Journal: Neural Regeneration Research
Article Title: Photobiomodulation repairs the blood–spinal cord barrier in a mouse model of spinal cord injury
doi: 10.4103/NRR.NRR-D-24-01098
Figure Lengend Snippet: Inhibition of mitophagy results in increased ROS accumulation in endothelial cells and reverses the therapeutic effects of PBM. (A) Immunoblotting analysis of LC3, PINK1, Parkin, and TJ protein (ZO-1, Claudin-5, and Occludin) expression in bEnd.3 cells from the Ctrl, OGD, OGD + PBM, and OGD + PBM + Mdivi-1 groups. (B–G) Quantitative analysis of LC3, PINK1, Parkin, and TJ protein (ZO-1, Claudin-5, and Occludin) expression in each group of cells ( n = 3 samples per group). (H) Immunofluorescence staining of PINK1 (green, Alexa Fluor 488) in cells from each group. Pretreatment with Mdivi-1 attenuated the PBM-induced increase in expression of the mitophagy-related proteins PINK1 and Parkin, as evidenced by a decrease in the LC3-II/I ratio. Scale bars: 50 μm. (I) Quantification of PINK1 fluorescence intensity in the OGD, OGD + PBM, and OGD + PBM + Mdivi-1 groups relative to the control group ( n = 3 samples per group). (J) JC-1 probe detection of the mitochondrial membrane potential of bEnd.3 cells in each group. Inhibition of mitophagy by Mdivi-1 counteracted the PBM-induced decrease in ΔΨm. Scale bars: 50 μm. (K) Ratio of JC-1 red to JC-1 green fluorescence ( n = 3 samples per group). (L) Dichlorodihydrofluorescein diacetate probe detection of ROS production in each group of bEnd.3 cells. Mdivi-1 treatment increased ROS levels in endothelial cells. (M) Quantification of ROS fluorescence intensity ( n = 3 samples per group). (N) Representative immunofluorescence images of ZO-1 (green, Alexa Fluor 488) in bEnd.3 cells from each group. Administration of Mdivi-1 blocked the PBM-induced increase in ZO-1 and Claudin-5 expression. Scale bars: 50 μm. (O) Quantification of ZO-1 fluorescence ( n = 3 samples per group). (P) Representative immunofluorescence images of Claudin-5 (red, Alexa Fluor 594) in bEnd.3 cells from each group. Pretreatment with Mdivi-1, a mitophagy inhibitor, blocked the PBM-induced increased in expression of the tight junction proteins ZO-1 and Claudin-5. Scale bars: 50 μm. (Q) Quantification of Claudin-5 fluorescence. ( n = 3 samples per group). Data are shown as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; OGD: oxygen-glucose deprivation; PBM: photobiomodulation; PINK1: PTEN-induced kinase 1.
Article Snippet: Cells were pretreated with the mitophagy inhibitor Mdivi-1 (1 μM, MedChem Express, Monmouth Junction, NJ, USA) for 2 hours prior to exposure to oxygen–glucose deprivation (OGD) and PBM (808 nm, 6 mW/cm 2 , 7 min/time, twice a day).
Techniques: Inhibition, Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Control, Membrane
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