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Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFκB signaling pathway.

Badshah H, Ali T, Kim MO - Sci Rep (2016)

Bottom Line: Osmotin improved synaptic functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95, SNAP-25, and syntaxin-1.Osmotin also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1 and caspase-3.Overall, our studies demonstrated that osmotin prevented neuroinflammation-associated memory impairment and neurodegeneration and suggest AdipoR1 as a therapeutic target for the treatment of neuroinflammation and neurological disorders, such as AD.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Life Science (BK 21), College of Natural Sciences (RINS), Gyeongsang National University, Jinju, 660-701, Republic of Korea.

ABSTRACT
Toll-like receptor 4 (TLR4) signaling in the brain mediates autoimmune responses and induces neuroinflammation that results in neurodegenerative diseases, such as Alzheimer's disease (AD). The plant hormone osmotin inhibited lipopolysaccharide (LPS)-induced TLR4 downstream signaling, including activation of TLR4, CD14, IKKα/β, and NFκB, and the release of inflammatory mediators, such as COX-2, TNF-α, iNOS, and IL-1β. Immunoprecipitation demonstrated colocalization of TLR4 and AdipoR1 receptors in BV2 microglial cells, which suggests that osmotin binds to AdipoR1 and inhibits downstream TLR4 signaling. Furthermore, osmotin treatment reversed LPS-induced behavioral and memory disturbances and attenuated LPS-induced increases in the expression of AD markers, such as Aβ, APP, BACE-1, and p-Tau. Osmotin improved synaptic functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95, SNAP-25, and syntaxin-1. Osmotin also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1 and caspase-3. Overall, our studies demonstrated that osmotin prevented neuroinflammation-associated memory impairment and neurodegeneration and suggest AdipoR1 as a therapeutic target for the treatment of neuroinflammation and neurological disorders, such as AD.

No MeSH data available.


Related in: MedlinePlus

Anti-inflammatory mechanism of osmotin against LPS-induced neuroinflammation in BV2 microglial cells (A) Shown are representative histograms for (a) Cell Viability, (b) Cytotoxicity, and (c) Caspase-3/7 assays performed under experimental conditions as mentioned in the Materials and Methods section. (B) Shown are representative Western blots probed with TLR4, p-NFκB, and TNF-α antibodies in microglial cells. (C) Shown are immunoprecipitation results followed by representative immunoblots as mentioned in the Materials and Methods section. AdipoR1 colocalizes with the TLR4/CD14 signaling complex in microglial cells. (D) Shown are representative Western blots probed with TLR4 and p-NFκB antibodies in microglial cells. AdipoR1 siRNA transfection significantly inhibited LPS-induced inflammation. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 per group). (E) The immunofluorescence images indicate localization of TLR4 (red panels), AdipoR1 (green panels) and merged TLR4 and AdipoR1 (merge panels). The images are representative of staining obtained in sections prepared from at least 5 animals per group (low magnification = 10x, scale bar: low zoom = 50 μm and high zoom = 20 μm). Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
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f2: Anti-inflammatory mechanism of osmotin against LPS-induced neuroinflammation in BV2 microglial cells (A) Shown are representative histograms for (a) Cell Viability, (b) Cytotoxicity, and (c) Caspase-3/7 assays performed under experimental conditions as mentioned in the Materials and Methods section. (B) Shown are representative Western blots probed with TLR4, p-NFκB, and TNF-α antibodies in microglial cells. (C) Shown are immunoprecipitation results followed by representative immunoblots as mentioned in the Materials and Methods section. AdipoR1 colocalizes with the TLR4/CD14 signaling complex in microglial cells. (D) Shown are representative Western blots probed with TLR4 and p-NFκB antibodies in microglial cells. AdipoR1 siRNA transfection significantly inhibited LPS-induced inflammation. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 per group). (E) The immunofluorescence images indicate localization of TLR4 (red panels), AdipoR1 (green panels) and merged TLR4 and AdipoR1 (merge panels). The images are representative of staining obtained in sections prepared from at least 5 animals per group (low magnification = 10x, scale bar: low zoom = 50 μm and high zoom = 20 μm). Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.

Mentions: Microglial cells are the primary LPS-responsive cells in the CNS. BV2 microglial cells were used for apoptotic, Western blot, immunoprecipitation, and immunofluorescence assays to elucidate the functional neuroprotective and anti-inflammatory roles of osmotin against LPS-induced neuroinflammation. An Apo-Tox Glo triplex assay kit was used to investigate microglial cell viability, toxicity, and caspase 3/7 activity in control, LPS-treated, osmotin-treated, and LPS and TAK242-treated groups. TAK242 is a well-known inhibitor of the TLR4 receptor, and it inhibits the downstream neuroinflammation following TLR4 activation13. Our results demonstrated that osmotin possessed potent neuroprotective activity following LPS treatment via the maintenance of cell viability, reduction of cytotoxicity, and inhibition of caspase 3/7 activation, similarly to LPS and TAK242 treatment. We also demonstrated that 0.4 μM osmotin was the optimum dose for adequate neuroprotective activity of the three doses used (i.e., 0.2 μM, 0.4 μM, and 0.8 μM) (Fig. 2A).


Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFκB signaling pathway.

Badshah H, Ali T, Kim MO - Sci Rep (2016)

Anti-inflammatory mechanism of osmotin against LPS-induced neuroinflammation in BV2 microglial cells (A) Shown are representative histograms for (a) Cell Viability, (b) Cytotoxicity, and (c) Caspase-3/7 assays performed under experimental conditions as mentioned in the Materials and Methods section. (B) Shown are representative Western blots probed with TLR4, p-NFκB, and TNF-α antibodies in microglial cells. (C) Shown are immunoprecipitation results followed by representative immunoblots as mentioned in the Materials and Methods section. AdipoR1 colocalizes with the TLR4/CD14 signaling complex in microglial cells. (D) Shown are representative Western blots probed with TLR4 and p-NFκB antibodies in microglial cells. AdipoR1 siRNA transfection significantly inhibited LPS-induced inflammation. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 per group). (E) The immunofluorescence images indicate localization of TLR4 (red panels), AdipoR1 (green panels) and merged TLR4 and AdipoR1 (merge panels). The images are representative of staining obtained in sections prepared from at least 5 animals per group (low magnification = 10x, scale bar: low zoom = 50 μm and high zoom = 20 μm). Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
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Related In: Results  -  Collection

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f2: Anti-inflammatory mechanism of osmotin against LPS-induced neuroinflammation in BV2 microglial cells (A) Shown are representative histograms for (a) Cell Viability, (b) Cytotoxicity, and (c) Caspase-3/7 assays performed under experimental conditions as mentioned in the Materials and Methods section. (B) Shown are representative Western blots probed with TLR4, p-NFκB, and TNF-α antibodies in microglial cells. (C) Shown are immunoprecipitation results followed by representative immunoblots as mentioned in the Materials and Methods section. AdipoR1 colocalizes with the TLR4/CD14 signaling complex in microglial cells. (D) Shown are representative Western blots probed with TLR4 and p-NFκB antibodies in microglial cells. AdipoR1 siRNA transfection significantly inhibited LPS-induced inflammation. The density values are expressed in arbitrary units as the mean ± SEM for the indicated proteins (n = 5 per group). (E) The immunofluorescence images indicate localization of TLR4 (red panels), AdipoR1 (green panels) and merged TLR4 and AdipoR1 (merge panels). The images are representative of staining obtained in sections prepared from at least 5 animals per group (low magnification = 10x, scale bar: low zoom = 50 μm and high zoom = 20 μm). Symbols for treatment groups and levels of significance are mentioned in the data analysis section of the Materials and Methods.
Mentions: Microglial cells are the primary LPS-responsive cells in the CNS. BV2 microglial cells were used for apoptotic, Western blot, immunoprecipitation, and immunofluorescence assays to elucidate the functional neuroprotective and anti-inflammatory roles of osmotin against LPS-induced neuroinflammation. An Apo-Tox Glo triplex assay kit was used to investigate microglial cell viability, toxicity, and caspase 3/7 activity in control, LPS-treated, osmotin-treated, and LPS and TAK242-treated groups. TAK242 is a well-known inhibitor of the TLR4 receptor, and it inhibits the downstream neuroinflammation following TLR4 activation13. Our results demonstrated that osmotin possessed potent neuroprotective activity following LPS treatment via the maintenance of cell viability, reduction of cytotoxicity, and inhibition of caspase 3/7 activation, similarly to LPS and TAK242 treatment. We also demonstrated that 0.4 μM osmotin was the optimum dose for adequate neuroprotective activity of the three doses used (i.e., 0.2 μM, 0.4 μM, and 0.8 μM) (Fig. 2A).

Bottom Line: Osmotin improved synaptic functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95, SNAP-25, and syntaxin-1.Osmotin also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1 and caspase-3.Overall, our studies demonstrated that osmotin prevented neuroinflammation-associated memory impairment and neurodegeneration and suggest AdipoR1 as a therapeutic target for the treatment of neuroinflammation and neurological disorders, such as AD.

View Article: PubMed Central - PubMed

Affiliation: Division of Applied Life Science (BK 21), College of Natural Sciences (RINS), Gyeongsang National University, Jinju, 660-701, Republic of Korea.

ABSTRACT
Toll-like receptor 4 (TLR4) signaling in the brain mediates autoimmune responses and induces neuroinflammation that results in neurodegenerative diseases, such as Alzheimer's disease (AD). The plant hormone osmotin inhibited lipopolysaccharide (LPS)-induced TLR4 downstream signaling, including activation of TLR4, CD14, IKKα/β, and NFκB, and the release of inflammatory mediators, such as COX-2, TNF-α, iNOS, and IL-1β. Immunoprecipitation demonstrated colocalization of TLR4 and AdipoR1 receptors in BV2 microglial cells, which suggests that osmotin binds to AdipoR1 and inhibits downstream TLR4 signaling. Furthermore, osmotin treatment reversed LPS-induced behavioral and memory disturbances and attenuated LPS-induced increases in the expression of AD markers, such as Aβ, APP, BACE-1, and p-Tau. Osmotin improved synaptic functionality via enhancing the activity of pre- and post-synaptic markers, like PSD-95, SNAP-25, and syntaxin-1. Osmotin also prevented LPS-induced apoptotic neurodegeneration via inhibition of PARP-1 and caspase-3. Overall, our studies demonstrated that osmotin prevented neuroinflammation-associated memory impairment and neurodegeneration and suggest AdipoR1 as a therapeutic target for the treatment of neuroinflammation and neurological disorders, such as AD.

No MeSH data available.


Related in: MedlinePlus