Journal: Neural Regeneration Research

Article Title: Hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect neurons from cardiac arrest–induced pyroptosis

doi: 10.4103/NRR.NRR-D-23-01922

Figure Lengend Snippet: PFKL mediates the neuroprotective and antioxidant effects of HP-BMSCs in CPR rats. (A) Neurological deficit scores (NDS) in four group (CPR + PBS, CPR + HP-BMSCs, CPR + si NC HP-BMSCs, CPR + si-PFKL HP-BMSCs, n = 8). The black line represents the NDS levels in the sham group. (B) Serum S100B levels after treatment as indicated ( n = 8). (C) Nissl staining of rat brains after CPR in different group (sham, CPR + PBS, CPR + HP-BMSCs, CPR + si NC HP-BMSCs, CPR + si-PFKL HP-BMSCs, n = 8). After 24 hours of resuscitation, the number of Nissl bodies in the rat cortex significantly decreased, and transplantation of HP-BMSCs could alleviate the reduction of Nissl bodies. However, this effect of HP-BMSCs was reversed after knocking down PFKL. Scale bars: 100 μm (upper) or 50 μm (lower). (D) The AOD of cortical neurons by Nissl staining. (E, F) Representative immunohistochemical staining of 4-HNE (E) and 8-OHdG (F) in cortex following ROSC. After transplantation of HP-BMSCs, the expression of 4-HNE and 8-OHdG in the cortex of CA/CPR rats significantly decreased, whereas this effect was attenuated after knocking down PFKL. The selected areas (black frame) were displayed at a higher magnification level. Scale bars: 70 μm (upper) or 50 μm (lower). (G, H) Percentage of 4-HNE-positive cells (G) and 8-OHdG-positive cells (H) in immunohistochemical images ( n = 8). (I–K) Histograms show the expression of SOD (I), GSH-Px (J) and MDA (K). n = 6 rats per group. (L) Representative images show iNOS (green, Alexa Fluor 488) immunostaining with NeuN (red, CY3) and DAPI (blue) in the cotex ( n = 8). The white boxed areas are shown magnified. HP-BMSCs significantly reduced the expression of iNOS in the neurons of CA-induced rats, while knocking down the expression of PFKL in HP-BMSCs did not lead to significant changes in the expression of iNOS. Scale bars: 50 μm (lower) or 1000 μm (upper). (M) Percentage of iNOS-positive neurons in immunofluorescence images. (N) ROS generation in cortex post-resuscitation was assayed by ROS assay kit. Compared with the si-NC HP-BMSCs group, the reduction effect of si-PFKL HP-BMSCs on post-resuscitation cortical ROS is significantly weakened. The fluorescence was detected by the fluorescence microplate reader ( n = 3). Scale bars: 100 μm. (O) The fluorescent intensity of ROS shown in N. Results expressed as median and interquartile range (A and D) or means ± SD (G–K, M and O). * P < 0.05, vs . sham group; # P < 0.05, vs . CPR + PBS group; & P < 0.05, vs . CPR + si-NC-BMSCs group (Kruskal–Wallis test followed by Dunn’s multiple comparisons test in A and D; one-way analysis of variance followed by Tukey’s multiple comparisons test in G–K, M and O). 4-HNE: 4-Hydroxynonenal; 8-OHdG: 8-hydroxy-2′-deoxyguanosine; AOD: average optical density; CPR: cardiopulmonary resuscitation; HP-BMSCs: hypoxia preconditioned bone marrow mesenchymal stem cells; iNOS: inducible nitric oxide synthase; MDA: malondialdehyde; PBS: phosphate-buffered saline; PFKL: liver isoform of phosphofructokinase; ROI: region of interest; ROS: reactive oxygen species; ROSC: return of spontaneous circulation; SOD: superoxide dismutase.

Article Snippet: For the second experiment, the cells were divided into five groups that were left untreated or treated with siRNAs, left untreated or treated with 10 μM mitochondria-targeted antioxidant (Mito-TEMPO, TargetMol, Shanghai.

Techniques: Staining, Transplantation Assay, Immunohistochemical staining, Expressing, Immunostaining, Immunofluorescence, ROS Assay, Fluorescence, Analysis, Saline, Immunohistochemistry, Knockdown