Diabetes-Mediated Exacerbation of Neuronal Damage and Inflammation After Cerebral Ischemia in Rat: Protective Effects of Water-Soluble Extract from Culture Medium of Ganoderma lucidum Mycelia

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

“…Apoptosis in the brain tissues was evaluated by the TUNEL method with the “Apoptosis in situ Detection Kit Wako” (Wako Laboratories) [ 8 , 33 ], which is based on the TUNEL (terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP nick end labeling) procedure, that is, the addition of fluorescein-dUTP to 3′-terminals of apoptotically fragmented DNA with TdT, followed by immunochemical detection using anti-fluorescein antibody conjugated with horseradish peroxidase and 3-3′-diaminobenzidine tetrachloride as a substrate. The coronal brain slices (8 μ m thick) were lightly counterstained with hematoxylin and observed under a microscope (BX51W1, Olympus).…”

“…The coronal brain slices (8 μ m thick) were lightly counterstained with hematoxylin and observed under a microscope (BX51W1, Olympus). Quantification of TUNEL-positive cells was achieved by cell counting in the hippocampal CA1 and the penumbral cortex region affected by H/I [ 33 , 40 ]. Three randomly chosen visual fields were counted in each region by an investigator without the knowledge of the experimental conditions.…”

“…Immunofluorescence was visualized using the fluorescence microscopy as described above. The percentage of cleaved caspase-3-positive cells was calculated as the number of the positive-staining nuclei to the total nuclei [ 33 , 40 ]. Regarding RIP3, because a lower level of expression of this protein was observed in every cell in the hippocampus, the cells having an enhanced expression of RIP3 under a laser beam at constant intensity were determined as RIP3-positive.…”

“…The functional food derived from this fungus, a water-soluble extract from the culture medium of G. lucidum mycelia (MAK), is a freeze-dried powder of a hot-water extract prepared from a solid culture medium composed of bagasse and defatted rice bran overgrown with G. lucidum mycelia; this food has a considerable background of 18 years of contributing to the health of consumers. Our previous reports revealed that chronic oral pretreatment with MAK relieves an exacerbation of cerebral injury induced by middle cerebral artery occlusion (MCAO) and reperfusion in streptozotocin- (STZ-) induced type 1 diabetic rats [ 32 , 33 ]. MAK suppressed the augmentation of oxidative stress and expression of a number of proinflammatory genes or proteins, such as TNF- α , interleukin- (IL-) 1 β , cyclooxygenase- (COX-) 2, and myeloperoxidase (MPO) in the STZ-induced diabetic rat brain, and decreased apoptotic cell death induced by MCAO/reperfusion in the ischemic penumbra [ 33 ].…”

“…Our previous reports revealed that chronic oral pretreatment with MAK relieves an exacerbation of cerebral injury induced by middle cerebral artery occlusion (MCAO) and reperfusion in streptozotocin- (STZ-) induced type 1 diabetic rats [ 32 , 33 ]. MAK suppressed the augmentation of oxidative stress and expression of a number of proinflammatory genes or proteins, such as TNF- α , interleukin- (IL-) 1 β , cyclooxygenase- (COX-) 2, and myeloperoxidase (MPO) in the STZ-induced diabetic rat brain, and decreased apoptotic cell death induced by MCAO/reperfusion in the ischemic penumbra [ 33 ]. These results imply that the cerebroprotective effects of MAK are mainly attributed to its antioxidant and anti-inflammatory properties.…”