nitric oxide assay kit  (Nanjing Jiancheng Bioengineering Research Institute Co Ltd)

Journal: Journal of Thoracic Disease

Article Title: Glycyrrhizic acid attenuates sepsis-induced acute lung injury by regulating macrophage M2 polarization via PRKCD

doi: 10.21037/jtd-2026-1327

Figure Lengend Snippet: GA promotes M2 polarization via PRKCD to alleviate SI-ALI. (A) Immunohistochemistry analysis of PRKCD expression. (B) Hematoxylin and eosin staining of lung tissues. (C) Assessment of pulmonary edema by W/D ratio. (D) Immunofluorescence analysis of iNOS and Arg1 expression in lung tissues. (E) ELISA analysis of IL-1β and TNF-α levels in BALF. Data are presented as mean ± SD (n=6 mice per group). For multiple-group comparisons, one-way ANOVA followed by Tukey’s post hoc test was applied. *, P<0.05; ***, P<0.001; ****, P<0.0001. ANOVA, analysis of variance; BALF, bronchoalveolar lavage fluid; DAPI, 4',6-diamidino-2-phenylindole; ELISA, enzyme-linked immunosorbent assay; GA, glycyrrhizic acid; IL-1β, interleukin-1 beta; NC, negative control; PRKCD, protein kinase C delta; SD, standard deviation; SI-ALI, sepsis-induced acute lung injury; TNF-α, tumor necrosis factor-alpha; W/D, wet-to-dry weight.

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies: inducible nitric oxide synthase (iNOS, A14031, ABclonal, Wuhan, China) and arginase 1 (Arg1, A1847, ABclonal).

Techniques: Immunohistochemistry, Analysis, Expressing, H&E Stain, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Negative Control, Standard Deviation

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