Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: Primer sequences used in qRT-PCR.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: NBP inhibited oxidative stress injury and triggered nuclear translocation of Nrf2 and upregulated its downregulated genes. Effects of NBP on (a) SOD activity, (b) MDA content ( n = 8 in each group); and (c) 8-iso PGF2 α level ( n = 10 in each group) after RCIR injury. (d)–(g) Representative immunoblots and densitometry analysis of nuclear-Nrf2, HO-1, and NQO1 in the hippocampus in four groups. (h)–(n) The mRNA levels of the Nrf2 and Nrf2 target genes in the hippocampus were evaluated. n = 6 in each group. (o) Localization of Nrf2 was performed by immunofluorescence staining in hippocampus 4 weeks after NBP treatment. Immunofluorescence labeling of Nrf2 (green) and nuclei was stained with DAPI (blue). The merged images showed the nuclear location of Nrf2 protein (400×, bar = 20 μ m). n = 3 in each group. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, the RCIR group vs. the sham group; # P < 0.05, ## P < 0.01, ### P < 0.001, the NBP80 group or the NBP120 group vs. the RCIR group; $ P < 0.05, $$ P < 0.01, the NBP80 group vs. NBP120 group. Values are expressed as the mean ± SD.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Translocation Assay, Activity Assay, Western Blot, Immunofluorescence, Staining, Labeling

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: NBP failed to promote nuclear-Nrf2 accumulation and mitigate oxidative stress injury in Nrf2 knockout (Nrf2 −/− ) mice. (a) The results of genotype identification by PCR. (b)–(d) NBP treatment failed to alleviate the activity of SOD, the content of MDA, and the level of 8-iso PGF2 α in Nrf2 −/− mice after RCIR injury. n = 8 or 10 per group. (e)–(h) Nuclear-Nrf2, HO-1, and NQO1 expressions were detected by Western blot in WT and Nrf2 −/− mice 4 weeks after RCIR. NBP treatment failed to upregulate the nuclear-Nrf2, HO-1, and NQO1 expressions in Nrf2 −/− mice compared to the WT group. n = 6 per group. β -Actin was used as an internal control. (i) The effect of NBP on Nrf2 nuclear accumulation was determined by immunofluorescent (400×, bar = 20 μ m) in WT and Nrf2 −/− mice 4 weeks after RCIR. n = 3 in each group. # P < 0.05, ### P < 0.001, the WT + NBP120 vs. the WT+ RCIR group; & P < 0.05, &&& P < 0.001, the Nrf2 −/− + RCIR group vs. the WT + RCIR group; ns: not significant, as indicated. Values are expressed as mean ± SD.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Knock-Out, Activity Assay, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: NBP significantly suppressed RCIR-induced microglial cell activation and regeneration, but not Nrf2 −/− mice. (a) Representative images of double immunofluorescence labeling of Iba-1 (red) and BrdU (green) (200×) in hippocampal CA1 regions of WT and Nrf2 −/− mice 4 weeks after RCIR. Iba-1 + /BrdU + cells represented microglial proliferation. Typical double-labeled areas were magnified. Bar = 50 μ m. n = 4 in each group. (b) The number of Iba-1 + /BrdU + cells in hippocampal CA1 regions. (c, d) Western blot analysis of Iba-1 expressions. n = 6 in each group. β -Actin was used as an internal control. ### P < 0.001, the WT + NBP120 vs. the WT + RCIR group; & P < 0.05, the Nrf2 −/− + RCIR group vs. the WT + RCIR group; ns: not significant, as indicated. Values are expressed as mean ± SD.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Activation Assay, Immunofluorescence, Labeling, Western Blot, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: NBP alleviated RCIR-induced inflammatory response, but with reduced inhibitory role in Nrf2 −/− mice. (a)–(c) ELISA analysis showing the expression of the inflammatory factors TNF- α , IL-6, and IL-1 β in the hippocampus of WT and Nrf2 −/− mice 4 weeks after RCIR. n = 10 per group. (d)–(g) Western blot analysis showing the expression of TLR4, MyD88, and p-NF- κ B in WT and Nrf2 −/− mice at 4 weeks after RCIR. n = 6 per group. β -Actin was used as an internal control. (h) Representative immunofluorescent micrographs of the hippocampus after staining with NF- κ B primary antibody in WT and Nrf2 −/− mice 4 weeks after RCIR injury. Green fluorescence, NF- κ B-positive cells; white arrow, nuclear translocation of NF- κ B after RCIR with or without NBP treatment. Scale bar = 20 μ m. n = 3 in each group. # P < 0.05, ## P < 0.01, ### P < 0.001, the WT + NBP120 vs. the WT + RCIR group; ns: not significant, as indicated. Values are expressed as mean ± SD.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Control, Staining, Fluorescence, Translocation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: The neuroprotective effects of NBP were abolished in Nrf2 −/− mice. (a)–(c) Quantification of time latency, swimming speed, and the percentage of time spent in the target quadrant was measured in WT and Nrf2 −/− mice using MWM. n = 9 per group. (d)–(f) Representative images of HE staining (400×, scale bar = 50 μ m), the ultrastructure of neurons (scale bar = 1 μ m), and TUNEL staining (400×, scale bar = 50 μ m) in the hippocampal CA1 region. (g) The quantitative graph for TUNEL staining of the hippocampus. n = 3 or 4 per group. (h)–(j) Western blot analysis of Bax and Bcl-2 expression. n = 6 per group. β -Actin was used as an internal control. ## P < 0.05, ### P < 0.001, the WT + NBP120 vs. the WT + RCIR group; ns: not significant, as indicated. Values are expressed as mean ± SD.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Staining, TUNEL Assay, Western Blot, Expressing, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dl-3-n-Butylphthalide Improves Neuroinflammation in Mice with Repeated Cerebral Ischemia-Reperfusion Injury through the Nrf2-Mediated Antioxidant Response and TLR4/MyD88/NF- κ B Signaling Pathway

doi: 10.1155/2022/8652741

Figure Lengend Snippet: Scheme summarizing the proposed mechanisms for the antioxidant, antineuroinflammatory, and neuroprotective effects of NBP in RCIR-stimulated VD mice. Mechanisms involve the promotion of antioxidant enzyme expression, reduction of PICs, inhibition of TLR4/MyD88/NF- κ B signaling and microglial proliferation, and the key role of NBP-mediated Nrf2 activation in the process.

Article Snippet: Nrf2 knockout CD1/ICR mouse line was generously provided by Dr. Thomas W. Kensler laboratory (Johns Hopkins University, Baltimore, America).

Techniques: Expressing, Inhibition, Activation Assay