ca inhibitor 1  (MedChemExpress)

Journal: bioRxiv

Article Title: CA inhibitor 1 treatment has only transient effects on estrous cyclicity in mice

doi: 10.64898/2026.01.12.699062

Figure Lengend Snippet: (A) CA inhibitor 1 administration resulted in a significant delay in the start of the next estrous cycle. (B) CA inhibitor 1 administration did not impact estrous cycle length across the 5 cycles. (C) CA inhibitor 1 did not impact average estrous cycle length. (D) CA inhibitor 1 significantly increased the proportion of normal length cycles compared to vehicle-treated mice. (E) CA inhibitor 1 did not impact ovarian weight.

Article Snippet: Adult female mice (n = 15) were subcutaneously injected with GS-6207 analog, CA inhibitor 1 (15mg/kg, MedChemExpress, Monmouth Junction NJ, HY-124594), a long-acting HIV capsid inhibitor, or 5% DMSO in sesame oil.

Techniques:

Journal: bioRxiv

Article Title: CA inhibitor 1 treatment has only transient effects on estrous cyclicity in mice

doi: 10.64898/2026.01.12.699062

Figure Lengend Snippet: (A) Schematics of location of mPOA. (B) Representative image of GFAP immunoreactivity in the mPOA of vehicle-treated mice. (C) Representative image of GFAP immunoreactivity in the mPOA of Ca inhibitor 1-treated mice. (D) CA inhibitor 1 did not impact GFAP immunoreactivity in the mPOA. (E) Representative image of ΔfosB immunoreactivity in the mPOA of vehicle-treated mice. (F) Representative image of ΔfosB immunoreactivity in the mPOA of CA inhibitor 1-treated mice. (G) CA inhibitor 1 did not impact ΔfosB immunoreactivity in the mPOA.

Article Snippet: Adult female mice (n = 15) were subcutaneously injected with GS-6207 analog, CA inhibitor 1 (15mg/kg, MedChemExpress, Monmouth Junction NJ, HY-124594), a long-acting HIV capsid inhibitor, or 5% DMSO in sesame oil.

Techniques:

Journal: iScience

Article Title: Bioluminescence imaging reveals enhanced SARS-CoV-2 clearance in mice with combinatorial regimens

doi: 10.1016/j.isci.2024.109049

Figure Lengend Snippet:

Article Snippet: VX-765, Caspase 1/4 inhibitor , InvivoGen , Cat# Inh-vx765i-1 CAS 273404-37-8.

Techniques: Blocking Assay, Virus, Variant Assay, Expressing, Luciferase, Recombinant, Modification, Saline, Lysis, Electron Microscopy, Isolation, Multiplex Assay, Staining, Antibody Labeling, Oligo Synthesis, Software, Real-time Polymerase Chain Reaction, Spectrophotometry, Membrane, Stripping Membranes, In Vivo Imaging

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Altered morphological features and phagocytic function in microglia of Iba-1 EGFP Tg mice brain after Pyk2-Inh infusion. A The experimental schedule for brain infusion assay in Iba1-EGFP Tg 7-week-old mice (n = 3/group). B Confocal images of GFP positive microglia (green) and β-amyloid (red). Obtained brain slices (thickness ≤ 1.0 mm) were treated with RapiClear 1.52 for tissue clearing and immunohistochemical assay was conducted. C , D Morphological changes were analyzed by IMARIS software. Cell surface, dendrite volume, and dendrite length were quantified. E Spot analysis by IMARIS software. F , G Quantification of the number of β-amyloid spots per cell and number of microglia deposits. H Representative confocal image of multinucleated microglia (indicated red arrow). Brain slices were stained with anti-NeuN (white) and nuclear DNA was stained with DAPI (white). Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Immunohistochemical staining, Software, Staining

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Inhibition of Pyk2 signaling induces multinucleation formation in microglia. A Morphological changes between mononuclear and multinucleated microglia. Immunocytochemistry of MG6 cells stained with anti-Iba1 (green) and anti-β-tubulin (magenta). Nuclear DNA was labeled with DAPI (white). Scale bar is 20 μm. B Cells were treated with Pyk2-Inh for 24 h. Phase contrast images (upper panel) and DAPI stained images (lower panel) were shown. Circles are indicated as multinucleated cells. C The quantification of the number of multinucleated cells/well (control: n = 4, Pyk2-Inh-500 nM: n = 4, Pyk2-Inh-1000 nM). D Cell proliferation was assessed by CCK-8. MG6 cells were treated with 100 μg/mL CSF1 and indicated concentration of Pyk2-Inh (n = 6 per group, One-way ANOVA, p < 0.05). E Representatives immunoblot images. The ratio of p-Pyk2/Pyk2, pFAK/FAK, p-ERK/ERK, p-Src/Src and Mannose receptor/Tubulin were described under the blot. Pyk2-Inh was administered as a pre-treatment two hours before stimulation with CSF1 (100 μg/mL). F Image quantification and analysis were performed. The same experiments were repeated three times. All full-length uncropped original western blots are included in a Sup. Fig. 4. Values are mean ± SD. #: negative control (without CSF1) vs control (with CSF1), ##: p < 0.01; *: control (with CSF1) vs Pyk2-Inh (with CSF1), *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Inhibition, Immunocytochemistry, Staining, Labeling, Control, CCK-8 Assay, Concentration Assay, Western Blot, Negative Control

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Multinucleated cells are generated from abscission failure in cytokinesis, not cell fusion. A Dynamics of cell division by time-lapse video microscopy. The phase contrast images of the microglia’s mitosis at different times. Nuclei were observed with Hoechst 33,342 staining. Arrowheads indicate representative images from anaphase to cytokinesis (control: n = 4, Pyk2-Inh: n = 4). B Cell division was quantified, categorizing it as either appropriate or inappropriate, in both control (n = 4) and Pyk2-Inh treated groups (n = 4). C Fluorescence images of microglia cells showing the stages of cell division. Immunocytochemistry of MG6 cells stained with anti-Pericentrin (red), anti-β-tubulin (green) and phalloidin (white). Nuclear DNA was labeled with DAPI (blue). Independent experiments were performed at least three times

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Generated, Microscopy, Staining, Control, Fluorescence, Immunocytochemistry, Labeling

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Comparison of phagocytotic function in multinucleated versus mononucleated microglia. A Immunofluorescence imaging depicting phagocytosis of pHrodo Red E.coli BioParticles by mononuclear and multinucleated MG6 cells after exposure to Pyk2-Inh. Cells were treated with pHrodo Red E. coli BioParticles for 1 h and fixed with paraformaldehyde. Actin cytoskeleton and nuclear DNA were stained with phalloidin (green) and DAPI (white), respectively. Engulfed red fluorescein were detected by microscopy and analyzed by IMARIS software (lower panel). B Bioparticles engulfed in each cell were quantified and compared. multi/n means divided by the number of nuclei in multinucleated microglia (n = 4 per group). C Mononuclear and multinucleated microglia were separated by fluorescence-activated cell sorting (FACS) and treated pHrodo-Red E. coli for 30 min. D , E Sorted Cells were labeled with pHrodo Red E. coli BioParticles for the indicated time and fluorescein was measured by fluorescence detector (n = 8 per group). On 4-h incubation with BioParticles, fluorescein intensity was indicated in bar graph. F CCK-8 assay was used to measure the proliferation of each type of microglia. Sorted cells were incubated for 24 h and proliferation assay were conducted (n = 4 per group). Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Comparison, Immunofluorescence, Imaging, Staining, Microscopy, Software, Fluorescence, FACS, Labeling, Incubation, CCK-8 Assay, Proliferation Assay

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Pyk2 inhibition stimulates β-amyloid-induced phagocytotic function in multinucleated microglia. A Immunoblot of microglia cells after treatment with 1000 nM Pyk2-Inh and 500 ng/mL β-amyloid oligomers (1–42). Cells were treated with 100 ng/mL CSF-1 and 1000 nM Pyk2-Inh for 24 h. Cell lysates were subjected to immunoblotting of p-Pyk2/Pyk2, Lamp1 and p-Erk/Erk. Tubulin was used as a loading control. B The quantification of immunoblotting data measured by ImageJ. The same experiments were repeated three times. All full-length uncropped original western blots are included in a Sup. Fig. 5. #: significance between negative control (without CSF1) and control (with CSF1) under β-amyloid absence, #: p < 0.05; ##: p < 0.01; *: significance between control (with CSF1) and Pyk2-Inh (with CSF1) under β-amyloid presence, *: p < 0.05; **: p < 0.01; ns; not significant. C Immunocytochemical imaging depicted phagocytosis of Aβ (1–42, 500 ng/mL)-Fluor 555 (red) labeled oligomers by microglia cells after exposure to Pyk2-Inh. Cells were stained with anti-Iba1 (cyan) and nuclear DNA was stained with DAPI (white). Scale bar is 20 μm. D The quantification of number of spots per cell. After obtaining three-dimensional images, β-amyloid are quantified by a spot analysis using ImageJ. E Lysosomal function was assessed using DQ ™ Green BSA. Intracellular accumulation of fluorescein derivatives (green) was observed. Scale bar is 100 μm. F Fluorescence intensities of dequenched BODIPY resulting from proteolysis activity were measured using a fluorescence plate reader. G The transcriptional expression of Trem2, Cd36, Cd74 and Apoe in control and Pyk2 inhibitor treated group in microglia cells. Values are mean ± SD. *: p < 0.05; **: p < 0.01; ***: p-value < 0.001; ****: p-value < 0.0001

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Inhibition, Western Blot, Control, Negative Control, Imaging, Labeling, Staining, Fluorescence, Activity Assay, Expressing

Journal: Journal of Neuroinflammation

Article Title: Enhanced phagocytosis associated with multinucleated microglia via Pyk2 inhibition in an acute β-amyloid infusion model

doi: 10.1186/s12974-024-03192-7

Figure Lengend Snippet: Pyk2 inhibition reduces LPS-induced inflammation in human microglia. A The transcriptional expression of IL1β, IL6 and TNF in control and Pyk2-Inh treated group in human microglia cells, HMC3. B The secreted protein level of IL-6 was measured by ELISA. C The time-course Immunoblotting images of phosphor-NF-kB and NF-kB in HMC3 cells after LPS stimulation and with or without Pyk2-Inh. β-actin was used for loading control. The same experiments were repeated three times. D Quantification of immunoblotting data was measured by ImageJ. All full-length uncropped original western blots are included in a Sup. Fig. 6. Values are mean ± SD. # : significance between negative control and LPS control, #: p < 0.05; ##: p < 0.01; *: significance between LPS control and Pyk2-Inh treated groups, *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant

Article Snippet: Pyk2 inhibitor (Pyk2-Inh, PF-431396, CAS no. 717906-29-1) was purchased from TOCRIS Biosciences/Bio-techne (Minneapolis, MN) and dissolved in dimethyl sulfoxide (DMSO) (Sigma, MO), then diluted to corresponding concentrations.

Techniques: Inhibition, Expressing, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: CNTNAP2 is cleaved by α-secretase and furin. a , b ADAM10 and ADAM17 cleaved mature CNTNAP2 (mCNT) in HEK cells. HEK cells were co-transfected with CNTNAP2 and empty vector, ADAM10 or ADAM17 plasmids, and harvested 24 h after transfection for Western Blot analysis. Non-transfected HEK (NT HEK) was used as a negative control. CTFα1 and CTFα2 were increased/generated by ADAM10 and ADAM17. n = 4 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. c , d ADAM10 is the major α-secretase that generates CTFα1. CNTNAP2 was co-transfected with GFP into HEK, ADAM10-knockout (KO), or ADAM17-KO HEK cells. The CTFα1/mCNT ratio was reduced in ADAM10-KO cells while increased in ADAM17-KO cells. n = 3 independent experiments, unpaired t -test, ** p < 0.01. e , f Furin cleaved mature CNTNAP2 in HEK cells. HEK cells were co-transfected with CNTNAP2 and vector or furin. CTFf at 55 kDa was increased by furin. n = 3 independent experiments, unpaired t -test, * p < 0.05. g CNTNAP2 on the cell membrane was cleaved in HEK and N2a cells. CNTNAP2 was co-transfected with vector (myc), ADAM10, ADAM17, or furin plasmids into HEK and N2a cells. Cells were fixed 24 h after transfection, and immunocytochemistry (ICC) was performed. CNTNAP2 was detected by an anti-CNTNAP2 antibody targeting the N-terminal 1001–1042 aa outside the cell membrane. CNTNAP2 was mainly expressed on the cell membrane and was reduced by co-transfected plasmids. Images were acquired using Zeiss Apotome to reflect the morphology. Scale bar represents 50 μm. h Quantification of ICC in HEK and N2a. CNTNAP2 mean intensity was measured using the corresponding conventional fluorescence images in Supplementary Fig. . n = 10 cells from 2 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Transfection, Plasmid Preparation, Western Blot, Negative Control, Generated, Knock-Out, Membrane, Immunocytochemistry, Fluorescence

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Sequential cleavages of furin, α- and γ-secretase. a Western blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. Exposure 1 was used to analyze CTFf in the ADAM17 overexpressing cells and C96 in the ADAM10 overexpressing cells; exposure 2 was used for assessing C79 and C96 in the ADAM17 overexpressing cells. TAPI-1 (10 μM) is an ADAM17-specific inhibitor; FI-2 (20 μM) stands for Furin inhibitor-2. b Quantification of the dynamic changes of mature CNTNAP2 and CTF levels in the ADAM10/ADAM17 transfected cells upon inhibitor treatments in ( a ). n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. c , d Dynamic changes of CTFs in the furin transfected cells, showing the furin-ADAM17 cleavages. n = 3 independent experiments, ordinary one-way ANOVA followed by Tukey’s multiple comparisons test, * p < 0.05; ** p < 0.01; *** p < 0.001. P values stand for comparisons with the Control group unless noted by the brackets. e Western Blot of control (Vector), ADAM10 overexpressing, and ADAM17 overexpressing HEK cells. GSI is γ-secretase inhibitor L-685,458 (20 nM). f Quantification of the sequential cleavage by α- and γ-secretase in ( e ). n = 3 independent experiments, unpaired t -test, * p < 0.05, ** p < 0.01. g Schematic of CNTNAP2 processing. Mature CNTNAP2 undergoes sequential cleavages by furin and α-, γ-secretase. Furin cleaves CNTNAP2 to generate CTFf, which is further processed by α-secretase. α-secretase ADAM10 and ADAM17 share the same cutting sites but have different site preferences. ADAM10 primarily cleaves at I79 to produce the predominant C79, and ADAM17 preferably cleaves at L96 to produce the weaker C96. C96 is subsequently processed by α-secretase into C79. γ-secretase further cleaves C79 at L53 within the transmembrane domain to generate CICD. All the results are expressed as mean ± SEM

Article Snippet: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Identification of α-secretase cleavage sites on CNTNAP2. a Peptide in vitro cleavage. Two peptides were incubated in the assay buffer with or without ADAM17 at 37 °C for 24 h and then were sent to Mass Spectrometry analysis. The extracted ion chromatogram showed two intact peptides (top) and cleaved complementary fragments (bottom). Peptide 1 (M108-G84) was cleaved by ADAM17 at 97H/L96. Peptide 2 (L96-N72) was cleaved at 80 A/79I. b N-terminal sequencing results identified the first 5 amino acids (aa) of CTFα1 as IRNGV and the first 5 aa of CTFα2 as LDSAS. The bottom sequence shows the last 108 to 72 aa of CNTNAP2 from the C-terminus and α-secretase cleavage sites at L96 and I79. c Protein ladders of the C-terminal CNTNAP2. C79 corresponded to the size of CTFα1, and C96 showed the same size as CTFα2. d , e Mutations at the α-secretase cleavage sites L96 and I79 affected CNTNAP2’s cleavage. Wildtype (WT) or mutant plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) into HEK cells. Alterations in the migration rate were observed in constructs containing D98R. Two upper/lower blots show samples from the same experiment. The parallel blots were processed in the same electrophoresis chamber and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: In Vitro, Incubation, Mass Spectrometry, Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Migration, Construct, Electrophoresis, Over Expression

Journal: Signal Transduction and Targeted Therapy

Article Title: Contactin-associated protein-like 2 (CNTNAP2) mutations impair the essential α-secretase cleavages, leading to autism-like phenotypes

doi: 10.1038/s41392-024-01768-6

Figure Lengend Snippet: Pathogenic mutations affect CNTNAP2 processing. a Schematic of CNTNAP2 protein and locations of pathogenic mutations found in ASD patients. Mutations predicted deleterious or at conserved sites are noted in red. ADAM10/17-mediated α-cleavage affected by the mutations was summarized in the brackets (10 = ADAM10, 17 = ADAM17; slight affections are noted in purple, while severe impacts are marked in red). SP signal peptide, DISC discoidin-like domain, L1-4 four laminin A-like G domains, FBG fibrinogen-like domain, E1-2 two epidermal growth factor (EGF)-like domains, TM transmembrane domain; 4.1b, 4.1 binding domain; PDZb, PSD-95/Discs large/zona occludens-1 (PDZ) binding domain. Braces represent three lobes of CNTNAP2 protein. The figure is to scale. b , c CNTNAP2 mutations affected expression patterns of CNTNAP2 full-length and CTFs. CNTNAP2 plasmids were co-transfected with GFP into HEK cells. n = 3 independent experiments, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test, * p < 0.05; ** p < 0.01; **** p < 0.0001. d , e Altered α-secretase cleavage in CNTNAP2 mutations. CNTNAP2 plasmids were co-transfected with empty vector (EV), ADAM10 (AD10), or ADAM17 (AD17) plasmids into HEK cells. Mutations showed variant cleavage patterns. Three blots show samples from the same experiment. The blots were processed in parallel and scanned together simultaneously. n = 3 independent experiments, two-way ANOVA followed by Dunnett’s multiple comparisons test (compare ADAM10 and ADAM17 with Control for each plasmid), row factor = mutation, column factor = ADAM10/17 overexpression. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. All the results are expressed as mean ± SEM

Article Snippet: ADAM17 inhibitor TAPI-1 (Cat# CAS 171235-71-5), Furin inhibitor 2 (Cat# SCP0148), ADAM10 inhibitor GI254023X (Cat# SML0789) and γ-secretase inhibitor L-685,458 (Cat# L1790) were purchased from Sigma-Aldrich.

Techniques: Binding Assay, Expressing, Transfection, Plasmid Preparation, Variant Assay, Mutagenesis, Over Expression

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: The increased expression of h-PGDS/PGD2/HO-1 was responsible for the inhibitory effects of RosA on H1N1 virus-induced NF-κB and P38 MAPK activation. A The expression of h-PGDS in H1N1 virus-infected cells was analyzed by Western blotting. B Relative protein expression of h-PGDS was normalized to GAPDH levels. C ELISA assay was performed to measure the levels of PGD 2 in the culture supernatant. D The levels of PGD 2 in the culture supernatant of h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection were quantified by ELISA assay. E The levels of P-p65 and P-p38 in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection were detected by Western blotting. F Relative protein expression of h-PGDS, P-IKBα, P-p65 and P-p38 was normalized to GAPDH levels. G Luminex assay was performed to measure the levels of pro-inflammatory cytokines (IL-6 and TNF-α) in the culture supernatant of h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection. H Western blot analysis of P-IKBα, P-p65 and P-p38 in H1N1 virus-infected cells treated with RosA alone or in combination with h-PGDS inhibitor. I The relative expression of P-IKBα, P-p65 and P-p38 expression was quantified relative to GAPDH. J Luminex assay were performed to measure the levels of pro-inflammatory cytokines (IL-6, IL-8, IP-10, TNF-α, MCP-1 and RANTES) in H1N1 virus-infected cells treated with RosA alone or in combination with h-PGDS inhibitor. K The expression of HO-1 in H1N1 virus-infected cells was detected by Western blotting. L HO-1 protein levels were quantified by normalizing to GAPDH levels. M Western blot analysis of HO-1 in H1N1 virus-infected cells treated with RosA alone or in combination with h-PGDS inhibitor. N Relative HO-1 expression was quantified relative to GAPDH. O The levels of HO-1 in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection were detected by Western blotting. P Relative HO-1 expression was quantified relative to GAPDH. Q Representative immunofluorescence images of h-PGDS (pink) and HO-1 (red) in lung SpC + (green) alveolar epithelial cells. R Quantitative analysis of fluorescence intensities for h-PGDS and HO-1 in SpC + alveolar epithelial cells. S The levels of PGD 2 in the lung homogenates were determined by ELISA assay. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: RosA (Fig. A) was purchased from MedChem Express (HY-N0529; Shanghai, China). h-PGDS inhibitor (h-PGDS inhibitor 1; CAS NO. 1033836-12-2) was acquired from AdooQ Bioscience (Irvine, CA, USA).

Techniques: Expressing, Virus, Activation Assay, Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation, Transfection, Luminex, Immunofluorescence, Fluorescence

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Effects of RosA on H1N1 virus-induced apoptosis. A The apoptosis of H1N1 virus-infected cells was harvested for flow cytometry analysis after RosA treatment for 24 h. B Apoptosis percentage in H1N1 virus-infected A549 cells treated with or without RosA. C Western blot analysis of cleaved PARP and cleaved caspase 3 in H1N1 virus-infected cells treated with RosA. D The relative expression of cleaved PARP and cleaved caspase 3 was quantified relative to GAPDH. E H1N1 virus-infected A549 cells were treated with RosA alone or in combination with h-PGDS inhibitor for 24 h. Flow cytometry was used to identify the apoptosis of these cells. F The percentage of apoptosis in A549 cells with H1N1 virus-infected A549 cells treated for 24 h with RosA alone or in conjunction with an h-PGDS inhibitor. G Western blot analysis of cleaved PARP and cleaved caspase 3 in H1N1 virus-infected cells treated with RA alone or in combination with h-PGDS inhibitor. H The relative expression of cleaved PARP and cleaved caspase 3 expression was quantified relative to GAPDH. I The apoptosis of cells in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus-infection were detected by flow cytometry. J The percentage of apoptosis in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection. K The levels of cleaved PARP and cleaved caspase 3 in h-PGDS overexpression (h-PGDS OE) plasmid-transfected A549 cells with or without H1N1 virus infection were detected by Western blotting. L The relative expression of cleaved PARP and cleaved caspase 3 expression was quantified relative to GAPDH. M Analysis of CD3 + CD8 + T lymphocytes by flow cytometry from peripheral blood. N Quantification of the proportions of CD3 + CD8 + T lymphocytes in the peripheral blood of H1N1 virus-infected mice. O Immunofluorescence staining of Granzyme B (pink) and TNF-α (red) in CD8 + T lymphocytes (green) of the lung tissues. P The relative fluorescence intensities of Granzyme B and TNF-α in CD8 + T lymphocytes were calculated. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: RosA (Fig. A) was purchased from MedChem Express (HY-N0529; Shanghai, China). h-PGDS inhibitor (h-PGDS inhibitor 1; CAS NO. 1033836-12-2) was acquired from AdooQ Bioscience (Irvine, CA, USA).

Techniques: Virus, Infection, Flow Cytometry, Western Blot, Expressing, Over Expression, Plasmid Preparation, Transfection, Immunofluorescence, Staining, Fluorescence

Journal: Chinese Medicine

Article Title: Rosmarinic acid treatment protects against lethal H1N1 virus-mediated inflammation and lung injury by promoting activation of the h-PGDS-PGD 2 -HO-1 signal axis

doi: 10.1186/s13020-023-00847-0

Figure Lengend Snippet: Effects of h-PGDS inhibition on RosA’s protective effects against H1N1 virus-mediated lung injury. Two days before infection with mouse-adapted H1N1 virus (5LD 50 ), mice in the group with the combination of RosA with h-PGDS inhibitor were treated intraperitoneally with h-PGDS inhibitor for 30 min, and then administrated intragastrically (i.g.) with RosA. A At day 7 p.i., gross pathology showed edema and hemorrhage (white arrows) in the lung tissues. B The severity of exudation and edema were elevated by the lung index (lung/body weight ratio). C At day 7 p.i., H&E staining was used to assess the lung histological alterations caused by H1N1 viruses. Black arrows show: (i) bronchi with epithelial sloughing; (ii) peribronchitis; (iii) perivasculitis; (vi) alveolar collapse and leukocyte infiltration. D Histological scoring of lung injury. E Representative immunofluorescence images of active caspase 3 (red) and TUNEL (green) assay in lung SpC + (pink) alveolar epithelial cells. F Quantitative analysis of fluorescence intensities for active caspase 3 and TUNEL in SpC + alveolar epithelial cells. G The expression of IL-6 and TNF-α in lung SpC + alveolar epithelial cells was detected by immunofluorescence. H Quantitative analysis of fluorescence intensities for IL-6 (pink) and TNF-α (red) in SpC + (green) alveolar epithelial cells. I Levels of pro-inflammatory mediators (IL-6, MCP-1, RANTES and TNF-α) in the lung homogenates were determined by Luminex assay. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: RosA (Fig. A) was purchased from MedChem Express (HY-N0529; Shanghai, China). h-PGDS inhibitor (h-PGDS inhibitor 1; CAS NO. 1033836-12-2) was acquired from AdooQ Bioscience (Irvine, CA, USA).

Techniques: Inhibition, Virus, Infection, Staining, Immunofluorescence, TUNEL Assay, Fluorescence, Expressing, Luminex