Journal: Materials Today Bio

Article Title: Age-mimicking hydrogel stiffness recapitulates the mechanical niche of the hippocampus to regulate neural stem cell senescence

doi: 10.1016/j.mtbio.2026.102985

Figure Lengend Snippet: Age-associated hippocampal stiffening and its replication via laminin-modified hydrogels . (a) The strategy of in vivo EdU labeling and marker immunostaining for analyzing NSC proliferation and neurogenesis across various mouse age groups. ( b ) Co-staining of GFAP, EdU, and DCX in the hippocampus across different ages. Representative images showing a reduction in active radial glia-like stem cells and neuroblasts/newborn neurons with increasing age. GFAP (green), DCX (red), EdU (gray), and DAPI (blue). Scale bar, 100 μm. ( c-e ) Quantification of active radial glia-like stem cells (GFAP + EdU + ) ( c ), neuroblasts (DCX + EdU + ) ( d ), and newborn neurons (DCX + ) (e) in the SGZ area as in ( b ). n = 3 or 4 mice per group. ( f ) Schematic showing the measurement of hippocampal tissue stiffness using the Pavone nanoindenter and the design of hyaluronic acid (HA)–laminin hydrogels with tunable stiffness to mimic hippocampal mechanical properties at different postnatal ages. Soft, medium, and stiff hydrogels correspond to the mechanical characteristics of hippocampal tissues from 1-, 4-, and 12-week-old mice, respectively. ( g ) Representative images of the dentate gyrus in mouse brain slices across age groups, captured under Pavone nanoindentation microscopy. The SGZ regions measured by the nanoindentation probe are demarcated by paired colorful dashed lines. ( h ) Quantification of Young’s modulus in the hippocampal SGZ region of mice at different ages using Pavone nanoindentation. Brain slices were obtained from four mice per age group. Measurements were taken from n = 227 spots (1 week), n = 149 spots (4 weeks), n = 241 spots (8 weeks), n = 157 spots (12 weeks). (i ) Schematic illustration of the synthesis of HA@HA and HA@HA–Laminin hydrogels. Hyaluronic acid (HA) was first crosslinked with adipic dihydrazide (ADH) using EDC/HCl activation under acidic conditions (pH 3–4) to form HA@HA. Subsequently, laminin was conjugated to the HA network via CDI-mediated coupling to generate HA@HA–Laminin hydrogels. ( j ) Quantification of Young’s modulus of Soft, Medium, and Stiff HA-laminin hydrogels using the same Pavone nanoindentation used for tissue (Soft hydrogel, n = 44 spots; Medium hydrogel, n = 37 spots; Stiff hydrogel, n = 29 spots). ( k ) Immunostaining of YAP1 protein shows the subcellular localization of YAP1 in NSCs cultured on HA-laminin hydrogels of varying stiffness. Dashed lines indicate representative cells with YAP1 predominantly in the cytoplasm (indicated by arrowheads), while solid lines represent cells with YAP1 primarily in the nucleus (indicated by arrows). Scale bar, 20 μm. ( l ) Quantification of YAP1 distribution in NSCs as in ( k ) (n = 4 wells). For all quantification data, statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison tests. Data are presented as mean ± SD (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: Proliferating cells were analyzed using a cell proliferation assay kit (Beyotime, C0075S) to detect the EdU signal in labeled cells.

Techniques: Modification, In Vivo, Labeling, Marker, Immunostaining, Staining, Microscopy, Activation Assay, Cell Culture, Comparison

Journal: Materials Today Bio

Article Title: Age-mimicking hydrogel stiffness recapitulates the mechanical niche of the hippocampus to regulate neural stem cell senescence

doi: 10.1016/j.mtbio.2026.102985

Figure Lengend Snippet: Hydrogels mimicking age-dependent hippocampal stiffness modulate the proliferation and differentiation potentials of NSCs from different ages. (a) The schematic of the EdU labeling assay used to evaluate the proliferation and differentiation capacity of young (1-week-old) and old (12-week-old) NSCs cultured on soft (112 Pa) and stiff (1048 Pa) HA-laminin hydrogels. ( b ) Representative images showing EdU incorporation in young NSCs cultured on 112 Pa (mimicking soft niche) and 1048 Pa (mimicking old niche) HA-laminin hydrogels for 3 and 5 days. ( c ) Quantification of the ratio of EdU + young NSCs as in ( b ) (n = 3 wells). ( d ) Representative images showing EdU incorporation in old NSCs cultured on 112 Pa (mimicking soft niche) and 1048 Pa (mimicking old niche) HA-laminin hydrogels for 3 and 5 days. ( e ) Quantification of the ratio of EdU + old NSCs as in ( d ) (n = 3 wells). ( f ) The strategy for assessing young and old NSCs differentiation potential on soft (112 Pa) and stiff (1048 Pa) HA-laminin hydrogels. All NSCs were induced for spontaneous differentiation for 7 days in DMEM/F12 medium without growth factors. ( g , i ) Immunostaining of neurons and astrocytes differentiated from young ( g ) and old ( i ) NSCs cultured on soft (112 Pa) and stiff (1048 Pa) HA-laminin hydrogels, respectively. Map2 + (red) and GFAP + (green) cells indicate neurons and astrocytes, respectively. Scale bar, 50 μm. ( h , j ) Quantification of the proportion of neurons (Map2 + ) and astrocytes (GFAP + ) differentiated from young ( g ) and old ( i ) NSCs. n = 3 or 4. Statistical significance was determined using two-way ANOVA, with Bonferroni’s Multiple Comparisons Test. Data are presented as mean ± SD ( c,e ). Statistical significance was determined using unpaired Student’s t tests. Data are presented as mean ± SD ( h, j ) (∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: Proliferating cells were analyzed using a cell proliferation assay kit (Beyotime, C0075S) to detect the EdU signal in labeled cells.

Techniques: Labeling, Cell Culture, Immunostaining

Journal: Materials Today Bio

Article Title: Age-mimicking hydrogel stiffness recapitulates the mechanical niche of the hippocampus to regulate neural stem cell senescence

doi: 10.1016/j.mtbio.2026.102985

Figure Lengend Snippet: Intervention of Piezo1 activity altered NSC proliferation on hydrogels mimicking different niche stiffness . (a) The mRNA expression level of Piezo1 in FACS-Young, FACS-Old, Hydrogel-Soft, and Hydrogel-Stiff NSCs groups (n = 3). ( b ) The strategy for treating 8-week-old adult NSCs cultured on soft and stiff hydrogels with the Piezo1 agonist Yoda1 and performing EdU labeling assays. ( c ) EdU detection in adult NSCs on soft and stiff hydrogels treated with 2 μm Yoda1 for 48 h, compared to DMSO mock. Scale bar, 50 μm. ( d ) Quantification of EdU + proliferating NSCs as in ( c ) (n = 3). ( e ) Piezo1 protein immunostaining in the scramble control and Piezo1 knockdown NSCs. Scale bars, 100 μm and 50 μm as indicated. ( f ) qPCR analysis of Piezo1 mRNA expression levels in the scramble control and Piezo1 knockdown NSCs (n = 3). ( g ) EdU detection in scramble control and Piezo1 knockdown NSCs cultured on stiff hydrogels. Scale bar, 100 μm. ( h ) Quantification of EdU + NSCs as in ( g ) (n = 3). ( i-l ) qPCR analysis of Cdk4, Haus1, Col6a4, and Col6a6 genes in the DMSO and 2 μM Yoda1 treatment groups, n = 3. ( m-p ) qPCR analysis of Cdk4, Haus1, Col6a4, and Col6a6 genes in the Scramble and Piezo1 knockdown groups, n = 3. Statistical significance was determined using unpaired Student’s t tests (a, f, h-p). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison tests (d). Data are presented as mean ± SEM (a) and mean ± SD(d, f, h, i-p) (∗p < 0.05, ∗∗p < 0.01).

Article Snippet: Proliferating cells were analyzed using a cell proliferation assay kit (Beyotime, C0075S) to detect the EdU signal in labeled cells.

Techniques: Activity Assay, Expressing, Cell Culture, Labeling, Immunostaining, Control, Knockdown, Comparison

Journal: Materials Today Bio

Article Title: Age-mimicking hydrogel stiffness recapitulates the mechanical niche of the hippocampus to regulate neural stem cell senescence

doi: 10.1016/j.mtbio.2026.102985

Figure Lengend Snippet: Hydrogels mimicking niche stiffness modulate hippocampal NSC potential and gene expression in monkeys of different ages . a, The schematic illustrates the experimental workflow, including the isolation of NSCs from the hippocampi of young (4-day-old) and old (16-year-old) monkeys, followed by their culture on soft or stiff HA-laminin hydrogels to assess proliferation and differentiation. b,c , The EdU labeling strategy to evaluate the proliferation capacity of young ( b ) and old ( c ) monkey hippocampal NSCs cultured on soft and stiff HA-laminin hydrogels. d , Representative images showing 4-h EdU incorporation in young monkey hippocampal NSCs cultured on soft and stiff HA-laminin hydrogels at Day 9. Scale bar, 50 μm. e , Quantification of EdU + young monkey NSCs as in ( d ) (n = 4). f , Representative images showing 24-h EdU incorporation in old monkey hippocampal NSCs cultured on soft and stiff HA-laminin hydrogels at Day 11. Scale bar, 50 μm g , Quantification of EdU + old monkey NSCs as in ( f ) (n = 4 or 5). h , The strategy for assessing the differentiation potential of young and old monkey NSCs on soft and stiff HA-laminin hydrogels. Monkey NSCs were induced for neuronal differentiation for 21 days in serum-free DMEM/F12 medium (growth factors withdrawn and supplemented with BDNF/GDNF/CNTF). i,j Immunostaining of neurons and astrocytes differentiated from young ( i ) and old ( j ) NSCs on soft and stiff HA-laminin hydrogels. Neurons are Map2 (gray) and Tuj1 (red) positive cells; astrocytes are GFAP (green) positive cells. Scale bars, 100 μm and 50 μm as indicated. k-n , Gene expression validation of PIEZO1 , HAUS1 , ITGAD , and COL6A6 in monkey NSCs derived from primary cultures of young and old monkeys, as well as old monkey NSCs cultured on soft and stiff hydrogels (n = 3). Statistical significance was determined using unpaired Student’s t tests. Data are presented as mean ± SD (∗p < 0.05, ∗∗p < 0.01).

Article Snippet: Proliferating cells were analyzed using a cell proliferation assay kit (Beyotime, C0075S) to detect the EdU signal in labeled cells.

Techniques: Gene Expression, Isolation, Labeling, Cell Culture, Immunostaining, Biomarker Discovery, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Hydroxysafflor yellow A attenuates sepsis-induced intestinal barrier dysfunction by modulating Bcl-2/SOD2-mediated mitochondrial apoptosis

doi: 10.3389/fphar.2026.1728183

Figure Lengend Snippet: Proteomic Analysis for Identifying Key Molecules Mediating Ferroptosis in Sepsis-Associated IEC-6 Cells (A) KEGG pathway enrichment analysis. (B) Immunofluorescence staining of ZO-1 (scale bar = 50 μm) and Western blot analysis of ZO-1 expression. (C) CCK-8 assay of cell viability (n = 8). (D) DCFH-DA showing ROS levels (scale bar = 20 μm, n = 8) (E) TUNEL staining of apoptotic cells. (F) Ki-67 immunofluorescence of proliferating cells (G) MitoTracker Red CMXRos staining showing mitochondrial morphology (scale bar = 50 μm, n = 3). (H) TEM images of mitochondrial ultrastructure. (I) Impact of LPS on mitochondrial respiration in IEC-6 cells (n = 3).

Article Snippet: DCFH-DA fluorescent probe (S0033S), MitoTracker Red CMXRos (C1049B), Ki67 cell proliferation detection kit (C2312S), TUNEL apoptosis detection kit (C1088), and ROS detection kit (S0033S) were supplied by Beyotime Biotechnology (Shanghai, China).

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

Journal: Frontiers in Pharmacology

Article Title: Hydroxysafflor yellow A attenuates sepsis-induced intestinal barrier dysfunction by modulating Bcl-2/SOD2-mediated mitochondrial apoptosis

doi: 10.3389/fphar.2026.1728183

Figure Lengend Snippet: In vitro effects of HSYA on IEC-6 cells following Bcl-2 inhibition. (A) Western blot analysis showing the relative expression level of Bcl-2 after treatment in Si-Bcl-2 of IEC-6 cells (n = 3). (B) Western blot analysis showing the effects of HSYA + Si-Bcl2 on the expression of Bcl2 in LPS-stimulated IEC-6 cells (n = 3). (C) TUNEL staining of apoptotic cells (scale bar = 50 μm, n = 3). (D) Ki-67 immunofluorescence for proliferating cells. (E,F) DCFH-DA (n = 3) and MitoTracker Red CMXRos staining (n = 8) showing intracellular ROS levels and mitochondrial morphology in IEC-6 cells (scale bar = 20 μm). (G) TEM images showing mitochondrial ultrastructure in IEC-6 cells following HSYA and HSYA + Si-Bcl-2 treatment. (H) Impact of HSYA + Si-Bcl-2 treatment on mitochondrial respiration in LPS-stimulated IEC-6 cells (n = 3).

Article Snippet: DCFH-DA fluorescent probe (S0033S), MitoTracker Red CMXRos (C1049B), Ki67 cell proliferation detection kit (C2312S), TUNEL apoptosis detection kit (C1088), and ROS detection kit (S0033S) were supplied by Beyotime Biotechnology (Shanghai, China).

Techniques: In Vitro, Inhibition, Western Blot, Expressing, TUNEL Assay, Staining, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Hydroxysafflor yellow A attenuates sepsis-induced intestinal barrier dysfunction by modulating Bcl-2/SOD2-mediated mitochondrial apoptosis

doi: 10.3389/fphar.2026.1728183

Figure Lengend Snippet: Effects of HSYA on intestinal epithelial cells under silence SOD-2. (A,B) Western blot analysis of SOD2 protein expression in IEC-6 cells (n = 3). (C) TUNEL staining for apoptotic cells (scale bar = 50 μm). (D) Ki-67 immunofluorescence for proliferating cells (scale bar = 50 μm, n = 8). (E,F) DCFH-DA (n = 8)and MitoTracker Red CMXRos staining (n = 3) showing intracellular ROS accumulation and mitochondrial morphology in IEC-6 cells (scale bar = 20 μm). (G) TEM images showing mitochondrial ultrastructure in IEC-6 cells treated with HSYA or HSYA + Si-SOD2 (scale bar = 1 μm). (H) Impact of HSYA + Si-SOD2 treatment on mitochondrial respiration in LPS-stimulated IEC-6 cells (n = 3).

Article Snippet: DCFH-DA fluorescent probe (S0033S), MitoTracker Red CMXRos (C1049B), Ki67 cell proliferation detection kit (C2312S), TUNEL apoptosis detection kit (C1088), and ROS detection kit (S0033S) were supplied by Beyotime Biotechnology (Shanghai, China).

Techniques: Western Blot, Expressing, TUNEL Assay, Staining, Immunofluorescence