Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: SASP and NRF2 expression in periodontal tissues from young and aged individuals with or without periodontitis. Periodontal tissues and PDLSCs were collected from young (≤25 years), middle-aged (35-45 years), and aged (≥60 years) individuals, with or without periodontitis. (A) Schematic diagram of the experimental procedure. (B, C) SA-β-gal (B) and P21 (C) staining in periodontal tissues of young (Young), young individuals with periodontitis (Young + PD), aged individuals (Aged), and aged individuals with periodontitis (Aged + PD). (D) RT-qPCR analysis of mRNA expression levels of IL1β , TNFα , IL6 , IL8 , MMP3 , and MMP13 in periodontal tissues. (E) IF staining for NFR2 in periodontal tissues. (F) Western blot analysis of NRF2 expression in PDLSCs collected from young (Young), middle-aged (Middle), and aged (Aged) individuals. (G) NRF2 expression levels in PDLSCs at different passages (Passage 3, 9, and 12) with or without LPS (10 μg/ml) treatment. ns, not significant; PD, periodontitis; IF, Immunofluorescence. Data are presented as means ± SEM, with n = 3-5 for each subgroup. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Expressing, Staining, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: Overexpression of Nrf2 suppresses SASP in senescent PDLSCs. PDLSCs were transduced with adenovirus carrying Nrf2 (Adv- NRF2 ) or control virus (Adv- GFP ), followed by treatment with LPS (10 μg/ml) with or without D-gal (30 mg/ml). (A) Representative images of SA-β-gal staining, followed by quantification. (B) Western blot analysis of P16 and P21 protein levels. (C) mRNA expression levels of IL1β , TNFα , IL6 , IL8 , TNFα , MMP3 , and MMP13 in PDLSCs. (D, E) ALP (D) and ARS (E) staining of PDLSCs. (F) Western blot analysis and quantification of COL-1α1, RUNX2, OPN, and ALP protein expression levels in PDLSCs after 7 days of osteogenic induction. ns, not significant. Data are presented as means ± SEM (n = 3 per subgroup). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Over Expression, Transduction, Control, Virus, Staining, Western Blot, Expressing

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: Nrf2 knockout aggravates SASP and alveolar bone resorption. (A) Schematic diagram of the experimental procedure. (B, C) IF staining of SA-β-gal (B) and P21 (C) in periodontal tissues from WT and Nrf2 −/− mice, settled with a periodontitis model and D-gal induced aged mouse model. (D) Relative mRNA level of Il1β in mice periodontal tissue. (E) Micro-CT reconstructions, followed by 3D and 2D views of maxillary molars and evaluation of the distance from the CEJ to the ABC. (F, G) Mice periodontal tissue sections prepared for H&E staining (F) and Masson's trichrome staining (G). ns, not significant. Data are presented as means ± SEM (n = 5 per subgroup). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Knock-Out, Staining, Micro-CT

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: NRF2 restores UPR function through upregulating IRE1α. (A, B) PDLSCs were transduced with an adenovirus vector or loaded with NRF2 (Adv- GFP / NRF2 ), followed by administrated with LPS and/or D-gal. Western blot for UPR markers (A) in PDLSCs. IF staining for IRE1α in PDLSCs (B). (C, D) PDLSCs were administered with NRF2 inhibitor (ML385), followed by treatment with LPS and/or D-gal. Protein levels of UPR markers (C). IF staining for IRE1α (D). (E) IF staining of IRE1α in WT and Nrf2 −/− mice, settled with the periodontitis model and D-gal induced aged mouse model. (F) Prediction of NRF2's binding motifs on the promoters of UPR markers in the JASPAR database ( https://jaspar.elixir.no/ ). (G) Prediction of the relationship between NRF2 and IRE1α mRNA expression in the GEPIA database ( http://gepia.cancer-pku.cn/ ). (H) CUT-RUN-qPCR assay and agarose gel electrophoresis for the binding of NRF2 to IRE1α promoter in PDLSCs. Data are presented as means ± SEM (n = 3 per subgroup). ∗P < 0.05.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Transduction, Plasmid Preparation, Western Blot, Staining, Binding Assay, Expressing, Agarose Gel Electrophoresis

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: Metformin suppresses SASP and upregulates NRF2 and UPR markers expression in senescent PDLSCs. (A) P16 and P21 expression in PDLSCs treated with metformin combined with LPS and D-gal. (B) SA-β-gal staining of PDLSCs. (C) mRNA expression level in PDLSCs. (D) Western blot for NRF2 and UPR markers in PDLSCs treated with metformin combined with LPS and D-gal. (E, F) IF staining for IRE1α (E) and NRF2 (F) in PDLSCs. (G, H) ALP (G) and ARS (H) staining of PDLSCs followed by osteogenic induction for 7 and 21 days, respectively. (I) Protein expression levels of osteogenic markers in PDLSCs were followed by 7 days of osteogenic induction. Data are presented as means ± SEM (n = 3 per subgroup). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Expressing, Staining, Western Blot

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: Metformin suppresses SASP and UPR via NRF2. (A) Schematic diagram of the experimental procedure. PDLSCs were pretreated with D-gal and LPS, followed by administration with metformin and/or NRF2 inhibitor (ML385). (B) Representative images of SA-β-gal staining for PDLSCs. (C) Protein expression level of P21 and P16. (D) Relative mRNA expression of IL1β , IL6 , IL8 , TNFα , MMP3 , and MMP13 in PDLSCs. (E) Western blot analysis of UPR markers expression levels in PDLSCs. (F) Transmission electron microscopy (TEM) images of ER morphology in PDLSCs. ns, not significant. Data are presented as means ± SEM (n = 3 per subgroup). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Staining, Expressing, Western Blot, Transmission Assay, Electron Microscopy

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: TAM-GM@Met suppresses SASP and upregulates NRF2 and UPR markers expression in senescent PDLSCs. (A) Schematic diagram of the co-culture of hydrogels with PDLSCs. (B) Western blot for P16 and P21 protein expression in PDLSCs treated with composite hydrogels combined with LPS and D-gal pre-treated with D-gal and LPS. (C) SA-β-gal staining of PDLSCs. (D) SASP mRNA expression level in PDLSCs. (E, F) ALP (E) and ARS (F) staining of PDLSCs followed by osteogenic induction for 7 and 21 days, respectively. (G) Protein expression levels of osteogenic markers in PDLSCs followed by 7 days of osteogenic induction. (H) Western blot for NRF2 and UPR markers in PDLSCs treated with metformin combined with LPS and D-gal. ns, not significant. Data are presented as means ± SEM (n = 3 per subgroup). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: Expressing, Co-Culture Assay, Western Blot, Staining

Journal: Bioactive Materials

Article Title: Metformin alleviates aging-associated periodontitis via NRF2-mediated restoration of the IRE1α dependent unfolded protein response

doi: 10.1016/j.bioactmat.2026.03.060

Figure Lengend Snippet: In vivo therapeutic effect of the TAM-GM@Met on mice with an aging-associated periodontitis model. (A) Schematic illustration of the therapeutic process on the D-gal induced aging mice periodontitis model. (B) Micro-CT reconstructions and buccal-palatal sectional views of maxillary molars and evaluation of the distance from the CEJ to the ABC. (C, D) Mice periodontal tissue sections prepared for H&E staining (C) and Masson's trichrome staining (D). (E) Schematic of TAM-GM@Met preparation. TAM hydrogel incorporates galactose-coated MPDA nanoparticles (GM) to target senescent cells for metformin delivery. Metformin activates NRF2, leading to transcriptional upregulation of IRE1α, restoration of the UPR, and suppression of SASP in PDLSCs. ns, not significant; TAM, tannic acid and Ag-MOFs based hydrogel; MPDA, mesoporous polydopamine; UPR, unfolded protein response. Data are presented as means ± SEM (n = 5 per subgroup). ∗∗∗P < 0.001.

Article Snippet: To assess the role of NRF2, PDLSCs were pretreated with ML385 ( HY100523 , MCE), an NRF2 inhibitor, for 24 h. For NRF2 overexpression, PDLSCs were transduced with an adenoviral vector encoding NRF2 (Adv- NRF2 ; titer: 7.11 × 10 10 ) or a control vector (Adv- GFP ; titer: 8.0 × 10 10 ) (OBiO Inc., Shanghai, China) for 24 h. To knock down IRE1α, PDLSCs were transfected with siRNA targeting IRE1α (sense: 5′-GUUUGAUCCCGGACUCAAATT-3′; antisense: 5′-UUUGAGUCCGGGAUCAAAC TT-3′) or a scrambled control siRNA.

Techniques: In Vivo, Micro-CT, Staining

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Transcriptomic and molecular analysis of the potential pathways involved in MMBOx-mediated BMSCs rejuvenation. (A) Circular heatmap showing differentially expressed genes (DEGs) associated with cell senescence, inflammation, and osteogenesis in senescent BMSCs treated with MMBOx@GPP compared to GPP. (B) Gene Ontology (GO) enrichment analysis of upregulated DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (D) Gene Set Enrichment Analysis (GSEA) plots of the glutathione metabolic process (ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate). (E) Heatmap of DEGs enriched in aging-related GO terms. (F) Western blot analysis of Keap1, Nrf2, Nqo1, Gclc, and GAPDH protein expression in BMSCs. (G) Quantitative analysis of protein band intensities ( n = 3). (H) Representative flow cytometry plots of ThiolTracker™ fluorescence staining indicating intracellular glutathione levels. (I) Quantification of intracellular GSH/GSSG ratio in BMSCs ( n = 3). (J) Schematic diagram illustrating the proposed mechanism by which MMBOx attenuates BMSCs senescence via Nrf2 pathway activation and glutathione metabolism enhancement. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Western blotting was employed to evaluate protein expression of key targets, including Keap1 (Affinity, AF5266; 1:1000), Nrf2 (Proteintech, 16396-1-AP; 1:1000), Nqo1 (Abcam, ab80588; 1:10,000), Gclc (Proteintech, 12601-1-AP; 1:25000) and GAPDH (Proteintech, 60004-1-Ig; 1:50,000).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining, Activation Assay

Journal: Bioactive Materials

Article Title: A multimodal ROS logic-gated therapeutic platform disrupts the vicious cycle of senescence to promote aged bone defect repair

doi: 10.1016/j.bioactmat.2026.02.002

Figure Lengend Snippet: Transcriptomic and molecular analysis of the potential pathways involved in MMBOx-mediated BMSCs rejuvenation. (A) Circular heatmap showing differentially expressed genes (DEGs) associated with cell senescence, inflammation, and osteogenesis in senescent BMSCs treated with MMBOx@GPP compared to GPP. (B) Gene Ontology (GO) enrichment analysis of upregulated DEGs. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of upregulated DEGs. (D) Gene Set Enrichment Analysis (GSEA) plots of the glutathione metabolic process (ES: enrichment score; NES: normalized enrichment score; FDR: false discovery rate). (E) Heatmap of DEGs enriched in aging-related GO terms. (F) Western blot analysis of Keap1, Nrf2, Nqo1, Gclc, and GAPDH protein expression in BMSCs. (G) Quantitative analysis of protein band intensities ( n = 3). (H) Representative flow cytometry plots of ThiolTracker™ fluorescence staining indicating intracellular glutathione levels. (I) Quantification of intracellular GSH/GSSG ratio in BMSCs ( n = 3). (J) Schematic diagram illustrating the proposed mechanism by which MMBOx attenuates BMSCs senescence via Nrf2 pathway activation and glutathione metabolism enhancement. Data are expressed as mean ± SD; ∗ P < 0.05, ∗∗∗ P < 0.001.

Article Snippet: Western blotting was employed to evaluate protein expression of key targets, including Keap1 (Affinity, AF5266; 1:1000), Nrf2 (Proteintech, 16396-1-AP; 1:1000), Nqo1 (Abcam, ab80588; 1:10,000), Gclc (Proteintech, 12601-1-AP; 1:25000) and GAPDH (Proteintech, 60004-1-Ig; 1:50,000).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Staining, Activation Assay

Journal: iScience

Article Title: Hypomorphic biliverdin reductase a mutations define bilirubin anti-malarial threshold

doi: 10.1016/j.isci.2026.115958

Figure Lengend Snippet: Expression analysis of BVRA missense mutants G17A and E97A in C57BL/6J mice (A) RT-qPCR analysis of Blvra mRNA expression in spleen, kidney, and liver from Blvra WT , Blvra −/− and Blvra G17A mice. Data are expressed as Blvra / Rplp0 (2 −ΔCt ) and presented as individual data points with bar graphs showing mean ± SD. n = 3 mice per genotype. (B) Western blot analysis of BVRA protein expression in spleen, kidney, and liver. Representative blots show BVRA (33 kDa) and β-actin (42 kDa) loading control. Quantification of BVRA protein levels normalized to β-actin is shown below each blot as individual data points with bar graphs showing mean ± SD. n = 3–5 per genotype. (C) RT-qPCR analysis of Blvra mRNA expression in spleen, kidney and liver tissues from Blvra WT , Blvra −/− , and Blvra E97A mice. Data are expressed as Blvra / Rplp0 (2 −ΔCt ) and presented as individual data points with bar graphs showing mean ± SD. n = 3 per genotype. (D) Western blot analysis of BVRA protein expression in spleen, kidney, and liver tissue. Representative blots and quantification of BVRA protein levels normalized to β-actin below each blot, shown as individual data points with bar graphs showing mean ± SD. n = 3–6 mice per genotype. Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; NS, not significant.

Article Snippet: C57BL/6J Nrf2 −/− , Jackson Laboratory , Cat#017009; RRID: IMSR_JAX:017009.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: iScience

Article Title: Hypomorphic biliverdin reductase a mutations define bilirubin anti-malarial threshold

doi: 10.1016/j.isci.2026.115958

Figure Lengend Snippet: BVRA oxidoreductase activity is dispensable for heme-induced NRF2 activation and insulin receptor signaling in BMDM (A) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with heme and NRF2 activation assessed by immunofluorescence. (B) Representative immunofluorescence images of NRF2 nuclear translocation in BMDM following heme stimulation. Images show confocal z stack cross-sections with orthogonal views. NRF2 is shown in red, DAPI nuclear stain in blue, and phalloidin (cytoskeleton marker) in white. Scale bars, 5 μm. (C) Quantification of cytoplasmic (left graph) or nuclear (right graph) NRF2 MFI in BMDM stimulated with (+) or without (−) heme. Each data point represents a technical replicate. n = 5–10 replicates. Data in (C) is representative from two independent experiments with a similar trend. (D) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with heme and NRF2-regulated gene expression assessed by qRT-PCR. (E) RT-qPCR analysis of NRF2 target genes ( Nqo1 , Fth , Gclc , Hmox1 , Nrf2 , and Blvra ) in BMDM stimulated with (+) or without (−) heme. Data expressed as gene/ Rplp0 (2 −ΔCt ). Each data point represents a technical replicate. n = 3–6 replicates from two independent experiments with a similar trend. (F) BMDM from Blvra WT , Blvra −/− , Blvra G17A , and Blvra E97A mice were stimulated with insulin and INSR signaling assessed by western blot. (G) Representative western blots showing IRS-1 phosphorylation at tyrosine 612 (IRS-1 Y612 ), at serine 307 (IRS-1 S307 ), total IRS-1, and vinculin as loading control in BMDM stimulated with insulin vs. control vehicle. (H) Quantification of IRS-1 Y612 normalized to IRS-1 S307 in BMDM stimulated with (+) or without (−) insulin. Each data point represents a technical replicate. n = 3 replicates from one experiment. Data in (C, E, and H) are presented as individual data points with bar graphs showing mean ± SD. Statistical comparisons were performed using two-way ANOVA with Šídák’s post hoc test. ∗ p < 0.05, ∗∗ p < 0.01; NS, not significant.

Article Snippet: C57BL/6J Nrf2 −/− , Jackson Laboratory , Cat#017009; RRID: IMSR_JAX:017009.

Techniques: Activity Assay, Activation Assay, Immunofluorescence, Translocation Assay, Staining, Marker, Gene Expression, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Control