Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells
<p>Effect of LPS stimulation on neuronal differentiation in NSCs. During the growth of isolated NSCs, bFGF was present (+bFGF, proliferation condition) to prevent differentiation and promote proliferation. In order to induce the neuronal differentiation, bFGF was removed (-bFGF, differentiation condition). (<b>A</b>) Cell viability in LPS-treated NSCs. NSCs were treated with LPS at 10 ng, 100 ng, 500 ng, 1 μg, 2 μg, or 5 μg/mL for 1 day in the absence of bFGF. The cells were collected for analysis for cell viability and performed using the Cell counting Kit-8. Absorbance was measured at 450 nm, and all experiments were performed in triplicate. Cytotoxic activity is expressed as the percentage of cell viability using the formula: cytotoxicity (%) = (1 − A450 of target cells/A450 of control cells) × 100; <span class="html-italic">n</span> = 3. Data are shown as means ± SD. (<b>B</b>,<b>C</b>) NSCs were treated with LPS (1 μg/mL) for 3 days in the absence of bFGF. They were stained with anti-Tuj1 (green) and DAPI (blue). Scale bar: 100 μm. (<b>C</b>) Neurite lengths were measured in randomly selected fields using three independent experiments, with <span class="html-italic">n</span> = 3 per group. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF control. (<b>D</b>) The cells were treated with LPS (1 μg/mL) for 12 h in the absence of bFGF. The Tuj1 mRNA level was analyzed using RT-PCR (upper panel) and real time-RT-PCR (graph); <span class="html-italic">n</span> = 3. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with–bFGF control. (<b>E</b>) The cells were treated with LPS (1 μg/mL) for 1 day in the absence of bFGF. Western blotting was performed using anti-Tuj1 or anti-calnexin antibodies to detect the respective protein bands (upper panel). Band intensity (graph) was quantified with Quantity Ones<sup>®</sup> software. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF control. (<b>F</b>) The cells were treated with LPS (1 μg/mL) for 12 h in the absence of bFGF. The mRNA levels of IL-6 and TNF-α were analyzed by real-time RT-PCR; <span class="html-italic">n</span> = 3. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF control, for IL-6 and TNF-α, respectively. Statistical significances were assessed by one-way ANOVA with a post hoc Tukey’s test.</p> "> Figure 2
<p>Effect of Bcl-2 overexpression on LPS-induced neuronal damage in NSCs. (<b>A</b>,<b>B</b>) NSCs were treated with LPS (1 μg/mL) for 1 day in the absence of bFGF. (<b>A</b>) Western blotting was performed using anti-Tuj1, anti-Bcl-2, or anti-calnexin antibodies to detect the respective protein bands. (<b>B</b>) Band intensity was quantified with Quantity Ones<sup>®</sup> software. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF control. (<b>C</b>,<b>D</b>) MSCV-IRES-EGFP or <span class="html-italic">rBcl-2</span>-MSCV-IRES-EGFP was transfected into the NSCs for 48 h, then the cells were treated with LPS (1 μg/mL) for 1 day in the absence of bFGF. (<b>C</b>) Western blotting was performed using anti-Tuj1, anti-Bcl-2, or anti-calnexin antibodies to detect the respective protein bands. (<b>D</b>) Band intensity was quantified with Quantity Ones<sup>®</sup> software. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector/LPS control. <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector control. (<b>E</b>,<b>F</b>) MSCV-IRES-EGFP or <span class="html-italic">rBcl-2</span>-MSCV-IRES-EGFP was transfected into the NSCs for 48 h, and the cells were treated with LPS (1 μg/mL) for 3 days in the absence of bFGF. The NSCs were stained with anti-Tuj1(green) and DAPI (blue). Scale bar: 100 μm. (<b>F</b>) Neurite lengths were measured in randomly selected fields using three independent experiments; <span class="html-italic">n</span> = 3 per group. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector/LPS control. <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector control. Statistical significances were assessed by one-way ANOVA with a post hoc Tukey’s test.</p> "> Figure 3
<p>Effect of Bcl-2 overexpression on LPS-induced IL-6 and TNF-α production in NSCs. (<b>A</b>) MSCV-IRES-EGFP or <span class="html-italic">rBcl-2</span>-MSCV-IRES-EGFP was transfected into the NSCs for 48 h, and the cells were treated with LPS (1 μg/mL) for 12 h in the absence of bFGF. The mRNA levels of <span class="html-italic">IL-6</span> and <span class="html-italic">TNF-α</span> were analyzed by real-time RT-PCR; <span class="html-italic">n =</span> 3. Data are shown as mean ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector/LPS control, for <span class="html-italic">IL-6</span> and <span class="html-italic">TNF-α</span> respectively. (<b>B</b>,<b>C</b>) MSCV-IRES-EGFP or <span class="html-italic">rBcl-2</span>-MSCV-IRES-EGFP was transfected into the NSCs for 48 h, and the cells were treated with LPS (1 μg/mL) for 24 h in the absence of bFGF. The levels of IL-6 (<b>B</b>) and TNF-α (<b>C</b>) were measured by ELISA. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/Vector/LPS control. Statistical significances were assessed by one-way ANOVA with a post hoc Tukey’s test.</p> "> Figure 4
<p>Effect of Bcl-2 depletion on LPS-induced neuronal damage in NSCs. (<b>A</b>,<b>B</b>) Control siRNA or Bcl-2 siRNA was transfected into the NSCs for 72 h, and then the cells were treated with LPS (1 μg/mL) for 1 day in the absence of bFGF. (<b>A</b>) Western blotting was performed using anti-Tuj1, anti-Bcl-2, or anti-calnexin antibodies to detect the respective protein bands. (<b>B</b>) Band intensity was quantified with Quantity Ones<sup>®</sup> software. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA/LPS. <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA. (<b>C</b>,<b>D</b>) Control siRNA or Bcl-2 siRNA was transfected into the NSCs for 72 h, and then the cells were treated with LPS (1 μg/mL) for 3 days in the absence of bFGF. The NSCs were stained with anti-Tuj1(green) and DAPI (blue). Scale bar: 100 μm. (<b>D</b>) Neurite lengths were measured in randomly selected fields using three independent experiments; <span class="html-italic">n</span> = 3 per group. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA/LPS. <sup>#</sup> <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA. Statistical significances were assessed by one-way ANOVA with a post hoc Tukey’s test.</p> "> Figure 5
<p>Effect of Bcl-2 depletion on LPS-induced IL-6 and TNF-α production in NSCs. (<b>A</b>) Control siRNA or Bcl-2 siRNA was transfected into the NSCs for 72 h, and then the cells were treated with LPS (1 μg/mL) for 12 h in the absence of bFGF. The mRNA levels of <span class="html-italic">IL-6</span> and <span class="html-italic">TNF-α</span> were analyzed by real-time RT-PCR; <span class="html-italic">n =</span> 3. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA/LPS, for <span class="html-italic">IL-6</span> and <span class="html-italic">TNF-α,</span> respectively. (<b>B</b>,<b>C</b>) Control siRNA or Bcl-2 siRNA was transfected into the NSCs for 72 h, and then the cells were treated with LPS (1 μg/mL) for 24 h in the absence of bFGF. The levels of IL-6 (<b>B</b>) and TNF-α (<b>C</b>) were measured by ELISA. Data are shown as means ± SD. * <span class="html-italic">p</span> < 0.05 compared with –bFGF/control siRNA/LPS. (<b>D</b>) The proposed model for Bcl-2-mediated neuronal differentiation in LPS-treated NSCs. The model suggests that Bcl-2 plays a neuroprotective role in LPS-induced neuroinflammation of NSCs, resulting in neuronal differentiation. Statistical significances were assessed by one-way ANOVA with a post hoc Tukey’s test.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Primary Culture of Neural Precursor Cells
2.3. Transient Transfection of NSCs
2.4. Real-Time PCR and Reverse Transcription (RT)-PCR
2.5. Cytotoxicity of NSCs Exposed to LPS
2.6. Western Blot Analysis
2.7. Immunofluorescence Staining
2.8. Measurement of Neurite Outgrowth
2.9. Enzyme-Linked Immunosorbent Assay (ELISA)
2.10. Statistical Analysis
3. Results
3.1. LPS-Induced Neuroinflammation Inhibits Neuronal Differentiation of NSCs
3.2. Bcl-2 Has a Neuroprotective Effect on LPS-Induced Neuroinflammation in NSCs
3.3. Knockdown of Bcl-2 Impairs Neuroprotective Function in LPS-Induced Neuroinflammation of NSCs
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
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Park, S.-Y.; Han, J.-S. Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells. Int. J. Mol. Sci. 2022, 23, 6399. https://doi.org/10.3390/ijms23126399
Park S-Y, Han J-S. Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells. International Journal of Molecular Sciences. 2022; 23(12):6399. https://doi.org/10.3390/ijms23126399
Chicago/Turabian StylePark, Shin-Young, and Joong-Soo Han. 2022. "Neuroprotective Effect of Bcl-2 on Lipopolysaccharide-Induced Neuroinflammation in Cortical Neural Stem Cells" International Journal of Molecular Sciences 23, no. 12: 6399. https://doi.org/10.3390/ijms23126399