Fig 1: A. muciniphila regulates the expression of immune factors. (A) The levels of MMP-1, MMP-9, CTSB, and CTSL in abdominal aortic tissue were analyzed by ELISA. (B,C) The expression of IL-33 in abdominal aortic tissue was analyzed by IHC (scale bar = 25 μm; the magnification is 400×). (D) qRT-PCR was used to analyze IL-33 gene expression in abdominal aortic tissue. (E) The expression of IL-33 protein in abdominal aortic tissue was analyzed by Western blot. (F) The levels of IL-4, IL-6, IL-10, IL-17A, IFN-γ, and C-reactive protein (CRP) in peripheral blood were determined by ELISA. (G) The expression of PCNA, OPN, and α-SMA protein in abdominal aortic aneurysm tissue were detected by Western blot. Data were shown as means ± SD and analyzed by two-way ANOVA (n = 10 mice/group). *Compared with the control group, P < 0.05; #compared with the AAA group, P < 0.05.
Fig 2: Overexpression of OSBPL10 attenuates lysosomal membrane permeabilization after SCI. A Volcano plot of transcriptome data from spinal cord tissue from SCI + AAV-NC and SCI + AAV-Osbpl10 group (n = 4). B Protein levels of CTSB, CTSL and CTSC in the cytoplasm and lysosomes extracted from the spinal cords of the indicated groups. The lysosomal membrane protein LAMP1 was used to identify the lysosomal fraction and as a loading control. GAPDH was used to identify the cytosolic fraction and as a loading control. C Quantitative analysis of the protein levels in (B) (n = 6; Ordinary one-way ANOVA). D Immunofluorescence staining and colocalization analysis of CTSB and LAMP1 with neurons in the ventral horn gray matter of spinal cord tissue in the indicated groups (scale bar: 25 μm for original pictures and 15 μm for enlarged pictures). E Quantitative analysis of the ratios of the diffuse CTSB cells in the anterior horn of the spinal cord tissue in the indicated groups (n = 6; Ordinary one-way ANOVA). F-G ELISA results of the activity of lysosomal enzymes CTSB and NAGLU in lysosomal or cytosolic part extracted from the spinal cord tissue (n = 6; Ordinary one-way ANOVA). H-I Immunofluorescence staining and colocalization analysis of OSBPL10 and LAMP1 with neurons in the ventral horn gray matter of spinal cord tissue in the indicated groups (scale bar: 25 μm for original pictures and 6 μm for enlarged pictures). J Quantitative analysis of the the ratio of OSBPL10 positive lysosomes to total lysosomes from each neuron in (H) (n = 6; Unpaired t-test). All data are presented as the means ± SD; Error bars represent SDs; ∗∗p < 0.01, ∗p < 0.05, ns indicates no significance
Fig 3: Overexpressing neuronal OSBPL10 alleviates LMP, impairment of autophagic flux and ferroptosis in a PS-dependent manner after SCI. A Binding affinities of OSBPL10 protein to different lipid isoforms by Auto Dock Vina v.1.2.0 software. B-C The Osbpl10 protein is shown in green cartoon and white surface representations, with the interacting amino acid residues colored orange and cholesterol or phosphatidylserine displayed as pink or white sticks. D Schematic diagram of lipid synthesis for phosphatidylserine and cholesterol, and the target enzymes of lipid synthesis enzyme inhibitors. E Immunofluorescence staining and colocalization analysis of CTSB and LAMP1 with neurons in the ventral horn gray matter of spinal cord tissue in the indicated groups (scale bar: 25 μm for original pictures and 15 μm for enlarged pictures). F Western blot analysis of ATG5, VPS34, BECLIN1, SQSTM1/P62 and LC3 expression levels in the indicated groups. G Quantitative analysis of protein expression of (F) (n = 6; Ordinary one-way ANOVA). H Western blot analysis of 4HNE, TP53, ACSL4, SLC7A11 and GPX4 expression levels in the indicated groups. I Quantitative analysis of protein expression of (H) (n = 6; Ordinary one-way ANOVA). J Immunofluorescence images of DHE and DAPI in spinal cords in the indicated groups (scale bar: 100 μm). K Colocalization analysis of the fluorescence intensity between DHE and DAPI in (J) (n = 6; Ordinary one-way ANOVA). All data are presented as the means ± SD; Error bars represent SDs; ∗∗p < 0.01, ∗p < 0.05, ns indicates no significance
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