Journal: Acta pharmaceutica Sinica. B

Article Title: Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis.

doi: 10.1016/j.apsb.2022.07.019

Figure Lengend Snippet: Figure 5 Honokiol activated the NRF2 anti-oxidant pathway in the SOD1-G93A cells. (A) Luminescence intensity of HEK293-ARE reporter cells after treatment with indicated concentrations of honokiol for 6, 12 and 24 h (n Z 3); (B) Representative images of NRF2 nuclear trans- localization after 24 h honokiol treatment. Scale bars, 8 mm; (C) Western blot analysis and quantification of NRF2 in nuclear and cytoplasmic cell lysates after 24 h honokiol treatment (n Z 4); (D) RT-qPCR analysis of transcription levels of Hmox1, Nqo1, Gclm, Gclc, Gss and Gsr after 12 h honokiol treatment (n Z 3e7); (E) Western blot analysis and quantification of HO-1, NQO1, GCLC, GCLM, GSS and GSR in total cell lysates after 24 h honokiol treatment (n Z 3e6). One-way ANOVA followed by post hoc LSD test was used for the comparison among three or more groups (SPSS 16.0). Data are presented as the mean SEM. #P < 0.05, ###P < 0.001 versus pEGFP control group; *P < 0.05, **P < 0.01, ***P < 0.001 versus SOD1-G93A model group.

Article Snippet: Membranes were blocked by the 5% skimmed milk solution for 2 h at room temperature and then incubated at 4 C overnight with antibodies against BCL2 (26539- 1-AP, 1:1000), BAX (50599-2-Ig, 1:2000), cytochrome c (10993- 1-AP, 1:2000), ATM (27156-1-AP, 1:1000), NRF2 (16396-1-AP, 1:1000), GCLC (12601-1-AP, 1:2000), GCLM (14241-1-AP, 1:2000), OPA1 (27733-1-AP, 1:1000), MFN2 (12186-1-AP, 1:2000), FIS1 (10956-1-AP, 1:1000), p62 (18420-1-AP, 1:1000), GFAP (26825-1-AP, 1:2000), HSP90 (13171-1-AP, 1:2000), GAPDH (60004-1-Ig, 1:5000) from Proteintech (Rosemont, IL, USA); GSR (sc-133245, 1:500), GSS (sc-166882, 1:500), VDAC1 (sc-390996, 1:500), PGC-1a (sc-518025, 1:500), NRF1 (sc515360, 1:500), TFAM (sc-166965, 1:500) from Santa Cruz Biotechnology (Dallas, TX, USA); PINK1 (BC100-494S, 1:500) from Novus biological (Littleton, CO, USA); 4-HNE (ab46545, 1:1000), HO-1 (ab68477, 1:2000), NQO1 (ab80588, 1:1000) from Abcam (Cambridge, UK).

Techniques: Western Blot, Quantitative RT-PCR, Comparison, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: Honokiol alleviated neurodegeneration by reducing oxidative stress and improving mitochondrial function in mutant SOD1 cellular and mouse models of amyotrophic lateral sclerosis.

doi: 10.1016/j.apsb.2022.07.019

Figure Lengend Snippet: Figure 9 Honokiol activated the NRF2eGSH pathway and improved mitochondrial function in the spinal cord and gastrocnemius muscle of SOD1-G93A mice. (A) and (D) Western blot analysis and quantification of HO-1, NQO1, GCLC, GCLM, GSS and GSR in the spinal cord (n Z 3e4); (B) and (E) Western blot analysis and quantification of PGC-1a, NRF1 and TFAM in the spinal cord (n Z 4); (C) and (F) Western blot analysis and quantification of OPA1, MFN2 and FIS1 in the spinal cord (n Z 4); (G) and (J) Western blot analysis and quantification analysis of HO-1, NQO1, GCLC, GCLM, GSS and GSR in the gastrocnemius muscle (n Z 4); (H) and (K) Western blot analysis and quantification of PGC-1a, NRF1 and TFAM in the gastrocnemius muscle (n Z 4e6); (I) and (L) Western blot analysis and quantification of OPA1, MFN2 and FIS1 in the gastrocnemius muscle (n Z 4). One-way ANOVA followed by post hoc LSD test was used for the comparisons among three or more groups (SPSS 16.0). Data are presented as the mean SEM (n Z 3e6, repeated independent experiments). #P < 0.05, ##P < 0.01 versus WT control group; *P < 0.05, **P < 0.01 versus SOD1-G93A model group.

Article Snippet: Membranes were blocked by the 5% skimmed milk solution for 2 h at room temperature and then incubated at 4 C overnight with antibodies against BCL2 (26539- 1-AP, 1:1000), BAX (50599-2-Ig, 1:2000), cytochrome c (10993- 1-AP, 1:2000), ATM (27156-1-AP, 1:1000), NRF2 (16396-1-AP, 1:1000), GCLC (12601-1-AP, 1:2000), GCLM (14241-1-AP, 1:2000), OPA1 (27733-1-AP, 1:1000), MFN2 (12186-1-AP, 1:2000), FIS1 (10956-1-AP, 1:1000), p62 (18420-1-AP, 1:1000), GFAP (26825-1-AP, 1:2000), HSP90 (13171-1-AP, 1:2000), GAPDH (60004-1-Ig, 1:5000) from Proteintech (Rosemont, IL, USA); GSR (sc-133245, 1:500), GSS (sc-166882, 1:500), VDAC1 (sc-390996, 1:500), PGC-1a (sc-518025, 1:500), NRF1 (sc515360, 1:500), TFAM (sc-166965, 1:500) from Santa Cruz Biotechnology (Dallas, TX, USA); PINK1 (BC100-494S, 1:500) from Novus biological (Littleton, CO, USA); 4-HNE (ab46545, 1:1000), HO-1 (ab68477, 1:2000), NQO1 (ab80588, 1:1000) from Abcam (Cambridge, UK).

Techniques: Western Blot, Control

Journal: Journal of Neurotrauma

Article Title: Advanced Age and Neurotrauma Diminish Glutathione and Impair Antioxidant Defense after Spinal Cord Injury

doi: 10.1089/neu.2022.0010

Figure Lengend Snippet: Glutathione synthesis and recycling. Glutathione is a recyclable tripeptide synthesized in two reactions. First, Glutamate-Cysteine Ligase ligates glutamate and cysteine, followed by glutathione synthetase, which adds glycine to finish the tripeptide structure. Two reduced glutathiones (GSH) are used as substrates for glutathione peroxidase (GPx) during the sequestration of reactive oxygen species (ROS). This oxidized glutathione (GSSG) is a dimer formed as a disulfide bridge that can be reduced by glutathione reductase (GR) back into two independent glutathione compounds using nicotinamide adenine dinucleotide phosphate (NADPH) as a substrate. GCLC, glutamate cysteine ligase catalytic domain.

Article Snippet: Blots were imaged using fluorescent development (Odyssey CLx; LI-COR Biosciences) and probed sequentially against GR (56 kD; 1:2,000; ab124995; Abcam Co.) and GCLC (70 kD; 1:1,000; H00002729-M01; Novus Biologicals), followed by GPx-1 (22 kD; 1:1,000; VPA00488; Bio-Rad) and GSH Synthetase (56 kD; 1:2,000; VMA00364; Bio-Rad), and finally against GAPDH (36 kD; 1:5,000; ab9484; Abcam Co.).

Techniques: Synthesized

Journal: Cells

Article Title: Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

doi: 10.3390/cells10081850

Figure Lengend Snippet: CDDO-Me activated Nrf2/ARE pathway predominantly in astrocytes in 6-OHDA rat brains. ( A ) Nrf2 immunostaining. Representative pictures show Nrf2 immunofluorescence in SNpc at week 2 after 6-OHDA injection, and nuclei were re-stained with Hoechst (200× magnification). The bar graph on the right side of the pictures is the quantitative analysis of optical density for fluorescence. The Nrf2-positive cells were few in the control group and mainly distributed in the cytoplasm. The Nrf2 signal increased in the 6-OHDA group compared with the control group (* p < 0.05, n = 5) and was mainly distributed in the nucleus. CDDO-Me increased Nrf2-positive cells in the 6-OHDA + CDDO group compared with the 6-OHDA group and control group (* p < 0.05,** p < 0.01, n = 5), and the Nrf2 signal was also mainly distributed in the nucleus in the 6-OHDA + CDDO group. ( B ) Co-immunostaining of Nrf2 with GFAP. Representative pictures show Nrf2 fluorescence was mainly distributed in neurons but not in GFAP-positive astrocytes in control group. Increased Nrf2 signal was colocalized with GFAP staining in the 6-OHDA group and 6-OHDA + CDDO group. The bar graph on the right side of the pictures is the quantitative analysis of optical density for Nrf2 fluorescence colocalization with GFAP-positive astrocytes. The results show more Nrf2 signal colocalized with GFAP fluorescence in the 6-OHDA group compared with the control group (* p < 0.05, n = 5). CDDO-Me further increased the Nrf2-positive signal in GFAP-positive astrocytes in the 6-OHDA + CDDO group compared with the 6-OHDA group (* p < 0.05, ** p < 0.01, n = 5). ( C ) Representative Western blots of Nrf2 and GCLC proteins in the substantia nigra and striatum homogenate. The bar graph below is the quantitative analysis of Western blot bands. Nrf2 did not significantly change in the substantia nigra (NS p > 0.05) and increased in the striatum in the 6-OHDA group compared with the control group (* p < 0.05). CDDO-Me increased Nrf2 in both substantia nigra and striatum in the 6-OHDA + CDDO group compared with in the 6-OHDA group (* p < 0.05). The Nrf2 downstream antioxidant GCLC also significantly increased in the substantia nigra and striatum in the 6-OHDA group compared with the control group (* p < 0.05). CDDO-Me further increased the GCLC level in the 6-OHDA + CDDO group compared with in the 6-OHDA group (* p < 0.05). ( D ) Representative Western blots of LC3 and quantitative analysis. The expression of the autophagy-related protein LC3-II did not significantly change in the substantia nigra and striatum in the 6-OHDA group compared with the control group (NS p > 0.05). CDDO-Me effectively increased LC3-II expression in the 6-OHDA + CDDO group compared with the 6-OHDA group (* p < 0.05).

Article Snippet: The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich).

Techniques: Immunostaining, Immunofluorescence, Injection, Staining, Fluorescence, Western Blot, Expressing

Journal: Cells

Article Title: Activation of Nrf2 in Astrocytes Suppressed PD-Like Phenotypes via Antioxidant and Autophagy Pathways in Rat and Drosophila Models

doi: 10.3390/cells10081850

Figure Lengend Snippet: Overexpression of Nrf2 in glial cells suppressed rotenone-induced PD-like phenotypes in flies. ( A ) survival curve and statistical analysis by Log-rank test and half survival time. Nrf2 overexpression in DMSO control drosophila did not significantly improve survival time in the DMSO/Elav;cncc or DMSO/Repo;cncc group compared with the DMSO/WT control group (NS p > 0.05). Rotenone treatment significantly shortened survival time compared with the corresponding DMSO control group ( ## p < 0.01). Nrf2 overexpression in neurons of PD model Drosophila significantly increased survival time in the rotenone/Elav;cncc group compared with the rotenone/WT group (* p < 0.05). Nrf2 overexpression in glial cells more significantly increased survival time in the rotenone/Repo;cncc group compared with the rotenone/Elav;cncc group and the rotenone/WT group (* p < 0.05 and ** p < 0.01). ( B ) The left graph shows climbing ability throughout lifetime. The right bar graph shows the climbing ability at ages of 3–5 weeks. Rotenone treatment significantly reduced climbing ability in the rotenone/WT group compared with the DMSO group ( ## p < 0.01). Nrf2 overexpression in glial cells in the rotenone/Repo;cncc group more significantly improved the motor ability than overexpression in neurons in the rotenone/Elav;cncc group (* p < 0.05 and ** p < 0.01). ( C ) Western blot analysis of Nrf2, GCLC, HO-1, and Ref(2)P proteins in Drosophila brain homogenates. The bar graph below is the quantitative analysis of Western blot bands. Nrf2 overexpression in neurons and glial cells increased GCLC and HO-1 in the Elav;cncc and Repo;cncc group compared with the WT control group (* p < 0.05 and ** p < 0.01). Nrf2 overexpression in glial cells further increased Nrf2, GCLC and HO-1 in the Repo;cncc group compared to overexpression in neurons in the Elav;cncc group (* p < 0.05). The Nrf2 overexpression significantly increased Ref(2)P in the rotenone/Elav;cncc and rotenone/Repo;cncc groups compared with the rotenone/WT group (* p < 0.05 and ** p < 0.01), but not in the DMSO control group. Nrf2 overexpression in glial cells enhanced autophagy more than in neurons (* p < 0.05). ( D ) Measure of redox state by DHE staining. Oxidative stress was relatively low in the DMSO control group. Rotenone treatment significantly increased oxidative stress in the rotenone/WT group compared with the DMSO control group (*** p < 0.001). Nrf2 overexpression slightly reduced oxidative stress in the DMSO control group and significantly decreased rotenone-induced oxidative stress in the rotenone/WT group (* p < 0.05 and ** p < 0.01). Nrf2 overexpression in glial cells in the rotenone/Repo;cncc group resulted in more DHE staining than such overexpression in neurons in the rotenone/Elav;cncc group (* p < 0.05).

Article Snippet: The primary antibodies used for the Western blot included anti-Nrf2 (1:1000, Assaybiotech), anti-GCLC (1:1000, BD), anti-HO-1 and anti-LC3I/II (Sigma), anti-Ref(2)P (1:1000, BD Pharmingen, San Jose, CA, USA), and anti-β-tubulin (1:5000, Sigma-Aldrich).

Techniques: Over Expression, Western Blot, Staining

Journal: Antioxidants

Article Title: Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/antiox12111999

Figure Lengend Snippet: Primer sequences used for PCR.

Article Snippet: The following primary antibodies were used in this study: anti-tyrosine hydroxylase (TH), anti-p-cAMP response element-binding protein (CREB), and anti-NQO1 from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-B-cell lymphoma 2 (Bcl-2) and anti-glial cell-derived neurotrophic factor (GDNF) from Abcam (Cambridge, UK); anti-ionized calcium-binding adapter molecule1 (Iba-1) from Wako (Osaka, Japan); anti-inducible nitric oxide synthase (iNOS) from BD Biosciences (San Jose, CA, USA); anti-tumor necrosis factor (TNF-α), anti-interleukin (IL)-6, anti-Toll-like receptor 2 (TLR2), anti-TLR4, anti-IL-10, anti-Nrf2, and anti-transforming growth factor-beta (TGF-β) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) and anti-neuronal nuclear (NeuN) from Merck-Millipore (Billerica, MA, USA); anti-HO-1 and anti-MnSOD from Enzo Life Sciences (Farmingdale, NY, USA); anti-4-hydroxy-2 E -nonenal (4-HNE) from Alpha diagnostic Intl Inc. (San Antonio, TX, USA); anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) from Antibodies-online (Aachen, German); anti-γ-glutamyl cysteine ligase-modulator subunit (GCLM) from GeneTex (Irvine, CA, USA); anti-cyclooxygenase-2 (COX-2), anti-IL-1β, and anti-γ-glutamyl cysteine ligase-catalytic subunit (GCLC) from MyBioSource (San Diego, CA, USA); and anti-glial fibrillary acidic protein (GFAP), anti-brain-derived neurotrophic factor (BDNF), and anti-β-actin from Sigma-Aldrich.

Techniques: Sequencing

Journal: Antioxidants

Article Title: Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/antiox12111999

Figure Lengend Snippet: Effects of NKT on the expressions of pro-inflammatory, anti-inflammatory, and antioxidant molecules in the brains of MPTP-treated mice. ( A ) Western blot data showing the effects of NKT on the protein expressions of iNOS, COX-2, pro-inflammatory cytokines, Iba-1, GFAP, TLR2/4 in the SN of MPTP mice (each group n = 6–7). ( B ) Quantification of Western blot data. ( C ) Effects of NKT on anti-inflammatory cytokines such as TGF-β and IL-10 in the SN of MPTP mice (each group n = 6–7). ( D ) Quantification of Western blot data. ( E ) Western blot data showing the effects of NKT on 4-HNE, Nrf2, HO-1, NQO1, MnSOD, GCLC, and GCLM in the SN of MPTP mice (each group n = 6–7). ( F ) Quantification of Western blot data. ( G ) Effect of NKT on intracellular GSH level in the SN of MPTP mice. The data are presented as the mean ± SEM. * p < 0.05 vs. control group; ** p < 0.01 vs. control group; # p < 0.05 vs. MPTP-treated group; ## p < 0.01 vs. MPTP-treated group.

Article Snippet: The following primary antibodies were used in this study: anti-tyrosine hydroxylase (TH), anti-p-cAMP response element-binding protein (CREB), and anti-NQO1 from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-B-cell lymphoma 2 (Bcl-2) and anti-glial cell-derived neurotrophic factor (GDNF) from Abcam (Cambridge, UK); anti-ionized calcium-binding adapter molecule1 (Iba-1) from Wako (Osaka, Japan); anti-inducible nitric oxide synthase (iNOS) from BD Biosciences (San Jose, CA, USA); anti-tumor necrosis factor (TNF-α), anti-interleukin (IL)-6, anti-Toll-like receptor 2 (TLR2), anti-TLR4, anti-IL-10, anti-Nrf2, and anti-transforming growth factor-beta (TGF-β) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) and anti-neuronal nuclear (NeuN) from Merck-Millipore (Billerica, MA, USA); anti-HO-1 and anti-MnSOD from Enzo Life Sciences (Farmingdale, NY, USA); anti-4-hydroxy-2 E -nonenal (4-HNE) from Alpha diagnostic Intl Inc. (San Antonio, TX, USA); anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) from Antibodies-online (Aachen, German); anti-γ-glutamyl cysteine ligase-modulator subunit (GCLM) from GeneTex (Irvine, CA, USA); anti-cyclooxygenase-2 (COX-2), anti-IL-1β, and anti-γ-glutamyl cysteine ligase-catalytic subunit (GCLC) from MyBioSource (San Diego, CA, USA); and anti-glial fibrillary acidic protein (GFAP), anti-brain-derived neurotrophic factor (BDNF), and anti-β-actin from Sigma-Aldrich.

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/antiox12111999

Figure Lengend Snippet: NKT inhibited ROS production and upregulated the antioxidant enzyme expression via Nrf2/ARE signaling pathway in rat primary astrocytes. ( A ) Rat primary astrocytes were treated with NKT prior to MPP + stimulation for 16 h, and intracellular ROS level was evaluated with CellROX staining (n = 3). Representative fluorescence images are shown in the left panel, and the quantification of fluorescence intensity is shown in the right panel. ( B ) Astrocyte cells were incubated with NKT for 6 h to determine the effects of NKT on the protein expression of HO-1, NQO1, and MnSOD (n = 3). ( C ) RT-PCR results demonstrating the influence of NKT on mRNA expression of antioxidant enzymes (n = 3). ( D ) Effect of NKT on intracellular GSH level in astrocytes (n = 3). ( E ) Effect of NKT on GCLC and GCLM protein expression (n = 3). Quantification data of Western blot analysis are shown in right panel. ( F ) RT-PCR results demonstrating the influence of NKT on mRNA expression of GCLC and GCLM (n = 3). ( G ) Western blot data showing the effect of NKT on nuclear translocation of Nrf2. ( H ) EMSA data showing the effect of NKT on Nrf2 DNA binding activity. ‘F’ indicates free probe. ( I ) Effect of NKT on ARE-luc reporter gene activity. The data are presented as the mean ± SEM. * p < 0.05 vs. control cells; ** p < 0.01 vs. control cells; ## p < 0.01 vs. MPP + -treated cells.

Article Snippet: The following primary antibodies were used in this study: anti-tyrosine hydroxylase (TH), anti-p-cAMP response element-binding protein (CREB), and anti-NQO1 from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-B-cell lymphoma 2 (Bcl-2) and anti-glial cell-derived neurotrophic factor (GDNF) from Abcam (Cambridge, UK); anti-ionized calcium-binding adapter molecule1 (Iba-1) from Wako (Osaka, Japan); anti-inducible nitric oxide synthase (iNOS) from BD Biosciences (San Jose, CA, USA); anti-tumor necrosis factor (TNF-α), anti-interleukin (IL)-6, anti-Toll-like receptor 2 (TLR2), anti-TLR4, anti-IL-10, anti-Nrf2, and anti-transforming growth factor-beta (TGF-β) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) and anti-neuronal nuclear (NeuN) from Merck-Millipore (Billerica, MA, USA); anti-HO-1 and anti-MnSOD from Enzo Life Sciences (Farmingdale, NY, USA); anti-4-hydroxy-2 E -nonenal (4-HNE) from Alpha diagnostic Intl Inc. (San Antonio, TX, USA); anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) from Antibodies-online (Aachen, German); anti-γ-glutamyl cysteine ligase-modulator subunit (GCLM) from GeneTex (Irvine, CA, USA); anti-cyclooxygenase-2 (COX-2), anti-IL-1β, and anti-γ-glutamyl cysteine ligase-catalytic subunit (GCLC) from MyBioSource (San Diego, CA, USA); and anti-glial fibrillary acidic protein (GFAP), anti-brain-derived neurotrophic factor (BDNF), and anti-β-actin from Sigma-Aldrich.

Techniques: Expressing, Staining, Fluorescence, Incubation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Translocation Assay, Binding Assay, Activity Assay, Control

Journal: Antioxidants

Article Title: Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/antiox12111999

Figure Lengend Snippet: Nrf2 siRNA or Nrf2-specific inhibitor, brusatol, reversed NKT-mediated upregulation of antioxidant molecules in rat primary astrocytes. ( A , B ) Cells were transfected with Nrf2 siRNA or control siRNA and then treated with NKT for 6 h to detect Nrf2 and antioxidant enzyme levels (n = 3). Western blot analysis was performed to confirm Nrf2 knockdown ( A ) and the expression of antioxidant enzymes ( B ). The left panel shows representative blots, and the right panel shows quantification data. * p < 0.05 vs. control cells; ** p < 0.01 vs. control cells; # p < 0.05 vs. control siRNA-transfected cells in the presence of NKT; ## p < 0.01 vs. control siRNA-transfected cells in the presence of NKT. ( C , D ) Cells were incubated with BT for 1 h, followed by NKT for 6 h to detect antioxidant enzymes ( C ) and GSH levels ( D ). The right panel depicts the quantification of Western blot data. ( E ) Quantitative RT-PCR data showing the effect of NKT on mRNA expressions of antioxidant enzymes and GCLC/GCLM. ( F ) Effect of NKT on transcriptional activities of ARE-luc, HO-1 E1-luc, or NQO1-luc (n = 3). The data are presented as the mean ± SEM. * p < 0.05 vs. control cells; ** p < 0.01 vs. control cells; & p < 0.05 vs. NKT-treated cells; && p < 0.01 vs. NKT-treated cells.

Article Snippet: The following primary antibodies were used in this study: anti-tyrosine hydroxylase (TH), anti-p-cAMP response element-binding protein (CREB), and anti-NQO1 from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-B-cell lymphoma 2 (Bcl-2) and anti-glial cell-derived neurotrophic factor (GDNF) from Abcam (Cambridge, UK); anti-ionized calcium-binding adapter molecule1 (Iba-1) from Wako (Osaka, Japan); anti-inducible nitric oxide synthase (iNOS) from BD Biosciences (San Jose, CA, USA); anti-tumor necrosis factor (TNF-α), anti-interleukin (IL)-6, anti-Toll-like receptor 2 (TLR2), anti-TLR4, anti-IL-10, anti-Nrf2, and anti-transforming growth factor-beta (TGF-β) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) and anti-neuronal nuclear (NeuN) from Merck-Millipore (Billerica, MA, USA); anti-HO-1 and anti-MnSOD from Enzo Life Sciences (Farmingdale, NY, USA); anti-4-hydroxy-2 E -nonenal (4-HNE) from Alpha diagnostic Intl Inc. (San Antonio, TX, USA); anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) from Antibodies-online (Aachen, German); anti-γ-glutamyl cysteine ligase-modulator subunit (GCLM) from GeneTex (Irvine, CA, USA); anti-cyclooxygenase-2 (COX-2), anti-IL-1β, and anti-γ-glutamyl cysteine ligase-catalytic subunit (GCLC) from MyBioSource (San Diego, CA, USA); and anti-glial fibrillary acidic protein (GFAP), anti-brain-derived neurotrophic factor (BDNF), and anti-β-actin from Sigma-Aldrich.

Techniques: Transfection, Control, Western Blot, Knockdown, Expressing, Incubation, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Astrocytic Nrf2 Mediates the Neuroprotective and Anti-Inflammatory Effects of Nootkatone in an MPTP-Induced Parkinson’s Disease Mouse Model

doi: 10.3390/antiox12111999

Figure Lengend Snippet: Brusatol reversed the effect of NKT on oxidative stress and astroglial Nrf2/antioxidant enzyme expression in MPTP-treated mice. ( A ) Effects of BT and NKT on the protein levels of 4-HNE, Nrf2, HO-1, NQO1, GCLC, GCLM in the SN of MPTP mice (each group n = 7–8). ( B ) Effects of BT and NKT on GSH level in the SN of MPTP-injected mice. ( C – E ) IF staining data showing 8-OHdG and GFAP expression in the SN of MPTP mice (n = 5–6 per group). Representative images ( C ) and high magnitude images ( D ) are provided and quantification of 8-OHdG expression and 8-OHdG + /GFAP + cells is shown ( E ). ( F ) IF staining data showing Nrf2 and GFAP expression in the SN (n = 5–6). The upper panel has representative images and the quantification of Nrf2 expression and Nrf2 + /GFAP + /DAPI + cells is shown in the bottom panel. The data are presented as the mean ± SEM. * p < 0.05 vs. control group; ** p < 0.01 vs. control group; # p < 0.05 vs. MPTP-treated group; ## p < 0.01 vs. MPTP-treated group; & p < 0.05 vs. MPTP + NKT-treated group; && p < 0.01 vs. MPTP + NKT-treated group.

Article Snippet: The following primary antibodies were used in this study: anti-tyrosine hydroxylase (TH), anti-p-cAMP response element-binding protein (CREB), and anti-NQO1 from Cell Signaling Technology, Inc. (Danvers, MA, USA); anti-B-cell lymphoma 2 (Bcl-2) and anti-glial cell-derived neurotrophic factor (GDNF) from Abcam (Cambridge, UK); anti-ionized calcium-binding adapter molecule1 (Iba-1) from Wako (Osaka, Japan); anti-inducible nitric oxide synthase (iNOS) from BD Biosciences (San Jose, CA, USA); anti-tumor necrosis factor (TNF-α), anti-interleukin (IL)-6, anti-Toll-like receptor 2 (TLR2), anti-TLR4, anti-IL-10, anti-Nrf2, and anti-transforming growth factor-beta (TGF-β) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1α) and anti-neuronal nuclear (NeuN) from Merck-Millipore (Billerica, MA, USA); anti-HO-1 and anti-MnSOD from Enzo Life Sciences (Farmingdale, NY, USA); anti-4-hydroxy-2 E -nonenal (4-HNE) from Alpha diagnostic Intl Inc. (San Antonio, TX, USA); anti-8-hydroxy-2′-deoxyguanosine (8-OHdG) from Antibodies-online (Aachen, German); anti-γ-glutamyl cysteine ligase-modulator subunit (GCLM) from GeneTex (Irvine, CA, USA); anti-cyclooxygenase-2 (COX-2), anti-IL-1β, and anti-γ-glutamyl cysteine ligase-catalytic subunit (GCLC) from MyBioSource (San Diego, CA, USA); and anti-glial fibrillary acidic protein (GFAP), anti-brain-derived neurotrophic factor (BDNF), and anti-β-actin from Sigma-Aldrich.

Techniques: Expressing, Injection, Staining, Control