3 independent experiments). h , Illustration of membrane tagged Lyn11-bPAC membrane confined cAMP formation and cAMP formation in hSCs after blue light stimulation (n=48 cells, n=3 independent experiments) or FSK (100 μM) (n=46 cells, n=3 independent experiments). i , Allodynia induced by blue light stimulation in Plp-Cre or Control mice infected with AAV- Lyn11-bPAC ( Plp-Lyn11-bPAC ) (n =8 mice per group). j , Concentration-dependent PKA activation induced by PGE 2 in hSCs (n=8 replicates). k , Membrane confined PKA activation in hSCs induced by PGE 2 (10 nM) in the presence of PF (1μM), BGC (1 μM) or Veh (cells number: PF=64, BGC=63, PGE 2 = 64, n=3 independent experiments). Data are mean ± s.e.m. a, e , h, Student’s t test , g, k 1-way or d, i 2-way ANOVA, Bonferroni correction. AUC, area under curve. ****P<0.0001 vs. Control/blue light.

Article Snippet: Human DRG (#0062-HP-240, Gentaur), mouse DRG, human sciatic nerve (#0062-HP-261, Gentaur) and mouse sciatic nerve formalin-fixed paraffin-embedded (FFPE) sections (5 µm) were incubated with the following primary antibodies EP2 (#ab167171, rabbit monoclonal, Abcam, 1:250), EP4 (#BS-8538R, rabbit polyclonal, Bioss, 1:200), S100B (#MA1-26621, mouse monoclonal, Invitrogen, 1:50), NeuN (#MAB377, mouse monoclonal, Merck, 1:250), diluted in fresh blocking solution (PBS, pH 7.4, 5% normal goat serum (NGS)), after antibody retrieval preformed with tri-sodium citrate buffer (pH 6) for EP2 and tris-EDTA buffer (pH 9) for EP4.

Techniques: Expressing, Sequencing, Infection, Activation Assay, Control, Membrane, Incubation, Concentration Assay

Journal: The Journal of Clinical Investigation

Article Title: Antibiotic pretreatment alleviates liver transplant damage in mice and humans

doi: 10.1172/JCI127550

Figure Lengend Snippet: (A–C) Intestine tissue and serum samples from portal veins of naive and Abx-treated (10 days) C57BL/6 mice were analyzed. (C, F, and G) BALB/c livers subjected to 18 hours of cold storage were transplanted into C57BL/6 mice pretreated or not with Abx for 10 days, followed by serum and liver sampling 6 hours after OLT. (D and E) Primary mouse hepatocytes were incubated with or without PGE2 (5 μM) for the indicted time periods. (A) Western blot detection and the relative intensity ratio of COX2 in intestines of naive and Abx-treated mice. β-Actin expression served as an internal control and was used for normalization (n = 3/group). (B) Representative immunohistochemical staining of COX2 in intestines from naive and Abx-treated mice (n = 3/group). Original magnification, ×100. (C) Serum PGEM levels were measured by ELISA (n = 4–6/group). *P < 0.05 versus peripheral/naive; #P < 0.05 versus portal/naive; §P < 0.05 versus peripheral/OLT. (D) qRT-PCR detection of mRNA coding for EP4 with TBP normalization (n = 4/group). †P < 0.05 versus hepatocytes without PGE2. (E) Western blot detection and relative intensity ratios of EP4 in hepatocytes. VCL expression served as an internal control and was used for normalization (n = 2/group). (F) Western blot detection of EP4 and its relative intensity ratio with VCL normalization in OLT (n = 4/group). (G) Representative immunohistochemical staining of EP4 in OLT with or without recipient Abx treatment (n = 3/group). Original magnification, ×200. Data indicate the mean ± SEM. Statistical significance was determined by Student’s t test or 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Primary Abs detecting COX2 (catalog 12282, clone D5H5), mouse CHOP (catalog 5554, clone D46F1), human CHOP (catalog 2895, clone L63F7), p-S6K (catalog 9234, clone Thr389, 108D2), LC3B (catalog 3868, clone D11), vinculin (VCL) (catalog 18799, clone E1E9V), β-actin (catalog 12620, clone D6A8) (all from Cell Signaling Technology), and EP4 (catalog 24895-1-AP; Proteintech) were used.

Techniques: Sampling, Incubation, Western Blot, Expressing, Control, Immunohistochemical staining, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Antibiotic pretreatment alleviates liver transplant damage in mice and humans

doi: 10.1172/JCI127550

Figure Lengend Snippet: (A) Primary mouse hepatocytes were incubated with DMSO, PGE2 (5 μM), or PGE2 (10 μM) for 60 minutes. Western blot detection and relative intensity ratios of CHOP and p-S6K. VCL expression served as an internal control and was used for normalization (n = 3/group). (B) Primary mouse hepatocytes were incubated with PGE2 (5 μM) for 60 minutes, and the EP4 antagonist (20 nM, ONO-AE3-208) was supplemented 15 minutes prior to PGE2 incubation in some cell cultures. Western blot detection and relative intensity ratios of CHOP and p-S6K with VCL normalization (n = 3/group). (C) Representative immunohistochemical images show detection of LC3B (red) in hepatocytes cultured with DMSO, PGE2, or PGE2 plus the EP4 antagonist (actin: green; DAPI: blue). n = 3. Original magnification, ×400. (D) BALB/c mouse livers subjected to 18 hours of cold storage were transplanted orthotopically into allogeneic C57BL/6 mice pretreated or not with Abx for 10 days, followed by OLT sampling 6 hours after reperfusion. Western blot detection of CHOP, p-S6K, and LC3B and relative quantifications with VCL normalization in OLT (n = 4/group). (E) Representative immunohistochemical detection of LC3B (green) in OLT, with or without recipient Abx pretreatment (DAPI: blue). n = 3. Original magnification, x400. Data indicate the mean ± SEM. *P < 0.05, Student’s t test or 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Primary Abs detecting COX2 (catalog 12282, clone D5H5), mouse CHOP (catalog 5554, clone D46F1), human CHOP (catalog 2895, clone L63F7), p-S6K (catalog 9234, clone Thr389, 108D2), LC3B (catalog 3868, clone D11), vinculin (VCL) (catalog 18799, clone E1E9V), β-actin (catalog 12620, clone D6A8) (all from Cell Signaling Technology), and EP4 (catalog 24895-1-AP; Proteintech) were used.

Techniques: Incubation, Western Blot, Expressing, Control, Immunohistochemical staining, Cell Culture, Sampling

Journal: The Journal of Clinical Investigation

Article Title: Antibiotic pretreatment alleviates liver transplant damage in mice and humans

doi: 10.1172/JCI127550

Figure Lengend Snippet: BALB/c livers subjected to 18 hours of cold storage were transplanted orthotopically into C57BL/6 mice, followed by serum and liver sampling 6 hours after OLT. Some OLT recipients were treated with Abx for 10 days before OLT, with or without an oral EP4 antagonist (5 mg/kg, ONO-AE3-208 at 0 and 3 hours after reperfusion). (A) Representative H&E staining (original magnification, ×100) and TUNEL staining (original magnification, ×200). Arrowheads indicate TUNEL+ dead cells. (B) Suzuki’s histological grading of liver IRI (n = 3–5/group). (C) Quantification of TUNEL+ cells/HPF (n = 3–5/group). (D) sAST levels were measured (n = 3–5/group). (E) Western blot detection of CHOP, p-S6K, and LC3B and relative intensity ratios with VCL normalization (n = 3–4/group). Data indicate the mean ± SEM. *P < 0.05 versus OLT plus Abx, by Student’s t test or 1-way ANOVA followed by Tukey’s HSD test.

Article Snippet: Primary Abs detecting COX2 (catalog 12282, clone D5H5), mouse CHOP (catalog 5554, clone D46F1), human CHOP (catalog 2895, clone L63F7), p-S6K (catalog 9234, clone Thr389, 108D2), LC3B (catalog 3868, clone D11), vinculin (VCL) (catalog 18799, clone E1E9V), β-actin (catalog 12620, clone D6A8) (all from Cell Signaling Technology), and EP4 (catalog 24895-1-AP; Proteintech) were used.

Techniques: Sampling, Staining, TUNEL Assay, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Antibiotic pretreatment alleviates liver transplant damage in mice and humans

doi: 10.1172/JCI127550

Figure Lengend Snippet: (A) Protocol liver Bx samples were collected 2 hours after portal reperfusion (prior to the abdominal closure) from 52 primary liver transplantation patients (October 2013–August 2015). OLT recipients were classified into control (recipients with pre-OLT Abx <10 days or without Abx; n = 24) and Abx treatment (recipients with pre-OLT Abx ≥10 days; n = 28) groups. (B) Representative Western blot detection of EP4 (cases 1 and 2: control group, cases 3 and 4: Abx group) and Western blot quantification of EP4 with β-actin normalization. Data indicate the mean ± SEM. #P < 0.05, by Mann-Whitney U test. (C) Representative EP4 staining pattern in human OLT. Original magnification, ×200.

Article Snippet: Primary Abs detecting COX2 (catalog 12282, clone D5H5), mouse CHOP (catalog 5554, clone D46F1), human CHOP (catalog 2895, clone L63F7), p-S6K (catalog 9234, clone Thr389, 108D2), LC3B (catalog 3868, clone D11), vinculin (VCL) (catalog 18799, clone E1E9V), β-actin (catalog 12620, clone D6A8) (all from Cell Signaling Technology), and EP4 (catalog 24895-1-AP; Proteintech) were used.

Techniques: Transplantation Assay, Control, Western Blot, MANN-WHITNEY, Staining

Journal: Cells

Article Title: GRK2 Mediated Abnormal Transduction of PGE2-EP4-cAMP-CREB Signaling Induces the Imbalance of Macrophages Polarization in Collagen-Induced Arthritis Mice

doi: 10.3390/cells8121596

Figure Lengend Snippet: The EP4 over-desensitization resulted in the abnormal transduction of PGE2-cAMP-CREB signaling in CIA mice. ( A ) EP subtype mRNA expression in PMs of CIA mice. Values are mean ± SEM, with three mice/group. ** P < 0.01 vs. normal. ( B ) EP subtype protein expression in PMs of CIA mice. Values are means ± SEM, with three mice/group. ## P < 0.01 vs. normal. ( C ) The expression of iNOS and Arg1 in PF-04217329-stimulated PMs of CIA mice. Values are mean ± SEM, with three mice/group. “+” means that the cells were grown in the presence of PF-04217329, “−” means that the cells were grown without PF-04217329. ( D ) The expression of iNOS and Arg1 in CAY10598-stimulated PMs of CIA mice. Values are mean ± SEM, with three mice/group. # P < 0.05, ## P < 0.01 vs. normal. * P < 0.05 vs. model. “+” means that the cells were grown in the presence of CAY10598, “−” means that the cells were grown without CAY10598. ( E ) Membrane localization of EP4 and GRK2 in PGE2 stimulated-PMs of CIA. Values are mean ± SEM, with three mice/group. ## P < 0.01 vs. normal. ** P < 0.01 vs. Model. “+” means that the cells were grown in the presence of PGE2, “−” means that the cells were grown without PGE2. ( F ) Membrane localization of EP4 and GRK2 in PGE2 stimulated-BMMs of CIA. Values are mean ± SEM, with three mice/group. # P < 0.05, ## P < 0.01 vs. normal. * P < 0.05 vs. Model. “+” means that the cells were grown in the presence of PGE2, “−” means that the cells were grown without PGE2.

Article Snippet: Rabbit anti-mouse GRK2, rabbit anti-mouse EP2, rabbit anti-mouse EP3, rabbit anti-mouse EP4 and protein A/G plus agarose: Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transduction, Expressing, Membrane

Journal: Cells

Article Title: GRK2 Mediated Abnormal Transduction of PGE2-EP4-cAMP-CREB Signaling Induces the Imbalance of Macrophages Polarization in Collagen-Induced Arthritis Mice

doi: 10.3390/cells8121596

Figure Lengend Snippet: GRK2-induced over-desensitization of EP4 involved in macrophage polarization. ( A ) Co-expression of GRK2 and EP4 in PMs. With three mice/group. ( B ) Co-expression of GRK2 and EP4 in RAW (M0, M1 and M2 macrophages). Values are mean ± SEM, with three datapoints/group. ** P < 0.01 vs. M0. ## P < 0.01 vs. M1. ( C ) The co-localization of GRK2 and EP4 in RAW (M0, M1 and M2 macrophages). Original magnification × 50. ( D ) The Pearson’s correlation of GRK2 and EP4 in RAW (M0, M1 and M2 macrophages). Values are mean ± SEM, with three samples/group. ** P < 0.01 vs. M0. ## P < 0.01 vs. M1. ( E ) The membrane localization of EP4 in GRK2 siRNA-transfected RAW. Values are mean ± SEM, with three datapoints/group. ** P < 0.01 vs. WT. ( F ) The membrane localization of EP4 in Raw264.7-GRK2 −/− . Values are mean ± SEM, with three datapoints/group. ** P < 0.01 vs. NC. ( G ) The ratio of CD86/CD206 in WT RAW treated by PGE2 at different time. Values are mean ± SEM, with three datapoints/group. * P < 0.05 vs. 0 min. ( H ) The ratio of CD86/CD206 in NC RAW treated by PGE2 at different time. Values are mean ± SEM, with three datapoints/group. * P < 0.05 vs. 0 min. ( I ) The ratio of CD86/CD206 in Raw264.7-GRK2 −/− treated by PGE2 at different time. Values are mean ± SEM, with three datapoints/group. * P < 0.05, ** P < 0.01 vs. 0 min. ( J ) The membrane localization of EP4 in PGE2-stimulated WT RAW, NC RAW and Raw264.7-GRK2 −/− . Values are mean ± SEM, with three datapoints/group. ** P < 0.01 vs. untreated group.

Article Snippet: Rabbit anti-mouse GRK2, rabbit anti-mouse EP2, rabbit anti-mouse EP3, rabbit anti-mouse EP4 and protein A/G plus agarose: Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Membrane, Transfection