PMC:7296049 / 18473-32793 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7296049","sourcedb":"PMC","sourceid":"7296049","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7296049","text":"Results\n\nNBP Alleviates Learning and Memory Deficits in CCH Rats\nThe degree of cognitive impairment in CCH rats and the effects of NBP were investigated by testing spatial learning and memory with the MWM. At 2 weeks after 2VO, there were no significant differences in performance between groups (Supplementary Figures S1A–D) but at 4 weeks, rats in the model group showed a decline in spatial learning and memory, which was alleviated by NBP treatment. The escape latency of CCH rats was significantly longer than that of the sham group on days 3–5 (day 3: P \u003c 0.05; day 4: P \u003c 0.01; day 5: P \u003c 0.001), while rats in the NBP-treated group had a shorter latency than untreated CCH rats (day 3: P \u003c 0.05, NBP120 vs. model group; day 4: P \u003c 0.05, NBP60 vs. model group and P \u003c 0.01, NBP120 vs. model group; day 5: P \u003c 0.05, NBP60 vs. model group and P \u003c 0.01, NBP120 vs. model group). Escape latency in each group gradually decreased in the final days of training (Figure 2A). In the spatial probe test on day 6, untreated CCH rats spent less time in the target quadrant and had fewer platform crossings than those in the sham group (all P \u003c 0.001). In contrast, rats in NBP-treated groups spent more time in the target quadrant and had a higher frequency of platform crossings (all P \u003c 0.05, NBP60 vs. model group and P \u003c 0.001, NBP120 vs. model group). Moreover, the NBP120 group spent more time in the target quadrant (P \u003c 0.05) and had more platform crossings (P \u003c 0.01) than the NBP60 group (Figures 2B–D).\nFigure 2 Dl-3-n-butylphthalide (NBP) alleviates learning and memory deficiency, and the pathologic changes in hippocampal CA 1 region at 4 weeks after 2VO (Morris water maze, n = 9 in each group; Hematox­ylin-eosin staining, n = 3 in each group). (A) Escape latency changes in different groups from day 1 to day 5. (B) Changes in time spent in the target quadrant (%) at day 6. (C) Changes in frequency of platform crossings in 2 min at day 6. (D) Swimming path of rats at day 6 in different groups. (E) Representative images of Hematoxylin-eosin staining in the hippocampal CA1 region. *p \u003c 0.05, **p \u003c 0.01, ***p \u003c 0.001, the model group vs. the sham group; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $p \u003c 0.05, $$p \u003c 0.01, the NBP60 group vs. NBP120 group. Values are expressed as mean ± SD.\n\nNBP Alleviates Pathologic Changes in the Hippocampal CA1 Region Following CCH\nNeurons in hippocampus CA1 region of rats in the sham group had a distinct shape and were of moderate size, with normal microstructure. In contrast, neuronal shrinkage and loss and pyknosis of the cytoplasm was observed in 2VO rats. Administration of NBP partly reversed these morphologic changes (Figure 2E).\n\nNBP Reduces Demyelination in the Corpus Callosum Following CCH\nAt 2 weeks after 2VO, CCH rats showed decreased myelin density in the corpus callosum by LFB staining and lower MBP protein and mRNA levels compared to the sham group (all P \u003c 0.001). NBP treatment alleviated demyelination in a dose-dependent manner, as evidenced by increased LFB staining (all P \u003c 0.001, NBP60 or NBP120 vs. model group), MBP mRNA level (P \u003c 0.05, NBP60 vs. model group and P \u003c 0.001, NBP120 vs. model group), and MBP protein level (P \u003c 0.05, NBP120 vs. model group; Figures 3A–E). At 4 weeks after the surgery, the model group still showed decreased myelin density and MBP protein and mRNA levels relative to the sham group (all P \u003c 0.001); moreover, demyelination was more severe in these rats compared to those sacrificed at 2 weeks (all P \u003c 0.05; Figures 3A–D), reflecting the progressive loss of myelin over time after CCH. By comparison, rats in NBP-treated groups showed less demyelination by LFB staining (all P \u003c 0.001, NBP60 or NBP120 vs. model group) and higher MBP protein level (P \u003c 0.05, NBP60 vs. model group and P \u003c 0.001, NBP120 vs. model group) and MBP mRNA level (P \u003c 0.01, NBP60 vs. model group and P \u003c 0.001, NBP120 vs. model group), with greater improvement observed in the NBP120 group than in the NBP60 group (LFB staining: P \u003c 0.05; protein levels of MBP: P \u003c 0.05; mRNA levels of MBP: P \u003c 0.001), demonstrating a dose-dependent protective effect (Figures 3A–E).\nFigure 3 Dl-3-n-butylphthalide (NBP) attenuates demyelination in corpus callosum after 2VO. (A) Representative images of luxol fast blue (LFB) staining in the corpus callosum at 2 weeks and 4 weeks after 2VO. Bar = 50 μm (n = 3 in each group). (B) Western blot analysis of the expressions of MBP (n = 6 in each group). (C) Myelin density in each group presented by the percentage of stained area in total area of corpus callosum. (D) Quantitative analysis of protein levels of MBP. β-actin was used as an internal control. (E) Quantitative analysis of mRNA levels of MBP. β-actin was used as an internal control (n = 6 in each group). ***p \u003c 0.001, the model group vs. the sham group; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $p \u003c 0.05, $$$p \u003c 0.001, the NBP60 group vs. NBP120 group; and \u0026p \u003c 0.05, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. MBP, myelin basic protein.\n\nNBP Reduces Oligodendrocyte Loss and Promotes Oligodendrocyte Regeneration in the Corpus Callosum\nTo confirm the specificity of antibody labeling, we performed immunohistochemistry on representative coronal brain sections and found that anti-Olig2 and -GFAP antibodies labeled oligodendrocytes and astrocytes, respectively, whereas BrdU labeling was restricted to the nucleus (Supplementary Figure S2).\nThe number of oligodendrocytes in the corpus callosum was quantified by Olig2 immunolabeling. Compared to the sham group, the number of Olig2+ cells decreased over time in untreated CCH rats (all P \u003c 0.001, model group sacrificed at 2 and 4 weeks vs. sham group and P \u003c 0.05, model group sacrificed at 2 weeks vs. at 4 weeks). However, the NBP120 group had more Olig2+ cells than the model group at 2 and 4 weeks (P \u003c 0.05 at 2 weeks, P \u003c 0.001 at 4 weeks; Figures 4A,C). We quantified the number of Olig2+/BrdU+ cells as an index of oligodendrocyte regeneration. Compared to untreated CCH rats, those treated with NBP had more Olig2+/BrdU+ cells at 2 and 4 weeks (all P \u003c 0.001, NBP60 or NBP120 vs. model group; Figures 4A,D). Additionally, while Olig2 protein and mRNA levels were lower in model rats than in sham rats at both postsurgery time points (protein level of Olig2: P \u003c 0.01; mRNA level of Olig2: P \u003c 0.001), higher levels were observed in the NBP120 group at 2 weeks (protein level of Olig2: P \u003c 0.05; mRNA level of Olig2: P \u003c 0.001) and at 4 weeks (protein level of Olig2: P \u003c 0.01; mRNA level of Olig2: p \u003c 0.05; Figures 4B,E,F).\nFigure 4 Dl-3-n-butylphthalide (NBP) promotes oligodendrocytes regeneration in corpus callosum after 2VO. (A) Representative images of Olig2 (red) and BrdU (green) immunofluorescence double labeling in corpus callosum at 2 weeks and 4 weeks after 2VO. Bar = 50 μm. Typical double-labeled areas are magnified (n = 3 in each group). (B) Western blot analysis of the expressions of Olig2 (n = 6 in each group). (C) The number of Olig2+ cells in corpus callosum. (D) The number of Olig2+/BrdU+ cells in corpus callosum. (E) Quantitative analysis of protein levels of Olig2. β-actin was used as an internal control. (F) Quantitative analysis of mRNA levels of Olig2. β-actin was used as an internal control (n = 6 in each group). **p \u003c 0.01, ***p \u003c 0.001, the model group vs. the sham group; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $p \u003c 0.05, $$$p \u003c 0.001, the NBP60 group vs. NBP120 group; and \u0026p \u003c 0.05, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. Olig2, oligodendrocyte lineage transcription factor 2.\n\nNBP Suppresses Astrocyte Proliferation Following CCH\nWe performed double immunofluorescence labeling of GFAP and BrdU to detect astrocyte regeneration. Compared to rats in the sham group, CCH rats showed an increased number of GFAP+/BrdU+ double-positive cells at 2 weeks (P \u003c 0.05) and 4 weeks (P \u003c 0.001). Treatment with NBP reduced astrocyte regeneration at 2 weeks (P \u003c 0.01 NBP60 vs. model group; P \u003c 0.001 NBP120 vs. model group) and 4 weeks (all P \u003c 0.001, NBP60 or NBP120 vs. model group; Figures 5A,B).\nFigure 5 Dl-3-n-butylphthalide (NBP) suppresses astrocytes regeneration after 2VO. (A) Representative images of GFAP (red) and BrdU (green) immunofluorescence double labeling in corpus callosum at 2 weeks and 4 weeks after 2VO. Bar = 50 μm. Typical double-labeled areas are magnified (n = 3 in each group). (B) The number of GFAP+/BrdU+ cells in corpus callosum. *p \u003c 0.05, ***p \u003c 0.001, the model group vs. the sham group; ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $$p \u003c 0.01, the NBP60 group vs. NBP120 group; \u0026\u0026\u0026p \u003c 0.001, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. GFAP, glial fibrillary acidic protein.\n\nNBP Suppresses Inflammation Induced by CCH\nTo investigate whether the protective effects of NBP against demyelination and cognitive dysfunction following CCH involve the suppression of inflammation, we measured the protein levels of TNF-α, p-STAT3, and NF-κB by western blotting and the mRNA level of TNF-α by qRT-PCR (Figures 6A–E). At 2 weeks after 2VO, CCH rats showed elevated expression of these inflammatory cytokines compared to the sham group (protein level of TNF-α: P \u003c 0.05; protein level of p-STAT3: P \u003c 0.001; protein level of NF-κB: P \u003c 0.001; mRNA level of TNF-α: P \u003c 0.01). However, treatment with NBP partly reversed this effect (protein level of TNF-α: P \u003c 0.05, NBP120 vs. model group; protein level of p-STAT3: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group; protein level of NF-κB: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group; mRNA level of TNF-α: P \u003c 0.05 NBP120 vs. model group). A similar trend was observed at 4 weeks after 2VO, with rats in the model group showing higher levels of proinflammatory cytokines than sham rats (protein level of TNF-α: P \u003c 0.01; protein level of p-STAT3: P \u003c 0.001; protein level of NF-κB: P \u003c 0.001; mRNA level of TNF-α: P \u003c 0.001). Compared to model rats sacrificed 2 weeks after 2VO in the same group, those sacrificed at 4 weeks had more severe inflammation (protein levels of TNF-α, p-STAT3, and NF-κB: all P \u003c 0.05; mRNA level of TNF-α: P \u003c 0.001). As expected, treatment with NBP reduced inflammation at 4 weeks (protein level of TNF-α: P \u003c 0.05, NBP120 vs. model group; protein level of p-STAT3: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group; protein level of NF-κB: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group; mRNA level of TNF-α: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group).\nFigure 6 Dl-3-n-butylphthalide (NBP) suppresses inflammation induced after 2VO. (A) Western blot analysis of the expressions of TNF-a, p-STAT3 and NF-KB (n = 6 in each group). (B) Quantitative analysis of protein levels of TNF-α. (C) Quantitative analysis of mRNA levels of TNF-α (n = 6 in each group). (D) Quantitative analysis of protein levels of p-STAT3. (E) Quantitative analysis of protein levels of NF-KB. β-actin was used as an internal control. *p \u003c 0.05, **p \u003c 0.01, ***p \u003c 0.001, the model group vs. the sham group; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $p \u003c 0.05, $$p \u003c 0.01, the NBP60 group vs. NBP120 group; \u0026p \u003c 0.05, \u0026\u0026\u0026p \u003c 0.001, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. TNF-α, tumor necrosis factor-α. p-STAT3, phosphorylated signal transducers and activators of transcription 3. NF-κB, nuclear factor κB.\n\nNBP Activates AMPK/SIRT1 Signaling\nTo investigate whether AMPK/SIRT1 signaling is involved in the protective effects of NBP, we measured the protein levels of p-AMPK and SIRT1 by western blotting and the mRNA level of SIRT1 by qRT-PCR (Figures 7A–D). At 2 weeks after 2VO, these factors were downregulated in CCH rats relative to sham rats (protein level of p-AMPK: P \u003c 0.001; protein level of SIRT1: P \u003c 0.01; mRNA level of SIRT1: P \u003c 0.01). However, treatment with NBP partly abrogated this decrease (protein level of p-AMPK: P \u003c 0.01, NBP120 vs. model group; protein level of SIRT1: P \u003c 0.05, NBP120 vs. model group; mRNA level of SIRT1, P \u003c 0.01, NBP120 vs. model group). At 4 weeks after 2VO, rats in the model group had lower levels of AMPK/SIRT1 pathway components compared to the sham group (protein level of p-AMPK: P \u003c 0.001; protein level of SIRT1: P \u003c 0.05; mRNA level of SIRT1: P \u003c 0.001). Moreover, compared to untreated CCH rats sacrificed 2 weeks after 2VO in the same group, those sacrificed at 4 weeks had decreased levels of AMPK/SIRT1 signaling factors (protein level of p-AMPK: P \u003c 0.05; protein and mRNA levels of SIRT1: all P \u003c 0.01). However, treatment with NBP increased their expression at 4 weeks after 2VO (protein level of p-AMPK: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.05, NBP60 vs. model group; protein level of SIRT1: P \u003c 0.01, NBP120 vs. model group and P \u003c 0.05, NBP60 vs. model group; mRNA level of SIRT1: P \u003c 0.001, NBP120 vs. model group and P \u003c 0.01, NBP60 vs. model group).\nFigure 7 Dl-3-n-butylphthalide (NBP) upregulates AMPK/SIRT1 signaling after 2VO. (A) Western blot analysis of the expressions of p-AMPK and SIRT1 (n = 6 in each group). (B) Quantitative analysis of protein levels of p-AMPK. (C) Quantitative analysis of protein levels of SIRT1. (D) Quantitative analysis of mRNA levels of SIRT1 (n = 6 in each group). β-actin was used as an internal control. *p \u003c 0.05, **p \u003c 0.01, ***p \u003c 0.001, the model group vs. the sham group; #p \u003c 0.05, ##p \u003c 0.01, ###p \u003c 0.001, the NBP60 group or NBP120 group vs. the model group; $p \u003c 0.05, $$$p \u003c 0.001, the NBP60 group vs. NBP120 group; and \u0026p \u003c 0.05, \u0026\u0026p \u003c 0.01, the model group sacrificed at 2 weeks vs. the model group sacrificed at 4 weeks. Values are expressed as mean ± SD. p-AMPK, phosphorylated AMP-activated protein kinase. 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