Journal: Redox Biology

Article Title: Reprogramming of the kynurenine pathway impairs NAD + homeostasis and mediates doxorubicin-induced cardiotoxicity in mice

doi: 10.1016/j.redox.2025.103957

Figure Lengend Snippet: The kynurenine pathway links NAD + metabolism and the TCA cycle. A , Schematic diagram of the KP pathway and its metabolites. B , Western blotting results of SIRT1, as well as KP pathway enzymes including IDO1, KYNU, HAAO, ACMSD and QPRT in the WT and IDO1KO cardiomyocytes treated with DOX, and β-actin as load control (n = 3). C–H , Quantification of (B). The protein level was standardized by β-actin and the value in WT-CTL group was designated as 1. I , IF staining of IDO1, ACMSD and QPRT in cardiomyocytes with or without DOX treatment. J , NAD + levels of each group (n = 5). K , ATP levels of each group (n = 5). The values are presented as means ± SD. Statistical analysis was performed by two-way ANOVA with Tukey's multiple comparisons test (C–H, J, K). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Adjusted P values were provided in the case of multiple groups. NAD + , nicotinamide adenine dinucleotide; TCA, tricarboxylic acid cycle; KP, kynurenine pathway; DOX, doxorubicin; KYNU kynureninase; HAAO, 3-hydroxyanthranilate 3,4-dioxygenase; ACMSD, α-amino-β-carboxy-muconate-semialdehyde decarbo-xylase; QPRT, quinolinate phosphoribosyl-transferase. SIRT1, sirtuin 1.

Article Snippet: To track the metabolic flux dynamics of the kynurenine pathway following ACMSD inhibition, cardiomyocytes were incubated with 13 C-tryptophan (MCE, China).

Techniques: Western Blot, Control, Staining

Journal: Redox Biology

Article Title: Reprogramming of the kynurenine pathway impairs NAD + homeostasis and mediates doxorubicin-induced cardiotoxicity in mice

doi: 10.1016/j.redox.2025.103957

Figure Lengend Snippet: Effect of KP pathway on DOX-induced cardiomyocytes damage and NAD + levels in vitro . Primary cardiomyocytes are treated with DOX along or DOX with Trp, NA, NAM for 24 h: A , NAD + levels decrease in the presence of different concentration DOX (n = 5). B , NAD + levels decrease after DOX treatment, whereas increase after co-treatment with Trp, NR and NMN (n = 5). C and D , IDO1 protein levels of WT and Ido1 −/− cardiomyocytes (n = 3). E and F , TUNEL staining of DOX, DOX + Trp treated WT and Ido1 −/− cardiomyocytes (n = 3). G , Cell survival rate of WT and Ido1−/− cardiomyocytes exposed to 1 μM DOX at different times using CCK-8 assay. H , Representative IF staining images of SIRT1. I , Representative images of JC-1 staining of each group (Scale bar = 50 μm). J , Representative flow cytometry histograms showing TMRE fluorescence intensity in cardiomyocytes treated under different conditions. K , Quantitative analysis of TMRE fluorescence intensity across experimental groups. H , ROS levels in different groups. Data are expressed as the means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons test (A, B, D) and two-way ANOVA with Tukey's multiple comparisons test (F, G, K, L). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗P < 0.0001. Adjusted P values were provided in case of multiple groups. IDO1, indoleamine 2,3-dioxygenase 1; KP, kynurenine pathway; DOX, doxorubicin; NAD + , nicotinamide adenine dinucleotide; Trp, tryptophan; NA, nicotinic acid; NAM, nicotinamide; ROS, reactive oxygen species. SIRT1, Sirtuin 1; WT, wild type.

Article Snippet: To track the metabolic flux dynamics of the kynurenine pathway following ACMSD inhibition, cardiomyocytes were incubated with 13 C-tryptophan (MCE, China).

Techniques: In Vitro, Concentration Assay, TUNEL Assay, Staining, CCK-8 Assay, Flow Cytometry, Fluorescence

Journal: Redox Biology

Article Title: Reprogramming of the kynurenine pathway impairs NAD + homeostasis and mediates doxorubicin-induced cardiotoxicity in mice

doi: 10.1016/j.redox.2025.103957

Figure Lengend Snippet: ACMSD inhibition increases NAD + levels in mice. A , Western blotting result of ACMSD, QPRT, SIRT1 and ACO2 in different groups of cardiac tissues from mice. B, Quantification of A (n = 3). The level of each protein was standardized by β-actin and the value in WT-CTL group was designated as 1. C , Representative images of DHE staining and D , Quantification of DHE staining (n = 3). E , levels of NAD + in mouse heart tissue of each group (n = 9). F , levels of ATP in mouse heart tissue of each group (n = 9). G-K , Representative concentrations of different metabolite measured by LS/MS/MS in mouse cardiac tissues treated with vehicle, DOX and DOX + TES-1025: Trp (G), Kyn (H), QA (I), 3-HAA (J) and KA (K) (n = 9). L , levels of Kyn/Trp ratio in each group. M , levels of QA/Kyn ratio in each group . N , levels of QA/3-HAA ratio in each group. O , levels of KA/Kyn ratio in each group. The values are presented as means ± SD. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparisons test (B, D-O). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Adjusted P values were provided in the case of multiple groups. ACMSD, α-amino-β-carboxy-muconate-semialdehyde decarboxylase; QPRT, quinolinate phosphoribosyl-transferase; Trp, tryptophan; Kyn, kynurenine; QA, quinolinic acid; KA, kynurenic acid; 3-HAA, 3-hydroxyanthranilic acid.

Article Snippet: To track the metabolic flux dynamics of the kynurenine pathway following ACMSD inhibition, cardiomyocytes were incubated with 13 C-tryptophan (MCE, China).

Techniques: Inhibition, Western Blot, Staining, Tandem Mass Spectroscopy

Journal: Journal of Translational Medicine

Article Title: Kynurenine-mediated redox regulation provides neuroprotection in central retinal artery occlusion

doi: 10.1186/s12967-025-07214-7

Figure Lengend Snippet: Alterations in tryptophan metabolism and IDO expression in the UPOAO model. ( A ) Metabolomics of tryptophan expression in serum from patients with CRAO and the controls. ( B ) Targeted retinal tryptophan metabolomics showing differential metabolites between sham and UPOAO groups (one pooled sample with 5 retinas). ( C ) Schematic of the tryptophan-kynurenine metabolic pathway. ( D ) qPCR analysis of Ido1, Kat1, Kat2a, Kat2b, Kmo, and Kynu mRNA expression in retinas ( n = 5, 1 technical replicate of 5 biological replicates for each group). ( E ) Western blot demonstrating reduced IDO1 protein levels in UPOAO retinas vs. sham controls ( n = 8, 2 technical replicates of 4 biological replicates for each group). ( F ) Immunofluorescence images showing decreased IDO1 expression (green) in UPOAO retinal sections compared to sham ( n = 3, 1 technical replicate of 3 biological replicates for each group). Nuclei counterstained with DAPI (blue). Scale bar, 20 µm. We employed ImageJ analysis software to measure the fluorescence intensity of IDO1-positive staining in the RGC layer of each sample, and then normalized these values to those of the control sample to obtain the relative intensity. The data in ( A - B ) and ( D - F ) were analyzed by unpaired Student’ s t-test

Article Snippet: Kynurenine powder (10 mg, MCE, China) was weighed and dissolved in 1 mL of sterile PBS solution.

Techniques: Expressing, Western Blot, Immunofluorescence, Software, Fluorescence, Staining, Control

Journal: Journal of Translational Medicine

Article Title: Kynurenine-mediated redox regulation provides neuroprotection in central retinal artery occlusion

doi: 10.1186/s12967-025-07214-7

Figure Lengend Snippet: Kynurenine treatment attenuates OGD-induced injury in R28 cells. ( A ) CCK-8 assay determining the optimal therapeutic concentration of kynurenine ( n = 6, 1 technical replicate of 6 biological replicates for each group). ( B ) Western blot analysis of apoptosis-related proteins BAX and cleaved caspase 3 expression across experimental groups ( n = 3, 1 technical replicate of 3 biological replicates for each group). ( C ) Apoptosis assessment using Propidium Iodide (PI)/Calcein-AM dual staining (1 technical replicate of 4 biological replicates for each group). Scale bar, 50 μm. ( D ) Quantitative apoptosis measurement by TUNEL assay (1 technical replicate of 3 biological replicates for each group). Scale bar, 50 μm. One-way ANOVA with Bonferroni post hoc analysis was used

Article Snippet: Kynurenine powder (10 mg, MCE, China) was weighed and dissolved in 1 mL of sterile PBS solution.

Techniques: CCK-8 Assay, Concentration Assay, Western Blot, Expressing, Staining, TUNEL Assay

Journal: Journal of Translational Medicine

Article Title: Kynurenine-mediated redox regulation provides neuroprotection in central retinal artery occlusion

doi: 10.1186/s12967-025-07214-7

Figure Lengend Snippet: Exogenous kynurenine supplementation mitigates visual impairment and RGC apoptosis in UPOAO model mice. ( A ) Experimental design: Mice were divided into three groups (PBS + Sham; PBS + UPOAO; Kyn + UPOAO). Intravitreal injection was administered in left eyes 1 day before modeling, followed by sham surgery or UPOAO induction. Analyses performed at day 7. ( B ) Representative ERG waveforms (stimulus intensity: 10 cd·s/m²) across groups. ( C ) Quantification of ERG b-wave amplitudes (1 technical replicate of 6 biological replicates for each group). ( D ) H&E-stained retinal sections showing structural integrity and RGC morphology and quantification of cells in ganglion cell layer (GCL) (1 technical replicate of 4 biological replicates for each group). Scale bar, 50 μm ( E ) Immunofluorescence identification of RGCs using RBPMS antibody and quantification of RGCs per field (1 technical replicate of 4 biological replicates for each group). Scale bar, 50 μm. ( F ) Representative whole-mount retinal preparations and RGC counts from retinal whole-mounts (1 technical replicate of 4 biological replicates for each group). Scale bar, 50 μm. ( G ) Retinal immunofluorescence staining of cleaved caspase 3 (CC3) expression (apoptotic cells shown by yellow arrows). Scale bar, 20 μm and quantification of CC3 expression levels ( n = 4, 1 technical replicate of 4 biological replicates for each group). The data in ( C ) were analyzed by Two-way ANOVA with Bonferroni’s post hoc test. One-way ANOVA with Bonferroni post hoc analysis was used in ( D - G )

Article Snippet: Kynurenine powder (10 mg, MCE, China) was weighed and dissolved in 1 mL of sterile PBS solution.

Techniques: Injection, Staining, Immunofluorescence, Expressing

Journal: Journal of Translational Medicine

Article Title: Kynurenine-mediated redox regulation provides neuroprotection in central retinal artery occlusion

doi: 10.1186/s12967-025-07214-7

Figure Lengend Snippet: Kynurenine alleviates oxidative stress in R28 cells following OGD. ( A ) Flow cytometry analysis of ROS levels. Quantification of ROS-positive cells using FlowJo software ( n = 3, 1 technical replicate of 3 biological replicates for each group). ( B ) Retinal ROS generation was quantitatively determined by measuring DHE fluorescence intensity ( n = 4, 1 technical replicate of 4 biological replicates for each group). ( C ) qPCR analysis of Nrf2, Sod1, Gstp1, and Gpx1 mRNA expression ( n = 6, 1 technical replicate of 6 biological replicates for each group). ( D ) Western blot showing NRF2 and AhR protein expression across groups ( n = 3, 1 technical replicate of 3 biological replicates for each group). ( E ) Retinal immunofluorescence staining detected AhR expression levels in UPOAO-treated mice and sham control groups ( n = 4, 1 technical replicate of 4 biological replicates for each group). ( F ) Western blot showing NRF2 and AhR protein in retinal tissue from UPOAO-treated mice and sham control groups ( n = 3, 1 technical replicate of 3 biological replicates for each group). ( G ) Immunofluorescence localization of AhR (green) and nuclei (DAPI, blue) in R28 cells with or without OGD treatment. Scale bar, 2 μm. One-way ANOVA with Bonferroni post hoc analysis was used in ( B - F )

Article Snippet: Kynurenine powder (10 mg, MCE, China) was weighed and dissolved in 1 mL of sterile PBS solution.

Techniques: Flow Cytometry, Software, Fluorescence, Expressing, Western Blot, Immunofluorescence, Staining, Control

Journal: Journal of Translational Medicine

Article Title: Kynurenine-mediated redox regulation provides neuroprotection in central retinal artery occlusion

doi: 10.1186/s12967-025-07214-7

Figure Lengend Snippet: Kynurenine mediates cytoprotection partial through the AhR-NRF2 pathway. ( A ) Western blot analysis of NRF2, apoptosis-related proteins BAX and cleaved caspase 3 expression across experimental groups ( n = 3, 1 technical replicate of 3 biological replicates for each group). ( B ) qPCR analysis of Nrf2, Sod1, Gstp1, and Gpx1 mRNA expression in treated R28 cells. ( n = 6, 1 technical replicate of 6 biological replicates for each group). ( C ) Quantitative apoptosis measurement by TUNEL assay ( n = 4, 1 technical replicate of 4 biological replicates for each group). One-way ANOVA with Bonferroni post hoc analysis was used in ( A - C )

Article Snippet: Kynurenine powder (10 mg, MCE, China) was weighed and dissolved in 1 mL of sterile PBS solution.

Techniques: Western Blot, Expressing, TUNEL Assay