Journal: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: Bioactive Materials

Article Title: Immunomodulatory effects of biodegradable Mg–Cu–Zn alloy in esophageal cancer

doi: 10.1016/j.bioactmat.2026.02.046

Figure Lengend Snippet: Distribution of CD163 + M2 TAMs in AKR-derived allograft tumor tissues from immunocompetent C57BL/6 mice. (a, b) Representative IHC staining images showing CD163 + M2 TAMs in the (a) peritumoral stroma and (b) tumor islets. Lower panels display higher-magnification views of the regions outlined by red dashed boxes. (c, d) Quantification of CD163 + cells in the (c) peritumoral stroma and (d) tumor islets. (e) Comparison of CD163 + cell density between the peritumoral stroma and tumor islets. (f) Total number of CD163 + cells in allograft tumors (peritumoral stroma and tumor islets combined). (g, h) Comparison of the density between iNOS + cells and CD163 + cells in the (g) peritumoral stroma and (h) tumor islets. (i, j) Quantification of iNOS + /CD163 + ratio in the (i) peritumoral stroma and (j) tumor islets. p < 0.05 (∗), p < 0.01 (∗∗), p < 0.001 (∗∗∗). A field of view is ∼0.086 mm 2 in (c−j).

Article Snippet: Tissue sections were then incubated with primary antibodies against iNOS (22226-1-AP, ProteinTech, China), CD163 (A26411PM, Abclone, China), CD8 (SP16, Maixin, China), CD4 (SP35, Maixin, China) or Ki-67 (12202S, Cell Signaling Technology) for 12 h at 4 °C, followed by secondary antibodies (Beyotime Biotechnology, Nantong, China).

Techniques: Derivative Assay, Immunohistochemistry, Comparison

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: Establishment of the APAP-induced liver injury in mice and cell models. (A) Paraffin-embedded liver sections underwent H&E staining as well as TNF-α, IL-6 and IL-1β immunostaining; scale bars, 100 μ m. (B) Serum ALT and AST levels were measured in APAP-induced mice (n=6). (C) Representative western blots and densitometry analysis of TNF-α, IL-6 and IL-1β expression in liver extracts prepared from APAP-induced mouse liver tissues. (D) Representative western blots and densitometry analysis of SREBP-1, PPAR-α and Fasn expression in liver extracts prepared from APAP-induced mouse liver tissues. (E) Representative western blots and densitometry analysis of iNOS and NOX4 expression in liver extracts prepared from APAP-induced mouse liver tissues. (F) Representative western blots and densitometry analysis of TNF-α, IL-6 and IL-1β expression in APAP-induced AML-12 cells. (G) Representative western blots and densitometry analysis of SREBP-1, PPAR-α and Fasn expression in APAP-induced AML-12 cells. (H) Representative western blots and densitometry analysis of iNOS and NOX4 expression in APAP-induced AML-12 cells. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01, **** P<0.0001. APAP, acetaminophen; H&E, hematoxylin and eosin; IHC, immunohistochemistry; ALT, alanine aminotransferase; AST, aspartate aminotransferase; SREBP-1, sterol regulatory element-binding protein 1; PPAR-α, peroxisome proliferator-activated receptor α; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Staining, Immunostaining, Western Blot, Expressing, Immunohistochemistry, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: p38γ knockdown inhibits oxidative stress and lipid accumulation in APAP-induced AML-12 cells. Intracellular (A) MDA, (B) GSH and (C) SOD activities were assayed in AML-12 cells transfected with p38γ siRNA and pEGFP-C1-p38γ according to the manufacturer's instructions (n=3). (D) Representative images of Oil red O staining of AML12 cells transfected with p38γ siRNA and pEGFP-C1-p38γ; scale bar, 100 μ m. (E) Reactive oxygen species production was detected by DCF, DHE and MitoSOX assay after overexpression and knockdown of p38γ. Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 expression in AML-12 cells following p38γ (F) knockdown and (G) overexpression. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01, *** P<0.001. APAP, acetaminophen; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; siRNA, small interfering RNA; NC, negative control; DCF, 2',7'-dichlorofluorescein; DHE, dihydroethidium; PPAR-α, peroxisome proliferator-activated receptor α; SREBP-1, sterol regulatory element-binding protein 1; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Knockdown, Transfection, Staining, Mitosox Assay, Over Expression, Western Blot, Expressing, Small Interfering RNA, Negative Control, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: p38γ is a direct target of miR-125. (A) TargetScan was used to predict that p38γ possesses binding sites for miR-125. (B) A luciferase reporter assay was performed to detect luciferase activity in the mimics NC + MUT p38γ, miR-125 mimics + MUT p38γ, mimics NC + WT p38γ and miR-125 mimics + WT p38γ groups. (C) The mRNA levels of p38γ in cells were determined by RT-qPCR. (D) Western blot analysis of p38γ expression in AML-12 cells treated with miR-125 inhibitor or miR-125 mimics. (E) Western blot analysis of iNOS, NOX4, TNF-α, IL-6 and IL-1β expression in AML-12 cells. (F) The miR-125 expression was determined by RT-qPCR in liver injury tissues and primary hepatocytes. (G) miR-125 expression were determined by RT-qPCR in AML-12 cells. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01, *** P<0.001. ns, not significant; miR-125, microRNA-125; NC, negative control; MUT, mutant; WT, wild type; RT-qPCR, reverse transcription quantitative PCR; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4; UTR, untranslated region; APAP, acetaminophen.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing, Negative Control, Mutagenesis, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: miR-125 inhibits oxidative stress and lipid accumulation by targeting p38γ. Intracellular levels of (A) MDA and (C) GSH and (B) activity of SOD were assessed in AML-12 cells. (D) Representative images of Oil red O staining of AML12 cells; scale bar, 100 μ m. (E) Reactive oxygen species production was detected using DCF, DHE and MitoSOX assays following overexpression and knockdown of miR-125. Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 activity in AML-12 cells treated with (F) miR-125 inhibitor and (G) miR-125 mimics. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01, *** P<0.001. APAP, acetaminophen; miR-125, microRNA-125; NC, negative control; siRNA, small interfering RNA; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; DCF, 2',7'-dichlorofluorescein; DHE, dihydroethidium; PPAR-α, peroxisome proliferator-activated receptor-α; SREBP-1, sterol regulatory element-binding protein 1; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Activity Assay, Staining, Over Expression, Knockdown, Western Blot, Negative Control, Small Interfering RNA, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: miR-125 reverses APAP-induced liver injury by targeting p38γ via activation of the PI3K/AKT signaling. (A) Kyoto Encyclopedia of Genes and Genomes enrichment of the potential signaling pathways associated with significantly differentially expressed genes between the control and APAP-treated groups. Western blot analysis of p-PI3K and p-AKT expression in (B) liver injury tissues and (C) AML-12 cells. Western blot analysis of p-PI3K and p-AKT expression in AML-12 cells transfected with (D) p38γ siRNA and (E) pEGFP-C1-p38γ. Western blot analysis of p-PI3K and p-AKT expression in AML-12 cells following (F) overexpression and (G) knockdown of miR-125. (H) Chemical structure of the PI3K/AKT signaling pathway inhibitor, LY294002. (I) Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 activity in AML-12 cells following p38γ knockdown and treatment with LY294002. (J) Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 activity in AML-12 cells following p38γ overexpression and treatment with LY294002. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01, *** P<0.001. APAP, acetaminophen; miR-125, microRNA-125; NC, negative control; siRNA, small interfering RNA; p-, phosphorylated; PPAR-α, peroxisome proliferator-activated receptor α; SREBP-1, sterol regulatory element-binding protein 1; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Activation Assay, Protein-Protein interactions, Control, Western Blot, Expressing, Transfection, Over Expression, Knockdown, Activity Assay, Negative Control, Small Interfering RNA, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: miR-125 reverses APAP-induced liver injury by targeting p38γ via activation of the PI3K/AKT signaling pathway. (A) Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 expression in AML-12 cells transfected with miR-125 inhibitor and treated with LY294002. (B) Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 expression in AML-12 cells transfected with miR-125 mimics and then treated with LY294002. Western blot analysis of TNF-α, IL-6 and IL-1β expression in AML-12 cells transfected with (C) p38γ siRNA and (D) pEGFP-C1-p38γ and then treated with LY294002. Western blot analysis of TNF-α, IL-6 and IL-1β expression in AML-12 cells transfected with (E) miR-125 inhibitor or (F) miR-125 mimics and then treated with LY294002. Data are presented as the mean ± SD of at least three repeats and were compared using a one-way ANOVA followed by Bonferroni's correction. * P<0.05, ** P<0.01. ns, not significant; APAP, acetaminophen; miR-125, microRNA-125; NC, negative control; siRNA, small interfering RNA; PPAR-α, peroxisome proliferator-activated receptor α; SREBP-1, sterol regulatory element-binding protein 1; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Activation Assay, Western Blot, Expressing, Transfection, Negative Control, Small Interfering RNA, Binding Assay

Journal: International Journal of Molecular Medicine

Article Title: Hepatic p38γ exacerbates acetaminophen-induced acute liver injury via PI3K/Akt-dependent mechanisms

doi: 10.3892/ijmm.2026.5855

Figure Lengend Snippet: p38γ knockdown attenuates liver injury in APAP-treated mice. (A) Small animal imaging analysis. (B) Fluorescence imaging analysis showed that AAV9-shRNA-p38γ was specifically located in the mouse liver tissues. (C) Immunofluorescence staining of p38γ in APAP-induced mice liver injury tissues; scale bar, 100 μ m. (D) Western blot analysis of p38γ expression in mouse liver tissues. (E) The H&E staining of liver sections; scale bar, 100 and 40 μ m. (F) IHC analysis of TNF-α, IL-6 and IL-1β expression in liver extracts prepared from mice liver tissues. Serum levels of (G) AST and (H) ALT were measured (n=6). Serum levels of (I) GSH, (J) MDA and (K) SOD were measured (n=3). (L) Representative images of Oil red O staining of AML-12 cells; scale bar, 100 and 40 μ m. (M) DCF analysis of reactive oxygen species production; scale bar, 100 and 40 μ m. (N) Western blot analysis of TNF-α, IL-6 and IL-1β expression in mouse liver tissues. (O) Western blot analysis of PPAR-α, SREBP-1, Fasn, iNOS and NOX4 expression in mouse liver tissues. Data are presented as the mean ± SD of at least three repeats and were compared using a two-tailed unpaired Student's t-test. * P<0.05, ** P<0.01, *** P<0.001. APAP, acetaminophen; AAV, adeno-associated virus; shRNA, short hairpin RNA; H&E, hematoxylin and eosin; IHC, immunohistochemistry; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GSH, glutathione; MDA, malondialdehyde; SOD, superoxide dismutase; DFH, dihydrofluorescein; DCF, 2',7'-dichlorofluorescein; PPAR-α, peroxisome proliferator-activated receptor alpha; SREBP-1, sterol regulatory element-binding protein 1; Fasn, fatty acid synthase; iNOS, inducible nitric oxide synthase; NOX4, NADPH oxidase 4.

Article Snippet: The anti-p38γ (cat. no. 20184-1-AP), anti-IL-6 (cat. no. 21865-1-AP), anti-IL-1β (cat. no. 16806-1-AP), anti-albumin (cat. no. 16475-1-AP), Akt (cat. no. 10176-2-AP), anti-phosphorylated (p-)Akt (cat. no. 66444-1-Ig), anti-TNF-α (cat. no. 17590-1-AP), anti-peroxisome proliferator-activated receptor (PPAR)-α (cat. no. 66826-1-Ig), anti-sterol regulatory element-binding protein (SREBP)-1 (cat. no. 14088-1-AP) and anti-Fasn (cat. no. 10624-2-AP) antibodies were purchased from Proteintech Group, Inc. Anti-NADPH oxidase 4 (NOX4) (cat. no. A11274) and anti-inducible nitric oxide synthase (iNOS) (cat. no. A14031) were purchased from ABclonal Biotech Co., Ltd. Anti-β-actin (cat. no. AF7018), anti-PI3K (cat. no. AF3241) and anti-p-PI3K (cat. no. AF6241) were purchased from Affinity Biosciences, Ltd. ELISA kits for IL-1β (cat. no. MLB00C-1), IL-6 (cat. no. M6000B-1) and TNF-α (cat. no. MTA00B-1) were purchased R&D Systems, Inc.

Techniques: Knockdown, Imaging, Fluorescence, shRNA, Immunofluorescence, Staining, Western Blot, Expressing, Two Tailed Test, Virus, Immunohistochemistry, Binding Assay

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: Discovery of TUS as a potent therapeutic agent for COPD. (A) High-throughput screening for CXCL1 inhibitory activity of compounds from FF. RAW 264.7 macrophages were treated with 1 μg/mL LPS and 20 μM diverse compounds from FF for 24 h, and the level of CXCL1 was detected with ELISA kit. (B) Structures of sesquiterpenoids from FF. (C) The H&E or Masson’s trichrome staining images of lung tissues of mice in different groups. (D) The protein levels of iNOS, COX-2, and IL-1β in the homogenate of lung tissues of mice in different groups. (E-F) The levels of MUC5AC and SP-D in the homogenate of lung tissues of mice in different groups. (G) The percentage of neutrophil in white blood cells of mice in different groups. (H-I) The levels of TNF-α and IL-1β in BALF of mice in different groups. The results were expressed as mean ± SD (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs CS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: High Throughput Screening Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: TUS inhibited NF-κB by activating Nrf2. (A) The protein levels of p -NF-κB, IκB, iNOS, and COX-2 in the RAW 264.7 cells treated with 1 μg/mL LPS and TUS at 2.5, 5, 10, and 20 μM. (B) The regulation of TUS on p -NF-κB levels in the cytoplasm and nucleus. RAW 264.7 cells treated with 1 μg/mL LPS and TUS at 10 and 20 μM for 8 h, and the protein level of p -NF-κB in the cytoplasm and nucleus were detected using Western blot. (C) Immunofluorescence indicated that TUS inhibited p -NF-κB translocation into the nucleus. RAW 264.7 cells were treated with 1 μg/mL LPS and TUS at 10 and 20 μM for 8 h, followed by incubation with p -NF-κB primary antibody and Alex 594 secondary antibody. The immunofluorescence of p -NF-κB was observed and imaged. (D-E) The regulatory effects of TUS on protein levels of p -NF-κB, IκB, iNOS, and COX-2 in the WT and Nrf2-silenced RAW 264.7 cells. WT and Nrf2-silenced RAW 264.7 cells were treated with 10 μM TUS, and the protein levels of p -NF-κB, iNOS, and COX-2 were detected using Western blot. (F) The regulatory effects of TUS on protein levels of iNOS and COX-2 in the WT or C434A mutant RAW 264.7 cells. WT and C434A mutant RAW 264.7 cells were treated with 10 μM TUS, and the protein levels of p -NF-κB, iNOS, and COX-2 were detected using Western blot. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Western Blot, Immunofluorescence, Translocation Assay, Incubation, Mutagenesis

Journal: Journal of Advanced Research

Article Title: Tussilagone attenuated cigarette smoke-induced chronic obstructive pulmonary disease through regulating Nrf2 and NF-κB/NLRP3 inflammasome via directly targeting cysteine 434 of KEAP1

doi: 10.1016/j.jare.2025.07.019

Figure Lengend Snippet: TUS inhibited lung inflammation by activating Nrf2. (A) The images of lung tissues stained by H&E in indicated groups. (B-E) The levels of TNF-α and IL-1β in BALF of different groups. (F) The protein levels of Nrf2, iNOS, p -NF-κB, and IL-1β in the homogenate of lung tissues in indicated groups. (G) The mRNA levels of NF-κB, iNOS, NLRP3, COX-2, TNF-α, and IL-1β in the homogenate of lung tissues in indicated groups. The results were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 treated vs LPS group.

Article Snippet: The primary antibodies for KEAP1 (10503-2-AP, 1:5000), Nrf2 (16396-1-AP, 1:2000), glutamate-cystine ligase, modifier subunit (GCLM, 14241-1-AP, 1:2000), cyclooxygenase-2 (COX-2, 27308-1-AP, 1:1000), IL-1β (16806-1-AP, 1:500), β-actin (20536-1-AP, 1:30000), inducible Nitric Oxide Synthase (iNOS, 22226-1-AP, 1:2000), HA (51064-2-AP, 1:2000), inhibitor of NF-κB (IκB, 10268-1-AP, 1:2000), Caspase-1 (22915-1-AP, 1:2000), NLRP3 (27458-1-AP, 1:2000), α-tubulin (11224-1-AP, 1:10000), and the secondary antibodies HRP-conjugated goat anti-mouse IgG (66031-1-Ig, 1:10000) and HRP-conjugated goat anti-rabbit IgG (66031-2-Ig, 1:10000) were purchased from Proteintech Group (Wuhan, China).

Techniques: Staining