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Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain.

Xuan M, Okazaki M, Iwata N, Asano S, Kamiuchi S, Matsuzaki H, Sakamoto T, Miyano Y, Iizuka H, Hibino Y - Evid Based Complement Alternat Med (2015)

Bottom Line: Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation.Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells.Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunobiochemistry, Faculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan ; Laboratory of Organic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan.

ABSTRACT
Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

No MeSH data available.


Related in: MedlinePlus

Effects of chronic pretreatment with MAK (1 g/kg) on apoptotic cell death in the ischemic penumbral regions. Representative photographs of TUNEL staining and cleaved caspase-3 immunostaining at 24 h of reoxygenation after H/I in the penumbral cortex (a), (e) and hippocampal CA1 region (c), (g) from the mice in each group. Scale bar = 50 μm. Quantification of the number of TUNEL-positive (arrow heads) or cleaved caspase-3-positive cells was achieved by cell counting in the penumbral cortex (b), (f) and hippocampal CA1 region (d), (h). Throughout, data are represented as means ± S.D. n = 3–5. ∗∗∗P < 0.001 compared with the respective sham-operated groups. †, ††P < 0.05, 0.01 compared with the H/I-treated control group.
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fig4: Effects of chronic pretreatment with MAK (1 g/kg) on apoptotic cell death in the ischemic penumbral regions. Representative photographs of TUNEL staining and cleaved caspase-3 immunostaining at 24 h of reoxygenation after H/I in the penumbral cortex (a), (e) and hippocampal CA1 region (c), (g) from the mice in each group. Scale bar = 50 μm. Quantification of the number of TUNEL-positive (arrow heads) or cleaved caspase-3-positive cells was achieved by cell counting in the penumbral cortex (b), (f) and hippocampal CA1 region (d), (h). Throughout, data are represented as means ± S.D. n = 3–5. ∗∗∗P < 0.001 compared with the respective sham-operated groups. †, ††P < 0.05, 0.01 compared with the H/I-treated control group.

Mentions: TUNEL staining of the ischemic penumbral region of the cortex (Figures 4(a) and 4(b)) and hippocampal CA1 region (Figures 4(c) and 4(d)) was performed to determine nucleosomal DNA fragmentation accompanied by apoptotic cell death. In both the sham-operated control and the MAK groups, no TUNEL-positive cells were detected in the regions. The number of TUNEL-positive cells was increased in the control group by H/I and subsequent reoxygenation and remarkably suppressed by pretreatment with MAK. The activation level of caspase-3 which directly activates DNase in the apoptotic final process was determined by immunostaining for cleaved caspase-3, an activated form of this enzyme. Similar to the result of TUNEL staining, the number of cells expressing cleaved caspase-3 was remarkably increased by H/I and subsequent reoxygenation in the control group (Figures 4(e)–4(h)). Pretreatment with MAK significantly inhibited the caspase-3 activation induced by H/I and subsequent reoxygenation.


Chronic Treatment with a Water-Soluble Extract from the Culture Medium of Ganoderma lucidum Mycelia Prevents Apoptosis and Necroptosis in Hypoxia/Ischemia-Induced Injury of Type 2 Diabetic Mouse Brain.

Xuan M, Okazaki M, Iwata N, Asano S, Kamiuchi S, Matsuzaki H, Sakamoto T, Miyano Y, Iizuka H, Hibino Y - Evid Based Complement Alternat Med (2015)

Effects of chronic pretreatment with MAK (1 g/kg) on apoptotic cell death in the ischemic penumbral regions. Representative photographs of TUNEL staining and cleaved caspase-3 immunostaining at 24 h of reoxygenation after H/I in the penumbral cortex (a), (e) and hippocampal CA1 region (c), (g) from the mice in each group. Scale bar = 50 μm. Quantification of the number of TUNEL-positive (arrow heads) or cleaved caspase-3-positive cells was achieved by cell counting in the penumbral cortex (b), (f) and hippocampal CA1 region (d), (h). Throughout, data are represented as means ± S.D. n = 3–5. ∗∗∗P < 0.001 compared with the respective sham-operated groups. †, ††P < 0.05, 0.01 compared with the H/I-treated control group.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4402482&req=5

fig4: Effects of chronic pretreatment with MAK (1 g/kg) on apoptotic cell death in the ischemic penumbral regions. Representative photographs of TUNEL staining and cleaved caspase-3 immunostaining at 24 h of reoxygenation after H/I in the penumbral cortex (a), (e) and hippocampal CA1 region (c), (g) from the mice in each group. Scale bar = 50 μm. Quantification of the number of TUNEL-positive (arrow heads) or cleaved caspase-3-positive cells was achieved by cell counting in the penumbral cortex (b), (f) and hippocampal CA1 region (d), (h). Throughout, data are represented as means ± S.D. n = 3–5. ∗∗∗P < 0.001 compared with the respective sham-operated groups. †, ††P < 0.05, 0.01 compared with the H/I-treated control group.
Mentions: TUNEL staining of the ischemic penumbral region of the cortex (Figures 4(a) and 4(b)) and hippocampal CA1 region (Figures 4(c) and 4(d)) was performed to determine nucleosomal DNA fragmentation accompanied by apoptotic cell death. In both the sham-operated control and the MAK groups, no TUNEL-positive cells were detected in the regions. The number of TUNEL-positive cells was increased in the control group by H/I and subsequent reoxygenation and remarkably suppressed by pretreatment with MAK. The activation level of caspase-3 which directly activates DNase in the apoptotic final process was determined by immunostaining for cleaved caspase-3, an activated form of this enzyme. Similar to the result of TUNEL staining, the number of cells expressing cleaved caspase-3 was remarkably increased by H/I and subsequent reoxygenation in the control group (Figures 4(e)–4(h)). Pretreatment with MAK significantly inhibited the caspase-3 activation induced by H/I and subsequent reoxygenation.

Bottom Line: Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation.Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells.Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Immunobiochemistry, Faculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan ; Laboratory of Organic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Josai University, Saitama 350-0295, Japan.

ABSTRACT
Type 2 diabetes mellitus has been known to increase systemic oxidative stress by chronic hyperglycemia and visceral obesity and aggravate cerebral ischemic injury. On the basis of our previous study regarding a water-soluble extract from the culture medium of Ganoderma lucidum mycelia (designed as MAK), which exerts antioxidative and neuroprotective effects, the present study was conducted to evaluate the preventive effects of MAK on apoptosis and necroptosis (a programmed necrosis) induced by hypoxia/ischemia (H/I) in type 2 diabetic KKAy mice. H/I was induced by a combination of unilateral common carotid artery ligation with hypoxia (8% O2 for 20 min) and subsequent reoxygenation. Pretreatment with MAK (1 g/kg, p.o.) for a week significantly reduced H/I-induced neurological deficits and brain infarction volume assessed at 24 h of reoxygenation. Histochemical analysis showed that MAK significantly suppressed superoxide production, neuronal cell death, and vacuolation in the ischemic penumbra, which was accompanied by a decrease in the numbers of TUNEL- or cleaved caspase-3-positive cells. Furthermore, MAK decreased the expression of receptor-interacting protein kinase 3 mRNA and protein, a key molecule for necroptosis. These results suggest that MAK confers resistance to apoptotic and necroptotic cell death and relieves H/I-induced cerebral ischemic injury in type 2 diabetic mice.

No MeSH data available.


Related in: MedlinePlus