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Cudrania cochinchinensis attenuates amyloid β protein-mediated microglial activation and promotes glia-related clearance of amyloid β protein.

Wang CJ, Chen CC, Tsay HJ, Chiang FY, Wu MF, Shiao YJ - J. Biomed. Sci. (2013)

Bottom Line: LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ.CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia.Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Geriatrics, Cheng Hsin General Hospital, Taipei, Taiwan.

ABSTRACT

Background: Microglial inflammation may significantly contribute to the pathology of Alzheimer's disease. To examine the potential of Cudrania cochinchinensis to ameliorate amyloid β protein (Aβ)-induced microglia activation, BV-2 microglial cell line, and the ramified microglia in the primary glial mixed cultured were employed.

Results: Lipopolysaccharide (LPS), Interferon-γ (IFN-γ), fibrillary Aβ (fAβ), or oligomeric Aβ (oAβ) were used to activate microglia. LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ. On the other hand, oAβ effectively activated the ramified microglia in mixed glial culture by observing the morphological alteration of the microglia from ramified to amoeboid. CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia. Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance. Results are expressed as mean ± S.D. and were analyzed by ANOVA with post-hoc multiple comparisons with a Bonferroni test.

Conclusions: The components of Cudrania cochinchinensis including CC-EW and CCB are potential for novel therapeutic intervention for Alzheimer's disease.

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Related in: MedlinePlus

Effect of Cudrania cochinchinensis on iNOS expression induced by IFN-γ combined with fAβ in BV-2 cells. BV-2 cell were treated with 0.2 ng/ml IFN-γ (I), 1 μM fAβ (A), IFN-γ combined with fAβ (IA). Alternatively, BV-2 cell were treated with 20 μg/ml CC-EW (IAC) or 20 μM CCB (IACB) for 2 h, and then were treated with IFN-γ combined with fAβ for 24 h. Cell treated with 10 ng/ml LPS (L) was used as positive control. (a) Cells were fixed, subjected to iNOS staining by anti-iNOS antibody (green) and nucleus staining using Hoechst 33258 (blue). (b) The levels of iNOS positive cells (%) are total count from three different fields. (c) The level of iNOS in cell lysate was determined by immunoblotting using β-actin as internal standard. (d) The relative level of iNOS in cell lysate exhibited as percentage of that in IA. Results are means ± S.D. from three independent experiments. Significant differences between the IA and the other treatments are indicated by ***, p < 0.001.
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Figure 4: Effect of Cudrania cochinchinensis on iNOS expression induced by IFN-γ combined with fAβ in BV-2 cells. BV-2 cell were treated with 0.2 ng/ml IFN-γ (I), 1 μM fAβ (A), IFN-γ combined with fAβ (IA). Alternatively, BV-2 cell were treated with 20 μg/ml CC-EW (IAC) or 20 μM CCB (IACB) for 2 h, and then were treated with IFN-γ combined with fAβ for 24 h. Cell treated with 10 ng/ml LPS (L) was used as positive control. (a) Cells were fixed, subjected to iNOS staining by anti-iNOS antibody (green) and nucleus staining using Hoechst 33258 (blue). (b) The levels of iNOS positive cells (%) are total count from three different fields. (c) The level of iNOS in cell lysate was determined by immunoblotting using β-actin as internal standard. (d) The relative level of iNOS in cell lysate exhibited as percentage of that in IA. Results are means ± S.D. from three independent experiments. Significant differences between the IA and the other treatments are indicated by ***, p < 0.001.

Mentions: Nitric oxide is produced by the activity of iNOS, which is expressed in activated inflammatory cells. To determine whether the modulation of nitric oxide production was attributable to the level of iNOS expression, the immunocytochemistry using anti-iNOS antibody was performed to detect iNOS expression in the treated BV-2 cells. IFN-γ significantly increases the percentage of iNOS-positive BV-2 cells (Figure 4a, b). fAβ did not increase the percentage of iNOS-positive BV-2 cells. However, it significantly enhances IFN-γ-induced iNOS expression. Alternatively, the positive control, LPS, extensively increase the percentage of iNOS-positive BV-2 cells. Use of IFN-γ combined with fAβ as stimuli, CC-EW and CCB significantly inhibits iNOS expression with IC50 at 40.83 ± 5.66 μg/ml and 11.37 ± 1.63 μM, respectively (Figure 4; Table 1). The results indicated that CCB is better than CC-EW on inhibiting the iNOS expression. The iNOS level was also determined from immunoblotting. The results show that IFN-γ combined with fAβ induced a significantly higher amount of iNOS expression than that induced by IFN-γ alone. Although the level is not as high as that stimulated by the positive control LPS. CC-EW reduced the iNOS expression induced by IFN-γ combined with fAβ to 35.24 ± 0.05% of that induced by IFN-γ plus fAβ. The results indicated that CC-EW significantly reduced the iNOS expression induced by IFN-γ combined with fAβ.


Cudrania cochinchinensis attenuates amyloid β protein-mediated microglial activation and promotes glia-related clearance of amyloid β protein.

Wang CJ, Chen CC, Tsay HJ, Chiang FY, Wu MF, Shiao YJ - J. Biomed. Sci. (2013)

Effect of Cudrania cochinchinensis on iNOS expression induced by IFN-γ combined with fAβ in BV-2 cells. BV-2 cell were treated with 0.2 ng/ml IFN-γ (I), 1 μM fAβ (A), IFN-γ combined with fAβ (IA). Alternatively, BV-2 cell were treated with 20 μg/ml CC-EW (IAC) or 20 μM CCB (IACB) for 2 h, and then were treated with IFN-γ combined with fAβ for 24 h. Cell treated with 10 ng/ml LPS (L) was used as positive control. (a) Cells were fixed, subjected to iNOS staining by anti-iNOS antibody (green) and nucleus staining using Hoechst 33258 (blue). (b) The levels of iNOS positive cells (%) are total count from three different fields. (c) The level of iNOS in cell lysate was determined by immunoblotting using β-actin as internal standard. (d) The relative level of iNOS in cell lysate exhibited as percentage of that in IA. Results are means ± S.D. from three independent experiments. Significant differences between the IA and the other treatments are indicated by ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Effect of Cudrania cochinchinensis on iNOS expression induced by IFN-γ combined with fAβ in BV-2 cells. BV-2 cell were treated with 0.2 ng/ml IFN-γ (I), 1 μM fAβ (A), IFN-γ combined with fAβ (IA). Alternatively, BV-2 cell were treated with 20 μg/ml CC-EW (IAC) or 20 μM CCB (IACB) for 2 h, and then were treated with IFN-γ combined with fAβ for 24 h. Cell treated with 10 ng/ml LPS (L) was used as positive control. (a) Cells were fixed, subjected to iNOS staining by anti-iNOS antibody (green) and nucleus staining using Hoechst 33258 (blue). (b) The levels of iNOS positive cells (%) are total count from three different fields. (c) The level of iNOS in cell lysate was determined by immunoblotting using β-actin as internal standard. (d) The relative level of iNOS in cell lysate exhibited as percentage of that in IA. Results are means ± S.D. from three independent experiments. Significant differences between the IA and the other treatments are indicated by ***, p < 0.001.
Mentions: Nitric oxide is produced by the activity of iNOS, which is expressed in activated inflammatory cells. To determine whether the modulation of nitric oxide production was attributable to the level of iNOS expression, the immunocytochemistry using anti-iNOS antibody was performed to detect iNOS expression in the treated BV-2 cells. IFN-γ significantly increases the percentage of iNOS-positive BV-2 cells (Figure 4a, b). fAβ did not increase the percentage of iNOS-positive BV-2 cells. However, it significantly enhances IFN-γ-induced iNOS expression. Alternatively, the positive control, LPS, extensively increase the percentage of iNOS-positive BV-2 cells. Use of IFN-γ combined with fAβ as stimuli, CC-EW and CCB significantly inhibits iNOS expression with IC50 at 40.83 ± 5.66 μg/ml and 11.37 ± 1.63 μM, respectively (Figure 4; Table 1). The results indicated that CCB is better than CC-EW on inhibiting the iNOS expression. The iNOS level was also determined from immunoblotting. The results show that IFN-γ combined with fAβ induced a significantly higher amount of iNOS expression than that induced by IFN-γ alone. Although the level is not as high as that stimulated by the positive control LPS. CC-EW reduced the iNOS expression induced by IFN-γ combined with fAβ to 35.24 ± 0.05% of that induced by IFN-γ plus fAβ. The results indicated that CC-EW significantly reduced the iNOS expression induced by IFN-γ combined with fAβ.

Bottom Line: LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ.CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia.Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Geriatrics, Cheng Hsin General Hospital, Taipei, Taiwan.

ABSTRACT

Background: Microglial inflammation may significantly contribute to the pathology of Alzheimer's disease. To examine the potential of Cudrania cochinchinensis to ameliorate amyloid β protein (Aβ)-induced microglia activation, BV-2 microglial cell line, and the ramified microglia in the primary glial mixed cultured were employed.

Results: Lipopolysaccharide (LPS), Interferon-γ (IFN-γ), fibrillary Aβ (fAβ), or oligomeric Aβ (oAβ) were used to activate microglia. LPS and IFN-γ, but not Aβs, activated BV-2 cells to produce nitric oxide through an increase in inducible nitric oxide synthase (iNOS) expression without significant effects on cell viability of microglia. fAβ, but not oAβ, enhanced the IFN-γ-stimulated nitric oxide production and iNOS expression.The ethanol/water extracts of Cudrania cochinchinensis (CC-EW) and the purified isolated components (i.e. CCA to CCF) effectively reduced the nitric oxide production and iNOS expression stimulated by IFN-γ combined with fAβ. On the other hand, oAβ effectively activated the ramified microglia in mixed glial culture by observing the morphological alteration of the microglia from ramified to amoeboid. CC-EW and CCB effectively prohibit the Aβ-mediated morphological change of microglia. Furthermore, CC-EW and CCB effectively decreased Aβ deposition and remained Aβ in the conditioned medium suggesting the effect of CC-EW and CCB on promoting Aβ clearance. Results are expressed as mean ± S.D. and were analyzed by ANOVA with post-hoc multiple comparisons with a Bonferroni test.

Conclusions: The components of Cudrania cochinchinensis including CC-EW and CCB are potential for novel therapeutic intervention for Alzheimer's disease.

Show MeSH
Related in: MedlinePlus