Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: ADGRG1 have the capacity to serve as potential target for IVDD treatment. (A) Primary human NPCs were treated with TBHP at concentrations of 50 μM, 100 μM, 200 μM for 12 h, and the cellular ferrous ion levels were assessed by confocal microscopy using FerroOrange probe. FerroOrange fluorescence intensity was quantified, n = 3. Scale bar, 50 μm. (B) GSEA enrichment plots of gene data sets associated with oxidative damage response, ferroptosis, permeabilize mitochondria and mitochondrial respiratory chain complex assembly in TBHP‐treated primary NPCs, compared to the control. (C) Primary NPCs were treated with TBHP for 12 h and cell lysates were subjected to immunoblotting with indicated ADGRG1, IVDD and ferroptosis-related antibodies. (D) Volcano plot of differentially expressed genes in NPCs between the TBHP‐treated group and the control. |log2FC| > 2, FDR < 0.05. (E-F) Primary NPCs were incubated with TBHP for 12 h, followed by immunofluorescent staining with anti-ADGRG1 (green) and anti-4-HNE (red) antibodies and examination by confocal microscopy. The fluorescence intensity was quantified. n = 3. Scale bar, 50 μm. (G) Magnetic resonance imaging (MRI) showed the IVDD Pfirrmann grade of needle puncture IVDD rat models with 25G (MI-IVDD), 21G(MOD-IVDD) and18G (SE-IVDD), and statistical analysis was performed, n = 3. (H) Immunoblotting analysis showed the expression of IVDD markers (COL1A1) and ADGRG1 in IVDD rat models with different degrees of needle-puncture degeneration, n = 3. (I) Feature plots depict the average expression of ADGRG1 (color-scaled) across each cell cluster. (J) Immunoblotting analysis showed the expression of IVDD markers (COL1A1) and ADGRG1 in the NP tissue of clinical MI/SE degenerative IVDs. The relative ADGRG1 grayscale was quantified in (L), n = 10. (K) Representative images for colocalization analysis of ADGRG1 (green), 4-HNE (red) fluorescence in MI and SE NP tissues. n = 5. Scale bar, 50 μm. (L) Relative ADGRG1 grayscale in (J) and fluorescence intensity in (K) were quantified. All data are expressed as the mean ± SD. For panels A) and F–H), data were analyzed using one-way ANOVA with Tukey's multiple comparisons, while panel L) was assessed using a two-tailed unpaired Student's t-test. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: Confocal Microscopy, Fluorescence, Control, Western Blot, Incubation, Staining, Magnetic Resonance Imaging, Expressing, Two Tailed Test

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: In vitro evaluation of the cargo transfer capacity and therapeutic potential of A1TP-HX-EVs. (A) Immunoblotting analysis confirmed the overexpression of GFP-tagged ADGEG1 protein in NPCs by retrovirus. (B) The live-cell workstation demonstrated the uptake of AIE-labeled A1TP-HX-EVs by NPCs in both the control group (GFP) and the ADGRG1 overexpression group (ADGRG1-GFP) within 24 h. (C) Representative images from the live-cell workstation showed the uptake of DPA-labeled HX-EVs at different concentrations by TBHP-treated NPCs within 24 h. Scale bar, 50 μm. (D) Representative images from the live-cell workstation showed the uptake of AIE-labeled A1TP-HX-EVs (DPA 10 μM) by primary NPCs within 24 h after treatment with different concentrations of TBHP for 12h. Scale bar, 50 μm. (E) AIE fluorescence intensity in (C-D) were quantified. n = 3. ∗ P < 0.05. ∗∗ P < 0.01. ns, not significant. (F) Primary NPCs were stained with FerroOrange probe and assessed by confocal microscopy. n = 3. Scale bar, 50 μm. (G) GSEA enrichment analysis of ferroptosis and oxidative damage response-related gene sets in A1TP-HX-EVs‐treated group and TBHP group NPCs. (H) Primary NPCs were induced with 100 μM TBHP for 12 h, and then treated with EVs, HX-EVs or A1TP-HX-EVs for 24 h. Cell lysates were immunoblotted with antibodies against IVDD markers and ferroptosis-related proteins and ADGRG1. (I) GSEA enrichment analysis of the mitochondrial respiratory chain complex assembly and transcriptional activation of mitochondrial biogenesis-related gene sets in A1TP-HX-EVs‐treated group and TBHP group NPCs. (J-K) Representative oxygen consumption traces of primary NPCs induced with TBHP and then treated as indicated for 24 h. Maximal respiration of NPCs were quantified. n = 3. (L) Primary NPCs were induced with TBHP, and then treated as indicated for 24 h, followed by immunofluorescent staining with anti-TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. All data are expressed as the mean ± SD. For B) and E), two‐way ANOVA with Tukey 's multiple comparison tests were used for statistical analysis. For panels F) and K-L), one‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001. ns, not significant.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: In Vitro, Western Blot, Over Expression, Labeling, Control, Fluorescence, Staining, Confocal Microscopy, Activation Assay, Comparison

Journal: Bioactive Materials

Article Title: ADGRG1-targeted hypoxia preconditioned extracellular vesicles ameliorate intervertebral disc degeneration by delivering taurine to disrupt the oxidative stress feedback loop-driven ferroptosis in nucleus pulposus cells

doi: 10.1016/j.bioactmat.2026.02.029

Figure Lengend Snippet: Taurine is the key small molecule in A1TP-HX-EVs that activated the AMPK/NRF2 pathway to regulate nucleus pulposus cell repair. (A) The LC-MS/MS analysis was used to detect the differential active small molecule components between placental HX-EVs and EVs. (B) The SMPDB enrichment analysis identified pathways related to small molecules that are up-expressed in HX-EVs compared to EVs. The metabolic pathways marked in red are related to ferroptosis inhibition and mitochondrial function. (C) Volcano plot of small molecule in HX-EVs versus EVs. |log2FC| > 0.5, FDR <0.05. (D) The content of taurine in placental MSC (pMSC), hypoxia-induced pMSC(HX-pMSC) and their derived EVs was detected by ELISA. n = 3. (E) Primary NPCs cells were induced with TBHP, and then treated with EVs, HX-EVs, and A1TP-HX-EVs for 24 h. The cell lysates were subjected to ELISA assay to detect taurine content. (F) Two shRNA lentiviruses were designed to knock down TAUT a key enzyme in taurine uptake in pMSC. (G) The content of taurine in TAUT-sh1-pMSC and TAUT-sh2-pMSC derived EVs (KD-HX-EVs) was detected by ELISA. n = 3. (H) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (I) Primary NPCs were induced with TBHP, and then treated with A1TP-HX-EVs and A1TP-KD-HX-EVs for 24 h, followed by immunofluorescent staining with anti- TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (J) A CDO1-overexpressing retrovirus was designed to overexpress CDO1 in pMSCs. (K) The content of taurine in CDO1-OE-pMSC derived EVs (OE-EVs) was detected by ELISA. n = 3. (L) Primary NPCs were induced with TBHP, and then treated with treated A1TP-EVs and A1TP-OE-EVs for 24 h. Cell lysates were immunoblotted with indicated antibodies. (M) Primary NPCs were induced with TBHP, and then treated with A1TP-EVs and A1TP-OE-EVs for 24 h, followed by immunofluorescent staining with anti-TOM20 (green) and anti-4-HNE (red) antibodies. n = 3. Scale bar, 50 μm. (N-O) Representative oxygen consumption traces of primary NPCs induced with TBHP and then treated with A1TP-HX-EVs, A1TP-KD-HX-EVs, or A1TP-OE-EVs for 24 h. Maximal respiration of NPCs were quantified. n = 3. All data are expressed as the mean ± SD. For E), I), M) and O), one‐way ANOVA with Tukey's multiple comparison tests were used for statistical analysis. For D), G) and K), two‐tailed unpaired Student's t‐tests were used for statistical analysis. ∗ P < 0.05. ∗∗ P < 0.01. ∗∗∗ P < 0.001. ns, not significant.

Article Snippet: After permeabilization and blocking with 10% goat serum containing 0.2% Triton X-100, the sections were incubated with primary antibodies against ADGRG1 (1:50; sc-390192, Santa Cruz Biotechnology), TOM20(11802-1-AP, Proteintech), 4-HNE(68538-1-Ig, Proteintech), 4-HNE(HY-P81208, MCE), Ferritin (Rockland 200-401-090-0100), LAMP1(65051-1-Ig, Proteintech), PAX1(sc-514352, Santa Cruz Biotechnology), FOXF1(PA5-83039, Thermo Fisher).

Techniques: Liquid Chromatography with Mass Spectroscopy, Inhibition, Derivative Assay, Enzyme-linked Immunosorbent Assay, shRNA, Knockdown, Staining, Comparison, Two Tailed Test

Journal: Redox Biology

Article Title: Ferroptosis-related mechanisms in prion diseases provide insights into neurodegeneration and reveal therapeutic implications

doi: 10.1016/j.redox.2026.104155

Figure Lengend Snippet: GPX4 and SLC7A11 expressions decreased in impaired astrocytes of ME7-infected mice, meanwhile, 4-HNE increased. (A-B) Representative immunofluorescence image and relative fluorescence intensity of GPX4 expression in the thalamus region of ME7-infected mice or non-infected controls (CTL), showing GPX4 (green) in astrocytes expressing the marker GFAP (red) (n = 3 mice per group). Nuclei stained with DAPI are shown in blue. Scale bar = 50 μm. (C-D) Representative immunofluorescence images and relative fluorescence intensity of SLC7A11 expression in the thalamus region of brains from ME7-infected mice or non-infected controls (CTL), showing SLC7A11 (green) in astrocytes expressing the marker GFAP (red) (n = 3 mice per group). Nuclei stained with DAPI are shown in blue. Scale bar = 50 μm. (E-F) Representative immunofluorescence images and relative fluorescence intensity of 4-HNE protein expression in the thalamus region of ME7-infected mice or non-infected controls (CTL), showing 4-HNE (green) in astrocytes expressing the marker GFAP (red) (n = 3 mice per group). Nuclei stained with DAPI are shown in blue. Scale bar = 50 μm. (G) Representative western blots showing GPX4, SLC7A11, and 4-HNE levels in the brain homogenates of ME7-infected and control mice. (H) Quantification of the immunoblots in (G). (I) Immunofluorescence image showing GPX4 (green) and NeuN (red) expression in the thalamus region of ME7-infected mice or non-infected controls (CTL). Scale bar = 50 μm. (J-K) Relative fluorescence intensity of NeuN and GPX4 compared to controls. Data are presented as the mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by Student's two-tailed t-test GPX4: Glutathione peroxidase 4, SLC7A11: Solute carrier family 7 member 11, 4-HNE: 4-Hydroxynonenal, NeuN: Neuronal nuclei, GPX4: Glutathione peroxidase 4.

Article Snippet: The sections were incubated overnight at 4 °C in a humidity chamber with glial fibrillary acidic protein (GFAP, 1:50, Santa Cruz Biotechnology, sc-33673), GPX4 (1:50, Santa Cruz Biotechnology, sc-166570), SLC7A11 (1:100, ABclonal, A13685), 4-HNE antibody (1:50, Bioss Inc., bs-6313R), and NeuN (1:50, Abcam, ab177487) overnight in a humidity chamber at 4 °C.

Techniques: Infection, Immunofluorescence, Fluorescence, Expressing, Marker, Staining, Western Blot, Control, Two Tailed Test

Journal: Molecules

Article Title: The Differential Redox Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress

doi: 10.3390/molecules31091454

Figure Lengend Snippet: ( A , B ) The predicted binding mode of Alvelestat (yellow sticks) within the wt HNE binding site (PDB ID: 2Ζ7F, gray surface/cartoon). Key residues are shown as gray lines; H-bonds are indicated by yellow dashed lines. ( C ) An interaction fraction plot between Alvelestat and selected HNE residues over the MD trajectory. ( D ) A 2D interaction diagram depicting the main contacts formed by Alvelestat within the active site. H-bonds are reported as red arrows. Atoms are colored coded: oxygen in red, nitrogen in blue, sulfur in yellow.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31091454/s1 , Figure S1: Superimposition of the Alvelestat predicted binding mode by docking calculations with the cognate ligand WQQ co-crystalized with HNE (PDB ID 5ABW): Figure S2: Molecular dynamics analysis of the Alvelestat (ALV) complex with HNE in the wild-type (wt) and oxidized (HNE_OX2) forms; Figure S3: 100 ns MD simulation of the wtHNE–Sivelestat pre-complex; Figure S4: Mass spectra of Alvelestat and Sivelestat under control and oxidative conditions; Figure S5: Surface representation of HNE highlighting the positions of the four disulfide bridges (yellow) relative to the catalytic site; Figure S6: Effect of disulfide bond overoxidation on HNE flexibility and catalytic geometry; Figure S7: Molecular dynamics analysis of the sivelestat (SIL)–HNE complex in wild-type (wt) and oxidized (HNE_OX2) forms.

Techniques: Binding Assay

Journal: Molecules

Article Title: The Differential Redox Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress

doi: 10.3390/molecules31091454

Figure Lengend Snippet: Induced-fit and covalent docking results for Sivelestat. ( A , B ) Most representative predicted pre-acylation complex of Sivelestat (red sticks) within the HNE binding site from MD simulation (PDB ID: 2Z7F, gray surface/cartoon with key residues shown as gray lines). ( C ) An interaction fraction plot between Sivelestat and selected HNE residues over the MD trajectory. ( D ) A 2D interaction diagram depicting the main contacts formed by Sivelestat within the active site. H-bonds are reported as red arrows. ( E ) The predicted acylated HNE complex with the pivaloyl moiety of Sivelestat from covalent docking. ( F ) Superposition of the predicted acylated complex with crystal structures of porcine pancreatic elastase acylated at Ser195 with an m-toluoylcarbonyl group (PDB ID: 8B53, yellow) and a cyclopropylcarbonyl group (PDB ID: 8B1Y, pale green). H-bonds and π−π stackings are indicated by yellow and blue dashed lines, respectively. Atoms are colored coded: oxygen in red, nitrogen in blue, sulfur in yellow.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31091454/s1 , Figure S1: Superimposition of the Alvelestat predicted binding mode by docking calculations with the cognate ligand WQQ co-crystalized with HNE (PDB ID 5ABW): Figure S2: Molecular dynamics analysis of the Alvelestat (ALV) complex with HNE in the wild-type (wt) and oxidized (HNE_OX2) forms; Figure S3: 100 ns MD simulation of the wtHNE–Sivelestat pre-complex; Figure S4: Mass spectra of Alvelestat and Sivelestat under control and oxidative conditions; Figure S5: Surface representation of HNE highlighting the positions of the four disulfide bridges (yellow) relative to the catalytic site; Figure S6: Effect of disulfide bond overoxidation on HNE flexibility and catalytic geometry; Figure S7: Molecular dynamics analysis of the sivelestat (SIL)–HNE complex in wild-type (wt) and oxidized (HNE_OX2) forms.

Techniques: Binding Assay

Journal: Molecules

Article Title: The Differential Redox Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress

doi: 10.3390/molecules31091454

Figure Lengend Snippet: IC 50 determination of Alvelestat (black circle) and Sivelestat (blue square) against HNE activity. Dose–response curves were obtained using an expanded set of inhibitor concentrations to improve curve fitting and definition of upper and lower plateaus. Values represent the mean ± standard deviation obtained from three separate experiments.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31091454/s1 , Figure S1: Superimposition of the Alvelestat predicted binding mode by docking calculations with the cognate ligand WQQ co-crystalized with HNE (PDB ID 5ABW): Figure S2: Molecular dynamics analysis of the Alvelestat (ALV) complex with HNE in the wild-type (wt) and oxidized (HNE_OX2) forms; Figure S3: 100 ns MD simulation of the wtHNE–Sivelestat pre-complex; Figure S4: Mass spectra of Alvelestat and Sivelestat under control and oxidative conditions; Figure S5: Surface representation of HNE highlighting the positions of the four disulfide bridges (yellow) relative to the catalytic site; Figure S6: Effect of disulfide bond overoxidation on HNE flexibility and catalytic geometry; Figure S7: Molecular dynamics analysis of the sivelestat (SIL)–HNE complex in wild-type (wt) and oxidized (HNE_OX2) forms.

Techniques: Activity Assay, Standard Deviation

Journal: Molecules

Article Title: The Differential Redox Resilience of Alvelestat and Sivelestat: A Mechanistic Hypothesis for Inhibitor Performance Under Oxidative Stress

doi: 10.3390/molecules31091454

Figure Lengend Snippet: Global reciprocal plot of data from HNE steady-state kinetics analysis in the presence of different concentrations (from 0.1 μM to 100 μM) of Alvelestat ( A ) or Sivelestat ( B ). Values represent the mean ± SD obtained from three separate experiments.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules31091454/s1 , Figure S1: Superimposition of the Alvelestat predicted binding mode by docking calculations with the cognate ligand WQQ co-crystalized with HNE (PDB ID 5ABW): Figure S2: Molecular dynamics analysis of the Alvelestat (ALV) complex with HNE in the wild-type (wt) and oxidized (HNE_OX2) forms; Figure S3: 100 ns MD simulation of the wtHNE–Sivelestat pre-complex; Figure S4: Mass spectra of Alvelestat and Sivelestat under control and oxidative conditions; Figure S5: Surface representation of HNE highlighting the positions of the four disulfide bridges (yellow) relative to the catalytic site; Figure S6: Effect of disulfide bond overoxidation on HNE flexibility and catalytic geometry; Figure S7: Molecular dynamics analysis of the sivelestat (SIL)–HNE complex in wild-type (wt) and oxidized (HNE_OX2) forms.

Techniques:

Journal: European Journal of Histochemistry : EJH

Article Title: Ketogenic diet regulates Uch-L1(C) to improve cerebral energy metabolism and cognitive function in Alzheimer's disease mice

doi: 10.4081/ejh.2026.4548

Figure Lengend Snippet: Detection of oxidative stress in the hippocampus and cortex of mice brain. A,B ) Expression of 8-OHdG and 4-HNE in hippocampus and temporal cortex in different groups examined by immunofluorescence images (n=3). C,D ) Analysis of fluorescence intensity of 8-OHdG in hippocampus and temporal cortex respectively. E,F ) Analysis of fluorescence intensity of 4HNE in hippocampus and temporal cortex, respectively. n=3, * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: 4-HNE , Stressmarq , SMC-511D , 1:200 (IF) 1:1000 (WB).

Techniques: Expressing, Immunofluorescence, Fluorescence