tlr4 inhibitor cli 095  (InvivoGen)

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin activates TLR signaling in cardiac fibroblasts (A) Cardiac fibroblasts were incubated with Hb (5 mg/mL) for the indicated times. Protein extracts were analyzed by immunoblotting for p-NFκB and p-MAPK. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB and p-MAPK band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). (B) Cardiac fibroblasts were incubated either vehicle or with varying concentrations of a TLR4 pharmacological inhibitor for 3 h. Hb (5 mg/mL) was then added. After 1 h, protein extracts were isolated and subsequently analyzed by immunoblotting for p-NFκB. Gapdh was used as a loading control. Representative immunoblots are shown on the left-hand side. Quantification was performed by normalizing p-NFκB band densities with those of the loading control. N = 4. One-sample t tests were used to compare groups to the control group (∗ p < 0.05; ns, not significant). (C) Cardiac fibroblasts were incubated with either LPS or Hb for 24 h, after which expression of the indicated pro-inflammatory cytokines was determined by qPCR. Expression values are shown relative to the housekeeping gene Gapdh. N = 6. ANOVA with Tukey post-hoc tests were used to determine significance (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Incubation, Western Blot, Control, Isolation, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Hemoglobin inhibits fibroblast-to-cardiomyocyte reprogramming via TLR2/TLR4-dependent chromatin compaction

doi: 10.1016/j.omtn.2026.102900

Figure Lengend Snippet: Hemoglobin mediates gene repression through TLR2 and TLR4 (A and B) A study was conducted to determine the effect of hemoglobin (Hb) on (A) fibroblast-to-cardiomyocyte reprogramming and (B) fibroblast gene expression. With respect to fibroblast-to-cardiomyocyte reprogramming, cardiac fibroblasts were transfected with either miR combo or a non-targeting control miR. 24 h later, the cells were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Fourteen days after miR transfection, cells were analyzed for expression of the indicated cardiomyocyte specific genes by qPCR. Expression values were normalized to the housekeeping gene Gapdh. N = 6–10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant). t test was used to determine the significance between the miR combo groups (## p < 0.01, # p < 0.05; ns, not significant). With respect to fibroblast gene expression, cardiac fibroblasts were incubated with vehicle, a TLR2 pharmacological inhibitor, a TLR4 pharmacological inhibitor, or a combination of both pharmacological inhibitors for 3 h. After incubation with the indicated pharmacological inhibitors, Hb was added (5 mg/mL) to the media. All media was replaced the next day. Expression of the indicated fibroblast-specific genes was determined by qPCR and normalized to the housekeeping gene Gapdh. N = 10. One-sample t tests were used to compare groups to the control group (∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05; ns, not significant).

Article Snippet: Hb (Millipore Sigma, H2625), TLR2 signaling inhibitor-TL2-C29 (InvivoGen, catalog no. inh-c29), and TLR4 inhibitor-CLI-095 (InvivoGen, catalog no. tlrl-cli95-4) were used.

Techniques: Gene Expression, Transfection, Control, Incubation, Expressing

Journal: Animal Models and Experimental Medicine

Article Title: Polydatin alleviates mitochondrial damage and apoptosis of lung epithelial cells by inhibiting toll‐like receptor 4‐dependent macrophage activation in asthma

doi: 10.1002/ame2.70100

Figure Lengend Snippet: Polydatin relieves ROS (reactive oxygen species) and inflammation in asthmatic mice. To investigate asthma pathology, we developed an asthma mouse model via sensitization of BALB/c mice with ovalbumin (OVA). After initial OVA exposure, animals received intraperitoneal injections of polydatin prior to subsequent aerosolized OVA challenges. (A) Structural representation of polydatin. (B) Experimental protocol diagram illustrating timeline and treatment schedules. (C) Lung resistance alterations in response to methacholine stimulation ( n = 8 per group). (D) Quantification of eosinophils (identified as CD45.2 + Siglec‐F + cells) within bronchoalveolar lavage fluid (BALF) using flow cytometry. (E) Representative histological lung sections stained with hematoxylin–eosin (HE) and periodic acid–Schiff (PAS). (F) Measurement of cytokine concentrations in BALF using ELISA (enzyme‐linked immunosorbent assay) assays ( n = 8 per group). (G) Fluorescent microscopic images demonstrating ROS (reactive oxygen species) production via dihydroethidium (DHE) staining in lung tissues. (H) WB (Western blotting) quantification of NOX2 (NADPH oxidase 2) and NOX4 (NADPH oxidase 4) protein expression in pulmonary tissues. (I) Identification of apoptotic cells using TUNEL (terminal deoxynucleotidyl transferase dUTP nick‐end labeling) staining in lung sections with corresponding quantitative analysis of positive cells ( n = 3). (J) WB detection of apoptosis‐related proteins within lung homogenates. (K) Immunohistochemical staining depicting the expression and localization of cleaved caspase‐3 and cleaved caspase‐9 proteins ( n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Prior to LPS and ATP stimulation, cells were pretreated for 1 h with polydatin (50 or 100 μmol/L), CLI‐095 (a TLR4‐specific inhibitor, CAS: 243984‐11‐4, 1 μg/mL, InvivoGen), or A438079 (a P2X7R‐specific antagonist, CAS: 899507‐36‐9, 10 μmol/L, Abcam).

Techniques: Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, TUNEL Assay, Immunohistochemical staining

Journal: Animal Models and Experimental Medicine

Article Title: Polydatin alleviates mitochondrial damage and apoptosis of lung epithelial cells by inhibiting toll‐like receptor 4‐dependent macrophage activation in asthma

doi: 10.1002/ame2.70100

Figure Lengend Snippet: Polydatin suppresses TLR4 (toll‐like receptor 4)/P2X7R/NLRP3 (NOD‐like receptor protein) activation in OVA (ovalbumin)–induced asthma. WB (Western blotting) measured (A) TLR4, IκBα, NF‐κB (nuclear factor kappa B) P65, and phosphorylation of IκBα and NF‐κB P65, and (B) P2X7R, NLRP3, ASC, and caspase‐1 in lung tissues. (C) Co‐expression of F4/80 (green) and (D) P2X7R (green) with TLR4 (red) in mouse lung tissues was assessed via immunofluorescence staining combined ( n = 3). (E) ELISA (enzyme‐linked immunosorbent assay) and real‐time PCR (polymerase chain reaction) were used to measure IL‐1β (interleukin‐1β) and IL‐18 protein levels in BALF (bronchoalveolar lavage fluid) and mRNA (messenger RNA) expression ( n = 8), and Dunnett's post hoc test (A–D) or Wilcoxon rank‐sum test (E) was performed subsequently. * p < 0.05 and ** p < 0.01.

Article Snippet: Prior to LPS and ATP stimulation, cells were pretreated for 1 h with polydatin (50 or 100 μmol/L), CLI‐095 (a TLR4‐specific inhibitor, CAS: 243984‐11‐4, 1 μg/mL, InvivoGen), or A438079 (a P2X7R‐specific antagonist, CAS: 899507‐36‐9, 10 μmol/L, Abcam).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Expressing, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, RNA Expression

Journal: Animal Models and Experimental Medicine

Article Title: Polydatin alleviates mitochondrial damage and apoptosis of lung epithelial cells by inhibiting toll‐like receptor 4‐dependent macrophage activation in asthma

doi: 10.1002/ame2.70100

Figure Lengend Snippet: Polydatin inhibits IL‐1β (interleukin‐1β) and IL‐18 by blocking the TLR4 (toll‐like receptor 4)/P2X7R pathway in macrophages. (A) Intracellular calcium concentrations were analyzed utilizing Fluo 3‐AM fluorescent staining ( n = 3). (B) WB (Western blotting) was performed to quantify the levels of TLR4, IκBα, NF‐κB (nuclear factor kappa B) p65, phosphorylated forms of IκBα and NF‐κB p65, P2X7R, NLRP3 (NOD‐like receptor protein) inflammasome components, IL‐1β, and IL‐18 ( n = 3). (C) Macrophage‐derived protein secretion levels of IL‐1β and IL‐18 were evaluated using ELISA (enzyme‐linked immunosorbent assay). (A and B) or Wilcoxon rank‐sum testing (C). * p < 0.05 and ** p < 0.01.

Article Snippet: Prior to LPS and ATP stimulation, cells were pretreated for 1 h with polydatin (50 or 100 μmol/L), CLI‐095 (a TLR4‐specific inhibitor, CAS: 243984‐11‐4, 1 μg/mL, InvivoGen), or A438079 (a P2X7R‐specific antagonist, CAS: 899507‐36‐9, 10 μmol/L, Abcam).

Techniques: Blocking Assay, Staining, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Animal Models and Experimental Medicine

Article Title: Polydatin alleviates mitochondrial damage and apoptosis of lung epithelial cells by inhibiting toll‐like receptor 4‐dependent macrophage activation in asthma

doi: 10.1002/ame2.70100

Figure Lengend Snippet: Polydatin suppresses ROS (reactive oxygen species) production and apoptosis of ECs (epithelial cells) by blockage of the TLR4 (toll‐like receptor 4)/P2X7R pathway in macrophages. (A) Intracellular ROS levels in BEAS‐2B cells were determined through DCFH‐DA fluorescence staining (10 μmol/L). (B) Alterations in MMP (mitochondrial membrane potential) were assessed using JC‐1 dye staining. (C) WB (Western blotting) analysis quantified protein expression of NOX2 (NADPH oxidase 2) and NOX4 (NADPH oxidase 4), and proteins related to apoptosis in BEAS‐2B ECs (epithelial cells). (D) Flow cytometry quantified apoptosis based on the proportion of annexin‐V + /7‐AAD + positive cells ( n = 3). (E) Additionally, WB was used to determine apoptosis‐related protein levels in BEAS‐2B cells. Histogram results represent the mean ± SEM (standard error of the mean) from three independent replicates. * p < 0.05 and ** p < 0.01.

Article Snippet: Prior to LPS and ATP stimulation, cells were pretreated for 1 h with polydatin (50 or 100 μmol/L), CLI‐095 (a TLR4‐specific inhibitor, CAS: 243984‐11‐4, 1 μg/mL, InvivoGen), or A438079 (a P2X7R‐specific antagonist, CAS: 899507‐36‐9, 10 μmol/L, Abcam).

Techniques: Fluorescence, Staining, Membrane, Western Blot, Expressing, Flow Cytometry

Journal: Animal Models and Experimental Medicine

Article Title: Polydatin alleviates mitochondrial damage and apoptosis of lung epithelial cells by inhibiting toll‐like receptor 4‐dependent macrophage activation in asthma

doi: 10.1002/ame2.70100

Figure Lengend Snippet: Schematic diagram of the mechanisms underlying the alleviation of OVA (ovalbumin)–induced asthma by polydatin. The alleviation of asthma by polydatin is dependent on the blockage of the TLR4 (toll‐like receptor 4)/P2X7R synergy in macrophages. The blockage of the TLR4/P2X7R synergy results in decreased release and secretion of IL‐1β (interleukin‐1β) and IL‐18In ECs, low IL‐1β and IL‐18 levels inhibit mitochondrial damage and apoptosis.

Article Snippet: Prior to LPS and ATP stimulation, cells were pretreated for 1 h with polydatin (50 or 100 μmol/L), CLI‐095 (a TLR4‐specific inhibitor, CAS: 243984‐11‐4, 1 μg/mL, InvivoGen), or A438079 (a P2X7R‐specific antagonist, CAS: 899507‐36‐9, 10 μmol/L, Abcam).

Techniques: