Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring chemicals caused BEAS-2B epithelial cell barrier dysfunction. BEAS-2B cells were grown in transwell inserts in complete medium. Once reached a monolayer and 80–85 % confluency, cells were serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM (A) 98 % Menthone. (B) L -Menthone, (C). Carvone (D) WS-23 (E) Acetoin, (F) Vanillin, (G) PG/VG, and (H) Benzoic Acid. Transepithelial electrical resistance (TEER) and voltage (mV) data were collected pretreatment (0 hr), 6, 8, 20, and 24 hrs. following the treatments and the correlation of TEER and mV vs. time ± SEM are represented. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. untreated control., two-way ANOVA. N = 3 wells per chemical treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Control

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents elicited an interleukin 6 cytokine response in lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100µM L -Menthone, 98 % Menthone, Carvone, WS-23, Vanillin, Acetoin, Benzoic Acid, and PG/VG. Apical conditioned media was collected after the 24-h time point and IL-6 was quantified. (A) control, PG/VG, and Benzoic Acid-induced response, and (B) L -Menthone, 98 % Menthone, Carvone, WS-23, Vanillin, and Acetoin response compared to untreated control. IL-6 concentration in pg/mL ± SEM is represented, *p < 0.05. vs. control, one-way ANOVA. N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Control, Concentration Assay

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents elicited an interleukin-8 cytokine response in lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. Apical conditioned media was collected after the 24-h time point and IL6 was quantified. (A) control, PG/VG, and Benzoic Acid-induced response, and (B) l-menthone, 98 % menthone, carvone, WS-23, vanillin, and acetoin response compared to untreated control. IL-8 concentration in pg/mL ± SEM is represented, *p < 0.05, and **p < 0.01 vs. untreated control. one-way ANOVA. N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Control, Concentration Assay

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents caused minimum cytotoxicity in BEAS-2B cells. BEAS-2B cells cultured in transwells in complete media, at 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, Cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. At the 24-h time point, cells were collected and stained with acridine orange and propidium iodide and the live, cell, and total cells were counted using CellDrop automatic cell counter. Cytotoxicity ± SEM is represented. *p < 0.05 vs. control, one-way ANOVA, N = 3 wells per treatment.

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, Staining, Control

Journal: Toxicology Reports

Article Title: Comparative toxicity of menthol- and tobacco-flavored electronic cigarette constituents inducing inflammation, epithelial barrier dysfunction, and nicotinic acetylcholine receptor modulation in the absence of nicotine

doi: 10.1016/j.toxrep.2026.102224

Figure Lengend Snippet: Menthol and tobacco flavoring constituents caused nicotinic acetylcholine receptor (nAchR) modulation in BEAS-2B lung epithelial cells. BEAS-2B cells cultured in transwells in complete media, 80–85 % confluency, and serum deprived overnight. Around 90–95 % confluency, cells were treated with 100 μM l-menthone, 98 % menthone, carvone, WS-23, vanillin, acetoin, benzoic acid, and PG/VG. At the 24-h time point, cells were collected, lysed, and after BCA protein estimation, 5 μg of protein were loaded to 10-well gel for SDS-gel electrophoresis. After cellulose membrane transfer and blocking, the membranes were probed with primary antibodies for nAchR1,4,5, and 7, with ß-actin loading control for normalization. The same membrane was sometimes re-probed up to 3 times with a different CHRNA. The blots with (A) Nicotinic Acetylcholine Receptors α1 expression with acetoin and PG/VG. (B) Nicotinic Acetylcholine Receptors α4 expression with carvone and WS-23. (C) Nicotinic Acetylcholine Receptors α5 expression with acetoin and PG/VG. (D) Nicotinic Acetylcholine Receptors α5 expression with l-menthone and 98 % menthone. (E) Nicotinic Acetylcholine Receptors α7 expression with carvone and WS-23. All respective CHRNA bands ß-actin are shown with their densitometry fold-change ± SEM. *p < 0.05 and ****p < 0.0001 vs. control, one-way ANOVA. N = 3 wells per chemical. Full blots are shown in the .

Article Snippet: Human bronchial epithelial cells (BEAS-2B) (ATCC) were seeded on the apical side of 12 mm diameter polyester membrane transwell inserts with 0.4 μM pore size and 1.12 cm 2 surface area (Corning #3460) in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 1:1 (Gibco, Cat# 11320033) supplemented with 5 % fetal bovine serum (FBS), 15 mM HEPES, 1 % L -glutamine, and 1 % antibiotic-antimycotic.

Techniques: Cell Culture, SDS-Gel, Electrophoresis, Membrane, Blocking Assay, Control, Expressing

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: Occludin modulates LPS-induced IL-8 secretion, barrier integrity, and cytoskeletal remodeling in human bronchial epithelial cells. (A) The BEAS-2b cells were transfected and were then incubated with LPS in a time-dependent manner before the generation of total cell lysates, and occludin transcripts were assessed by qRT-PCR. ∗ p < 0.05 compared to the control. (B) The cells were treated with LPS in a time-dependent manner. The occludin-specific antibody was assessed by Western blot analysis. β-actin was used as a loading control. (C) A construct expressing wild-type occludin or siRNA-occludin was transiently transfected into BEAS-2b cells. The cells were washed and serum-starved overnight. They were subsequently treated with LPS for 2 h and a cytokine assay was performed with cultured media and cell lysates were harvested for qRT-PCR of IL-8 (D) . The density of the resulting spots of IL-8 was measured using a densitometric analysis. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT occludin-treated transfectants. (E) The cells were transfected with either the wild-type occludin construct or siRNA-occuldin before incubation with LPS for various times, and then TEER testing was performed. Error bars represent the SEM of at least three independent experiments. (F) Cells were then treated with LPS for 2 h. F-actin staining was performed using ActinRed 555 ReadyProbe reagent (Molecular Probes) following the manufacturer's instructions. Cell nuclei were stained with diluted Deep Red (1:300). The fluorescence intensity was analyzed and statistically evaluated (right panel). ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT occludin-treated transfectants. All data are representative of at least three independent experiments.

Article Snippet: Human bronchial epithelial (BEAS-2b) cells were purchase from ATCC (CRL-9609), and cells were cultured in BEBM (Lonza, Basel, Switzerland) with a BEGM kit at 37 °C in a humidified incubation with 5% CO 2 .

Techniques: Transfection, Incubation, Quantitative RT-PCR, Control, Western Blot, Construct, Expressing, Cytokine Assay, Cell Culture, Staining, Fluorescence

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: The C-terminal domain of occludin reduces IL-8 expression and F-actin formation in BEAS-2b cells (A) The occludin deletion mutants were imaged. (B) The cells were transfected with each occludin deletion mutant construct and treated with LPS for 2 h, the IL-8 transcripts were assessed by qRT-PCR and TEER testing was performed (C) . ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with other deletion mutant construct-treated transfectants. (D) After transfected with deletion mutant construct, cells were then treated with LPS for 2 h. F-actin staining was performed using ActinRed 555 ReadyProbe reagent (Molecular Probes) following the manufacturer's instructions. Cell nuclei were stained with diluted Deep Red (1:300). The fluorescence intensity was analyzed and statistically evaluated (lower panel). ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT occludin-treated transfectants. All data are representative of at least three independent experiments.

Article Snippet: Human bronchial epithelial (BEAS-2b) cells were purchase from ATCC (CRL-9609), and cells were cultured in BEBM (Lonza, Basel, Switzerland) with a BEGM kit at 37 °C in a humidified incubation with 5% CO 2 .

Techniques: Expressing, Transfection, Mutagenesis, Construct, Quantitative RT-PCR, Control, Staining, Fluorescence

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: A peptide based on the core sequence of the occludin C-terminal domain modulates LPS-induced inflammatory responses. (A) Peptides were synthesized with a Tat region (italic amino acids) based on the C-terminal domain core sequence of occludin and also tagged with FITC for tracing. A mutant peptide was synthesized by substituting the core sequence YTT with AAA. (B) The BEAS-2b cells were treated with wild-type occludin peptide (pepWT OCLN) or mutant occludin peptide (pepMut OCLN) for 30 h and incubated with LPS for 2 h. The occludin transcripts were assessed by qRT-PCR (B) , TEER testing (C) , and F-actin formation (D) were performed (C) . ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with wild-type occludin peptide-treated cells. The fluorescence intensity was analyzed and statistically evaluated (right panel). All data are representative of at least three independent experiments.

Article Snippet: Human bronchial epithelial (BEAS-2b) cells were purchase from ATCC (CRL-9609), and cells were cultured in BEBM (Lonza, Basel, Switzerland) with a BEGM kit at 37 °C in a humidified incubation with 5% CO 2 .

Techniques: Sequencing, Synthesized, Mutagenesis, Incubation, Quantitative RT-PCR, Control, Fluorescence

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: Comparison of mRNA expression in the BEAS-2b cells overexpressed occludin. BEAS-2B cells were transfected with wild-type occludin or siRNA-occludin for 24 h and then incubated with LPS for 2 h, after which RNA sequencing analysis was performed. (A) The heat map shows the general regulation pattern of inflammation-related genes that are differentially expressed when WT occludin and siRNA-occludin were treated with LPS, an inflammatory substance. Each row represents a gene, and the columns represent the values of the control group treated only with LPS and the WT occludin and siRNA-occludin groups with LPS. This was used by converting each log value into a fold change value. All genes were adjusted to have the same mean and standard deviation, the unit of change is the standard deviation from the mean, and the color value range of each row is the same. (B) Significant genes were selected using Gene category chat (Fold change value of 2.00 and normalized data (log2) value of 4.00). The above pie chart shows the distribution of four gene categories when comparing LPS versus control, WT occludin + LPS/LPS, and siRNA-occludin + LPS/control. The bar graph below shows RED = upregulated, GREEN = downregulated for each gene category, and shows the number of upregulated and downregulated genes in each gene category. (C) The most markedly altered categories were summarized based on the results shown in panel B. (D) The protein-protein interaction network constructed by the STRING database differentially displays commonly occurring genes by comparing WT occludin + LPS/LPS, siRNA-occludin + LPS/LPS, and LPS/control. These nodes represent proteins associated with inflammation, and these connecting lines denote interactions between two proteins. Different line thicknesses indicate types of evidence used in predicting the associations.

Article Snippet: Human bronchial epithelial (BEAS-2b) cells were purchase from ATCC (CRL-9609), and cells were cultured in BEBM (Lonza, Basel, Switzerland) with a BEGM kit at 37 °C in a humidified incubation with 5% CO 2 .

Techniques: Comparison, Expressing, Transfection, Incubation, RNA Sequencing, Control, Standard Deviation, Construct

Journal: Redox Biology

Article Title: Prophylactic C-terminal occludin–derived peptide attenuates LPS-induced airway inflammation via barrier preservation and mitochondrial ROS regulation

doi: 10.1016/j.redox.2026.104119

Figure Lengend Snippet: The peptide regulates mitochondrial dysfunction and ROS production by inhibiting LPS-induced p38 activation. (A) The BEAS-2b cells were treated with wild-type occludin peptide (pepWT OCLN) or mutant occludin peptide (pepMut OCLN) and incubated with LPS for 15, 30 min. The phospho-specific and total antibodies were assessed by Western blot analysis. β-actin was used as a loading control. (B) The BEAS-2b cells were transfected with p38 overexpression construct (WT p38) or siRNA-p38 for 24 h and incubated with LPS for 4 h siRNA-scramble was used as a negative control. The proinflammatory cytokine transcripts were assessed by qRT-PCR. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT p38-transfected cells. (C) The mitochondrial membrane potential of LPS-induced BEAS-2b cells treated with either WT OCLN peptide or mut peptide was stained with JC-1 dye. Images are representative results of 3 independent experiments. (D) The mitochondria fission was stained using phospho-Drp1 antibody and visualized. The fluorescence intensity was analyzed and statistically evaluated (right panel). ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS treatment; ∗∗∗ p < 0.05 compared with WT OCLN peptide-treated transfectants. (E) After the BEAS-2b cells were harvested, cell lysates were used for MTT assay. (F) After mitochondria from the cells was isolated, the mitochondria lysates were used for mtROS measurement. ∗ p < 0.05 compared with control; ∗∗ p < 0.05 compared with LPS only; ∗∗∗ p < 0.05 compared with LPS- and WT occludin peptide-treated cells. All data shown are representative of three independent experiments.

Article Snippet: Human bronchial epithelial (BEAS-2b) cells were purchase from ATCC (CRL-9609), and cells were cultured in BEBM (Lonza, Basel, Switzerland) with a BEGM kit at 37 °C in a humidified incubation with 5% CO 2 .

Techniques: Activation Assay, Mutagenesis, Incubation, Western Blot, Control, Transfection, Over Expression, Construct, Negative Control, Quantitative RT-PCR, Membrane, Staining, Fluorescence, MTT Assay, Isolation