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Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion.

Liu Y, Tang G, Li Y, Wang Y, Chen X, Gu X, Zhang Z, Wang Y, Yang GY - J Neuroinflammation (2014)

Bottom Line: MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05).Compound C, a selective AMPK inhibitor, eliminated this promotional effect.Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China. yongting.wang@gmail.com.

ABSTRACT

Background: Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.

Methods: Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.

Results: Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.

Conclusions: Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

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Related in: MedlinePlus

Metformin promoted ZO-1, occludin and claudin-5 rearrangement and lessened IgG and Evans blue extravasation. (A) Occludin, ZO-1 and claudin-5 expression in sham group, NS and metformin-treated mice at 3 days following tMCAO. Scale bar =10 μm. (B) Representative result of occludin, ZO-1 and claudin-5 expression at 1 and 3 days after tMCAO. Bar graphs show a quantification of occludin, ZO-1 and claudin-5 expression. (C) IgG leakage at 3 days following tMCAO in saline and metformin-treated group. Higher magnifications are shown below. Boxes displayed representative IgG staining. Scale bar =1 mm (upper) and 100 μm (lower). Bar graph shows a semi-quantification of integrated optical density (IOD) of IgG at 1 and 3 days after tMCAO (n =3 per group). (D) Images show Evans blue extravasation in sham (a), saline (b) and metformin (c) group at 3 days after tMCAO. Blue area indicates extravasation of Evans blue. Bar graph shows a quantification analysis of Evans blue contents in brain tissue (n =3 per group). Data were mean ± SD, *P < 0.05, metformin versus saline group, **P < 0.01, metformin versus saline group.
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Fig4: Metformin promoted ZO-1, occludin and claudin-5 rearrangement and lessened IgG and Evans blue extravasation. (A) Occludin, ZO-1 and claudin-5 expression in sham group, NS and metformin-treated mice at 3 days following tMCAO. Scale bar =10 μm. (B) Representative result of occludin, ZO-1 and claudin-5 expression at 1 and 3 days after tMCAO. Bar graphs show a quantification of occludin, ZO-1 and claudin-5 expression. (C) IgG leakage at 3 days following tMCAO in saline and metformin-treated group. Higher magnifications are shown below. Boxes displayed representative IgG staining. Scale bar =1 mm (upper) and 100 μm (lower). Bar graph shows a semi-quantification of integrated optical density (IOD) of IgG at 1 and 3 days after tMCAO (n =3 per group). (D) Images show Evans blue extravasation in sham (a), saline (b) and metformin (c) group at 3 days after tMCAO. Blue area indicates extravasation of Evans blue. Bar graph shows a quantification analysis of Evans blue contents in brain tissue (n =3 per group). Data were mean ± SD, *P < 0.05, metformin versus saline group, **P < 0.01, metformin versus saline group.

Mentions: To evaluate endothelial cell permeability after metformin treatment, we conducted occludin/CD31, ZO-1/CD31 and claudin-5/CD31 double staining to observe tight junction distribution in situ at 3 days after tMCAO. Result indicated that occludin and ZO-1 were continuously located on the margin of ECs in sham group, claudin-5 was continuously located along ECs, and fewer gaps were formed in the metformin-treated group (Figure 4A). Gap formation and rearrangement were used to evaluate tight junction disruption after injury. To evaluate tight junction rearrangement, Western blot was adopted and we found that metformin-treated mice demonstrated occludin, ZO-1 and claudin-5 hyper-expression (Figure 4B). In addition, we performed IgG immunostaining and Evans blue extravasation to evaluate endothelial permeability and found that there was significantly reduced IgG and Evans blue leakage at 3 days after tMCAO in metformin-treated mice (Figure 4C-D).Figure 4


Metformin attenuates blood-brain barrier disruption in mice following middle cerebral artery occlusion.

Liu Y, Tang G, Li Y, Wang Y, Chen X, Gu X, Zhang Z, Wang Y, Yang GY - J Neuroinflammation (2014)

Metformin promoted ZO-1, occludin and claudin-5 rearrangement and lessened IgG and Evans blue extravasation. (A) Occludin, ZO-1 and claudin-5 expression in sham group, NS and metformin-treated mice at 3 days following tMCAO. Scale bar =10 μm. (B) Representative result of occludin, ZO-1 and claudin-5 expression at 1 and 3 days after tMCAO. Bar graphs show a quantification of occludin, ZO-1 and claudin-5 expression. (C) IgG leakage at 3 days following tMCAO in saline and metformin-treated group. Higher magnifications are shown below. Boxes displayed representative IgG staining. Scale bar =1 mm (upper) and 100 μm (lower). Bar graph shows a semi-quantification of integrated optical density (IOD) of IgG at 1 and 3 days after tMCAO (n =3 per group). (D) Images show Evans blue extravasation in sham (a), saline (b) and metformin (c) group at 3 days after tMCAO. Blue area indicates extravasation of Evans blue. Bar graph shows a quantification analysis of Evans blue contents in brain tissue (n =3 per group). Data were mean ± SD, *P < 0.05, metformin versus saline group, **P < 0.01, metformin versus saline group.
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Related In: Results  -  Collection

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Fig4: Metformin promoted ZO-1, occludin and claudin-5 rearrangement and lessened IgG and Evans blue extravasation. (A) Occludin, ZO-1 and claudin-5 expression in sham group, NS and metformin-treated mice at 3 days following tMCAO. Scale bar =10 μm. (B) Representative result of occludin, ZO-1 and claudin-5 expression at 1 and 3 days after tMCAO. Bar graphs show a quantification of occludin, ZO-1 and claudin-5 expression. (C) IgG leakage at 3 days following tMCAO in saline and metformin-treated group. Higher magnifications are shown below. Boxes displayed representative IgG staining. Scale bar =1 mm (upper) and 100 μm (lower). Bar graph shows a semi-quantification of integrated optical density (IOD) of IgG at 1 and 3 days after tMCAO (n =3 per group). (D) Images show Evans blue extravasation in sham (a), saline (b) and metformin (c) group at 3 days after tMCAO. Blue area indicates extravasation of Evans blue. Bar graph shows a quantification analysis of Evans blue contents in brain tissue (n =3 per group). Data were mean ± SD, *P < 0.05, metformin versus saline group, **P < 0.01, metformin versus saline group.
Mentions: To evaluate endothelial cell permeability after metformin treatment, we conducted occludin/CD31, ZO-1/CD31 and claudin-5/CD31 double staining to observe tight junction distribution in situ at 3 days after tMCAO. Result indicated that occludin and ZO-1 were continuously located on the margin of ECs in sham group, claudin-5 was continuously located along ECs, and fewer gaps were formed in the metformin-treated group (Figure 4A). Gap formation and rearrangement were used to evaluate tight junction disruption after injury. To evaluate tight junction rearrangement, Western blot was adopted and we found that metformin-treated mice demonstrated occludin, ZO-1 and claudin-5 hyper-expression (Figure 4B). In addition, we performed IgG immunostaining and Evans blue extravasation to evaluate endothelial permeability and found that there was significantly reduced IgG and Evans blue leakage at 3 days after tMCAO in metformin-treated mice (Figure 4C-D).Figure 4

Bottom Line: MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05).Compound C, a selective AMPK inhibitor, eliminated this promotional effect.Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200025, China. yongting.wang@gmail.com.

ABSTRACT

Background: Metformin, a widely used hypoglycemic drug, reduces stroke incidence and alleviates chronic inflammation in clinical trials. However, the effect of metformin in ischemic stroke is unclear. Here, we investigated the effect of metformin on ischemic stroke in mice and further explored the possible underlying mechanisms.

Methods: Ninety-eight adult male CD-1 mice underwent 90-minute transient middle cerebral artery occlusion (tMCAO). Metformin (200 mg/kg) was administrated for up to 14 days. Neurobehavioral outcomes, brain infarct volume, inflammatory factors, blood-brain barrier (BBB) permeability and AMPK signaling pathways were evaluated following tMCAO. Oxygen glucose deprivation was performed on bEND.3 cells to explore the mechanisms of metformin in inhibiting inflammatory signaling pathways.

Results: Infarct volume was reduced in metformin-treated mice compared to the control group following tMCAO (P < 0.05). Neurobehavioral outcomes were greatly improved in metformin-treated mice (P < 0.05). MPO+ cells, Gr1+ cells, MPO activity and BBB permeability were decreased after metformin administration (P < 0.05). In addition, metformin activated AMPK phosphorylation, inhibited NF-κB activation, down-regulated cytokine (IL-1β, IL-6, TNF-α) and ICAM-1 expression following tMCAO (P < 0.05). Furthermore, metformin activated AMPK signaling pathway and alleviated oxygen-glucose deprivation-induced ICAM-1 expression in bEND.3 cells (P < 0.05). Compound C, a selective AMPK inhibitor, eliminated this promotional effect.

Conclusions: Metformin down-regulated ICAM-1 in an AMPK-dependent manner, which could effectively prevent ischemia-induced brain injury by alleviating neutrophil infiltration, suggesting that metformin is a promising therapeutic agent in stroke therapy.

Show MeSH
Related in: MedlinePlus