Journal: Investigative Ophthalmology & Visual Science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: Inhibiting ONC-induced ER stress with 4PBA alleviates microglial activation, promotes RGC survival, and preserves retinal structure. ( A ) RT-qPCR analysis of ER stress–related genes ( Atf4 , Ddit3 [CHOP], Ppp1r15a [GADD34], and Hspa5 [GRP78]) demonstrates significant mRNA elevation in ONC + PBS retinas versus the ONC + 4PBA group. Data: mean ± SEM ( n = 4–6). P < 0.05, * P < 0.01, ** P < 0.001, *** P < 0.0001 (one-way ANOVA). ( B , C ) Western blot quantification shows 4PBA-mediated reduction of CHOP and GRP78 protein levels in ONC retinas compared to PBS controls. Data: mean ± SEM ( n = 5). P < 0.05, * P < 0.01, ** P < 0.001 (one-way ANOVA). ( D , E ) Iba1⁺ microglial activation analysis by retinal whole-mount immunofluorescence. 4PBA treatment significantly reduces microglia density (30.7% decrease vs. PBS) and promotes ramified morphology. Data: mean ± SEM ( n = 5). * P < 0.01, *** P < 0.0001 (one-way ANOVA). Scale bar : 50 µm. ( F ) Western blot analysis confirms downregulation of pro–IL-1β, cleaved IL-1β, and iNOS protein expression in 4PBA-treated versus PBS-treated ONC retinas. ( G , H ) RBPMS⁺ retinal ganglion cell survival assessment by whole-mount immunofluorescence. 4PBA treatment mitigates RGC loss compared to PBS controls. Data: mean ± SD ( n ≥ 4). ** P < 0.001. Scale bar : 50 µm. ( I , J ) Representative image of retinal thickness determined by OCT in the sham, ONC + PBS, and ONC + 4PBA groups. Two vertical calipers were placed on each side of the optic nerve head, 800 and 1000 µm away from the center of the optic nerve head. The combined thickness (µm) of the nerve fiber layer, ganglion cell layer, and inner plexiform layer was measured. The ONC + 4PBA group exhibited significantly less thinning compared to the ONC + PBS group. For each treatment group, n ≥ 3. Data are presented as mean ± SD. ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were treated with one or more of the following reagents: 4PBA (2.5 mM, T5886; TargetMol), a chemical chaperone and ER stress inhibitor, to investigate the role of ER stress in microglial activation and inflammatory responses; tunicamycin (TUN, 2 µg/mL, MB5419-1; Meilunbio, Dalian, China), a well-known inducer of ER stress, to establish an ER stress model and study its interplay with inflammation; or lipopolysaccharide (LPS, 1 µg/mL; Sigma Aldrich, St, Louis, MO, USA), a dual activator of inflammation and ER stress, to explore the relationship between ER stress and inflammatory responses.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Immunofluorescence, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: Inhibiting ER stress with 4PBA alleviates inflammation in BV2 cells in vitro. ( A ) RT-qPCR analysis of Tnfa , Il6 , and Nos2 mRNA levels in TUN-treated BV2 cells reveals ER stress–induced proinflammatory activation versus untreated controls (NC). Data: mean ± SEM ( n = 6). P < 0.05, ** P < 0.001 (Student's t -test). ( B ) Volcano plot illustrating downregulation of mRNA expression associated with proinflammatory responses in BV2 cells treated with 4PBA under LPS-induced inflammatory conditions, as assessed through bulk RNA-seq analysis. ( C ) GO analysis of the downregulated differentially expressed genes (DEGs) between the LPS group and LPS + 4PBA group in BV2 cells. ( D ) KEGG pathway analysis confirms suppression of LPS-induced inflammatory cascades, including NOD-like receptor and NF-κB signaling (top 15 pathways shown). ( E ) Intersection analysis identifies 24 shared genes between ER stress–associated genes (GeneCards) and LPS_4PBA DEGs, including key mediators Nos2 , Lcn2 , Il1b , and Il6 . ( F , G ) Western blot quantification demonstrates 4PBA-mediated downregulation of LCN2, pro–IL-1β, cleaved IL-1β, and iNOS protein levels versus LPS controls. Data: mean ± SEM ( n = 4). P < 0.05, ** P < 0.001 (one-way ANOVA).

Article Snippet: Cells were treated with one or more of the following reagents: 4PBA (2.5 mM, T5886; TargetMol), a chemical chaperone and ER stress inhibitor, to investigate the role of ER stress in microglial activation and inflammatory responses; tunicamycin (TUN, 2 µg/mL, MB5419-1; Meilunbio, Dalian, China), a well-known inducer of ER stress, to establish an ER stress model and study its interplay with inflammation; or lipopolysaccharide (LPS, 1 µg/mL; Sigma Aldrich, St, Louis, MO, USA), a dual activator of inflammation and ER stress, to explore the relationship between ER stress and inflammatory responses.

Techniques: In Vitro, Quantitative RT-PCR, Activation Assay, Expressing, RNA Sequencing, Western Blot

Journal: Investigative Ophthalmology & Visual Science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: Mutual promotion between LCN2 and ER stress. ( A ) RT-qPCR results indicate elevated mRNA levels of Lcn2 in the TUN group compared to the NC group. Data are shown as mean ± SEM with n = 6 biological repeats. *** P < 0.001, calculated by Student’s t -test. ( B , C ) Immunoblot analysis demonstrates a significant decrease in LCN2 protein expression in the TUN + 4PBA group compared to the TUN group in BV2 cells. Data are shown as mean ± SEM with n = 4. * P < 0.05, *** P < 0.001, calculated by Student’s t -test. ( D , E ) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the TUN group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 5. * P < 0.05, ** P < 0.01, *** P < 0.001, calculated by one-way ANOVA. ( F , G ) Immunoblot analysis shows a significant upregulation of GRP78 and CHOP protein expression in the LPS group compared to the negative control group in BV2 cells, which was alleviated by Lcn2 knockdown using siRNA. Data are shown as mean ± SEM with n = 4. ** P < 0.01, *** P < 0.001, calculated by one-way ANOVA.

Article Snippet: Cells were treated with one or more of the following reagents: 4PBA (2.5 mM, T5886; TargetMol), a chemical chaperone and ER stress inhibitor, to investigate the role of ER stress in microglial activation and inflammatory responses; tunicamycin (TUN, 2 µg/mL, MB5419-1; Meilunbio, Dalian, China), a well-known inducer of ER stress, to establish an ER stress model and study its interplay with inflammation; or lipopolysaccharide (LPS, 1 µg/mL; Sigma Aldrich, St, Louis, MO, USA), a dual activator of inflammation and ER stress, to explore the relationship between ER stress and inflammatory responses.

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

Journal: Investigative Ophthalmology & Visual Science

Article Title: Endoplasmic Reticulum Stress Drives Neuroinflammation Through Lipocalin 2 Upregulation in Retinal Microglia After Optic Nerve Injury

doi: 10.1167/iovs.66.5.12

Figure Lengend Snippet: LCN2 exacerbates neuroinflammation and retinal ganglion cell degeneration post-ONC. ( A–C ) Microglial phagocytic activation in retinal flat mounts visualized by CD68 + /Iba1 + dual immunofluorescence across experimental groups at 7 days post-ONC. Scale bar : 50 µm. Quantification of ( B ) Iba1 + cell density and ( C ) CD68 + /Iba1 + activation index. Data represent mean ± SEM; n = 4 biological replicates; * P < 0.05, *** P < 0.001 (one-way ANOVA with Tukey's post hoc test). ( D ) Transcriptional profiling of microglial-associated inflammatory mediators in retinal tissues. RT-qPCR analysis of Fcgr3 , Tnfa , Il1b , and Il6 mRNA levels normalized to Gapdh . Data represent mean ± SEM; n = 6; ** P < 0.01, *** P < 0.001 (one-way ANOVA with Tukey's post hoc test). ( E , F ) RGC degeneration quantification by RBPMS + cell counting in retinal whole mounts. ( E ) Representative images showing RGC loss patterns. ( F ) Survival rates normalized to sham controls. Scale bars : 50 µm. Data represent mean ± SEM; n = 4–6; * P < 0.05, *** P < 0.001 (one-way ANOVA with Tukey's post hoc test). ( G , H ) Therapeutic modulation of LCN2 by 4PBA treatment. ( G ) Quantification of LCN2 protein levels normalized to β-tubulin and ( H ) representative immunoblots. Data represent mean ± SEM; * P < 0.05, calculated by one-way ANOVA.

Article Snippet: Cells were treated with one or more of the following reagents: 4PBA (2.5 mM, T5886; TargetMol), a chemical chaperone and ER stress inhibitor, to investigate the role of ER stress in microglial activation and inflammatory responses; tunicamycin (TUN, 2 µg/mL, MB5419-1; Meilunbio, Dalian, China), a well-known inducer of ER stress, to establish an ER stress model and study its interplay with inflammation; or lipopolysaccharide (LPS, 1 µg/mL; Sigma Aldrich, St, Louis, MO, USA), a dual activator of inflammation and ER stress, to explore the relationship between ER stress and inflammatory responses.

Techniques: Activation Assay, Immunofluorescence, Quantitative RT-PCR, Cell Counting, Western Blot