Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: Measurement of SH-SY5Y cell viability after ( A ) treatment with different concentrations of 6-OHDA (0–500 µM) for 2 h and ( B ) 300 µM 6-OHDA treatment for 2 h and post-treatment with vitamin K2 for 6 h. The data are presented as mean ± SD ( n = 3).

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques:

Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: Effect of vitamin K2 on SH-SY5Y cell apoptosis in the presence of 6-OHDA. ( A ) Apoptotic cells were detected by flow cytometry. ( B ) Images of cells were obtained by fluorescence microscopy. All images were taken with a 20× objective. Images were taken in red modes, where red staining represents apoptosis cells, and representative graphs of the mean fluorescence intensity (MFI) from three different fields of view, using ImageJ software. ( C ) Measurement of Bax and Bcl-2 mRNA expression changes by RT-qPCR. ( D ) Changes in protein expression levels of Bax and Bcl-2 detected by Western blot; images were quantified by using the ImageJ software. The data are presented as mean ± SD ( n = 3). Significant difference * p < 0.5, ** p < 0.05, and *** p < 0.01 vs. control.

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques: Flow Cytometry, Fluorescence, Microscopy, Staining, Software, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: Effect of vitamin K2 on mitochondrial dynamic balance in 6-OHDA-injured cells. ( A ) Measurement of MFN1, MFN2, and DRP1 mRNA expression changes by using RT-qPCR. ( B ) Changes in protein expression levels of MFN1, MFN2, and DRP1 detected by Western blot; images were quantified by using the ImageJ software. The data are presented as mean ± SD ( n = 3). Significant difference * p < 0.5, ** p < 0.05, and *** p < 0.01 vs. control.

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: Effects of vitamin K2 on mitophagy and mitochondrial biogenesis in 6-OHDA-injured cells. ( A ) Measurement of p62, LC3A, PGC-1α, NRF1, and TFAM mRNA expression changes by RT-qPCR. ( B ) Changes in protein expression levels of p62, LC3A, PGC-1α, NRF1, and TFAM assessed by Western blot; images were quantified by using the ImageJ software. The data are presented as mean ± SD ( n = 3). Significant difference * p < 0.5, ** p < 0.05, and *** p < 0.01 vs. control.

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: PINK1/Parkin signaling pathway was involved in the regulation of mitophagy by vitamin K2. ( A ) Measurement of PINK1 and Parkin mRNA expression changes by RT-qPCR. ( B ) Changes in protein expression levels of PINK1 and Parkin detected by Western blot; images were quantified by using the ImageJ software. The data are presented as mean ± SD ( n = 3). Significant difference * p < 0.5 vs. control. (ns: no significance)

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Control

Journal: Nutrients

Article Title: Vitamin K2 Modulates Mitochondrial Dysfunction Induced by 6-Hydroxydopamine in SH-SY5Y Cells via Mitochondrial Quality-Control Loop

doi: 10.3390/nu14071504

Figure Lengend Snippet: Schematic illustration of the protective role of vitamin K2 in 6-OHDA-induced cytotoxicity in SH-SY5Y cells. The 6-OHDA triggered ROS, causing oxidative stress in SH-SY5Y cells, which leads to mitochondrial depolarization (mitochondrial membrane potential reduction) and induction of mitochondria-mediated apoptosis, resulting in neuronal cell death. However, post-treatment with vitamin K2 maintains the balance of mitochondrial fission/fusion through the expression of regulatory proteins MFN1/2 and DRP1; promotes mitophagy by recruiting autophagy molecules p62 and LC3 through the PINK1/Parkin signaling pathway; and promotes mitochondrial biogenesis through the expression of regulatory proteins PGC-1α, NRF1, and TFAM. Thus, the normal operation of the mitochondrial quality-control loop (mitochondrial fusion, fission, autophagy, and biogenesis) is maintained, the intracellular oxidative stress is relieved, the cell membrane potential is restored, and the mitochondrial dysfunction is relieved. Moreover, by upregulating the expression of Bcl-2 protein and downregulating the expression of Bax protein to play the role of the main switch of apoptosis, the cell damage caused by mitochondrial dysfunction is reduced. Note: ROS, reactive oxygen species; 6-OHDA, 6-Hydroxydopamine; Ψm, mitochondrial membrane potential.

Article Snippet: To verify the mitigating effect of vitamin K2 (MK-7, extracted from Bacillus subtilis (natto) in our laboratory) on neuronal toxicity induced by 6-OHDA (MCE, Shanghai, China), we administered different concentrations of vitamin K2 (10, 20, 30, and 40 µM) and 6-OHDA (100, 200, 300, 400, and 500 µM) in a time-dependent dose-response manner.

Techniques: Membrane, Expressing, Control

Journal: Biomolecules

Article Title: DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

doi: 10.3390/biom15070907

Figure Lengend Snippet: ATM inhibition improves cell viability and reduces etoposide-induced DNA damage in cell cultures. ( A ) Effect of AZD1390 on SH-SY5Y cell viability following etoposide-induced DNA damage. SH-SY5Y cells were treated with increasing concentrations of the ATM inhibitor AZD1390 (0, 5, 10, and 20 nM) in the presence of etoposide. Cell viability was assessed using the MTT assay. Data are presented as the mean ± SEM from three independent experiments. Data were compared between groups using one-way ANOVA. * p < 0.05, ** p < 0.01, AZD1390 vs. etoposide; *** p < 0.001 etoposide vs. control. NS = non-significant. ( B ) Representative immunofluorescence images of mouse neuronal cultures showing the colocalization of DDSB marker [γ-H2AX (Ser139); red] and neuronal marker (MAP2; green). Quantification of γ-H2AX foci in MAP2-positive neurons was performed to assess DDSB accumulation. The solid box shows a magnified view of the area marked by the white dashed box in the main image. Data were compared between groups using one-way ANOVA and are presented as the mean ± SEM * p < 0.05; AZD1390 vs. etoposide. Scale bar = 100 µM; magnification = 200×.

Article Snippet: In Group 3, synucleinopathy mice were treated daily with the ATM inhibitor AZD1390 (20 mg/kg, orally: Cat. No.: HY-109566; MedChem Express, Monmouth Junction, NJ, USA) for 6 weeks.

Techniques: Inhibition, MTT Assay, Control, Immunofluorescence, Marker

Journal: Biomolecules

Article Title: DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

doi: 10.3390/biom15070907

Figure Lengend Snippet: ATM inhibition reduces DNA damage in the midbrain of a mouse model of α-synucleinopathy. ( A ) Representative immunofluorescence images showing α-synuclein (HA-Tag; green) localized in dopaminergic neurons labeled with tyrosine hydroxylase (TH; red) in the substantia nigra (SN), six weeks after AAV1/2-A53T-α-Syn vector injection into the WT mouse brain. (N = 4/group). Magnification 100×; Scale bar, 200 μm. ( B ) Immunofluorescence staining for phospho-ATM (p-ATM; red) in midbrain sections of WT mice infused with AAV1/2-A53T-αSyn vector. Elevated p-ATM signal was observed in dopaminergic neurons, identified by co-labeling with tyrosine hydroxylase (TH; green), indicating activation of the DNA damage response in α-synuclein-expressing neurons. Scale bar, 50 μm. (N = 4/group). ( C ) quantification of TH-positive neurons. ( D , E ) Immunoblot analysis of γ-H2A.X (Ser139) levels in the midbrain of AAV-EV and AAV-A53T-Syn-treated WT and AAV-A53T-Syn-treated WT followed by AZD1390 treatment (N = 6 mice/group). Data were compared between groups using one-way ANOVA with a Holm–Sidak post hoc test. The values are represented as mean ± SEM * p < 0.05; ** p < 0.01.

Article Snippet: In Group 3, synucleinopathy mice were treated daily with the ATM inhibitor AZD1390 (20 mg/kg, orally: Cat. No.: HY-109566; MedChem Express, Monmouth Junction, NJ, USA) for 6 weeks.

Techniques: Inhibition, Immunofluorescence, Labeling, Plasmid Preparation, Injection, Staining, Activation Assay, Expressing, Western Blot

Journal: Biomolecules

Article Title: DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

doi: 10.3390/biom15070907

Figure Lengend Snippet: AZD1390 reduces senescence in the brains of α-synucleinopathy mice. ( A – C ) Western blot analysis was conducted to assess phosphorylated p53 (P-p53) and phosphorylated NF-κB (P-NF-κB) in midbrain tissue lysates from AAV-EV control, α-synuclein-expressing, and α-synuclein + AZD1390-treated WT mice. Band intensities were quantified by densitometry and normalized to GAPDH. Statistical significance was determined using one-way ANOVA followed by Holm–Sidak post hoc test. Data are presented as the mean ± SEM (N = 6/group). * p < 0.05, ** p < 0.01. ( D , E ) The mRNA expression of senescence markers Cdkn1a and Cdkn2a assessed by RT-qPCR. Data were analyzed using one-way ANOVA followed by a Holm–Sidak post hoc test; * p < 0.05 or ** p < 0.01 was considered statistically significant (N = 5/group).

Article Snippet: In Group 3, synucleinopathy mice were treated daily with the ATM inhibitor AZD1390 (20 mg/kg, orally: Cat. No.: HY-109566; MedChem Express, Monmouth Junction, NJ, USA) for 6 weeks.

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

Journal: Biomolecules

Article Title: DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

doi: 10.3390/biom15070907

Figure Lengend Snippet: AZD1390 reduces neuroinflammatory responses in the brains of α-synucleinopathy mice. RT-qPCR as employed to assess the mRNA expressions of cytokines Il-6 ( A ) and Tnf -α ( B ) and chemokines Ccl2 ( C ), and Cxcl10 ( D ) in midbrains of controls, AAV-A53T-Syn-treated mice and AAV-A53T-Syn-injected mice treated with AZD1390 post 6 weeks of vector infusion. Data were analyzed using one-way ANOVA followed by the Holm–Sidak post hoc test. The values are expressed as the mean ± SEM * p < 0.05 (N = 5/group).

Article Snippet: In Group 3, synucleinopathy mice were treated daily with the ATM inhibitor AZD1390 (20 mg/kg, orally: Cat. No.: HY-109566; MedChem Express, Monmouth Junction, NJ, USA) for 6 weeks.

Techniques: Quantitative RT-PCR, Injection, Plasmid Preparation

Journal: Biomolecules

Article Title: DNA Damage Response Regulation Alleviates Neuroinflammation in a Mouse Model of α-Synucleinopathy

doi: 10.3390/biom15070907

Figure Lengend Snippet: Inhibition of ATM alleviates behavioral deficits in a mouse model of α-synucleinopathy. ( A ) The cylinder test was performed to evaluate forelimb asymmetry in AAV-A53T-αSyn-expressing mice, with or without treatment with the AZD1390. The frequency of contact with the cylinder wall using the impaired versus non-impaired forelimb was quantified to assess motor asymmetry. Data were analyzed using one-way ANOVA followed by the Holm–Sidak post hoc test. The values are expressed as the mean ± SEM * p < 0.05. ( B , C ) Gait analysis was conducted using the CatWalk system to evaluate walking duration and average speed in α-synuclein-expressing mice, with or without AZD1390 treatment. Data were analyzed using one-way ANOVA followed by the Holm–Sidak post hoc test. The values are expressed as the mean ± SEM * p < 0.05; ** p < 0.01. ( D , E ) Raised beam task was used to assess the ability of mice to walk on narrow transverse beam to enter a dork box and number of slips during walking on 9 mm square and round beams. Motor performance was assessed by measuring beam latency and counting the number of slips during traversal. Beam latency data were analyzed using one-way ANOVA, while the number of slips, a non-parametric behavioral measure, was assessed using the Kruskal–Wallis test. The values are expressed as mean ± SEM * p < 0.05; ** p < 0.01. N = 6–7 mice/group.

Article Snippet: In Group 3, synucleinopathy mice were treated daily with the ATM inhibitor AZD1390 (20 mg/kg, orally: Cat. No.: HY-109566; MedChem Express, Monmouth Junction, NJ, USA) for 6 weeks.

Techniques: Inhibition, Expressing