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The purpose of this study was to evaluate the effects of diosgenin on the D-galactose-induced cerebral cortical widely dispersed apoptosis. Male 12-week-old Wistar rats were divided into four groups: Control (1mg/kg/day of saline, i.p.), DD0 (150mg/kg/day of D-galactose, i.p.), DD10, and DD50 (D-galactose$+10$ or 50mg/kg/day of diosgenin orally). After eight weeks, histopathological analysis, positive TUNEL and Western blotting assays were performed on the excised cerebral cortex from all four groups. The TUNEL-positive apoptotic cells, the components of Fas pathway (Fas, FADD, active caspase-8 and active caspase-3), and mitochondria pathway (t-Bid, Bax, cytochrome $c$, active caspase-9 and active caspase-3) were increased in the DD0 group compared with the control group, whereas they were decreased in the DD50 group. The components of survival pathway (p-Bad, Bcl-2, Bcl-xL, IGF-1, p-PI3K and p-AKT) were increased in the DD50 group compared to the control group, whereas the levels of Bcl-xL, p-PI3K, and p-AKT were also compensatorily increased in the DD0 group compared to the control group. Taken together, diosgenin suppressed D-galactose-induced neuronal Fas-dependent and mitochondria-dependent apoptotic pathways and enhanced the Bcl-2 family associated pro-survival and IGF-1-PI3K-AKT survival pathways, which might provide neuroprotective effects of diosgenin for prevention of the D-galactose-induced aging brain.

Oxidative stress is considered as a major factor in aging and exacerbates aging process through a variety of molecular mechanisms. D-galactose, a normal reducing sugar with high dose can cause the accumulation of reactive oxygen species (ROS) or stimulate free radical production indirectly by the formation of advanced glycation end products in tissues, finally resulting in oxidative stress. 20(R)-ginsenoside Rg3 (20(R)-Rg3), a major and representative component isolated from red ginseng (Panax ginseng C.A Meyer), has been shown to observably have an anti-oxidative effect. We thereby investigated the beneficial effects of 20(R)-Rg3 on D-galactose-induced oxidative stress injury and its underlying mechanisms. Our results showed that continuous injection of D-galactose with 800mg/kg/day for 8 weeks increased the levels of alanine aminotransferase (ALT) and blood urea nitrogen (BUN). However, such increases were attenuated by the treatment of 20(R)-Rg3 for 4 weeks. Meanwhile, 20(R)-Rg3 markedly inhibited D-galactose-caused oxidative stress in liver and kidney. The anti-oxidants, including catalase (CAT) and superoxide dismutase (SOD), were elevated in the mice from 20(R)-Rg3-treated group compared with that from D-galactose group. In contrast, a significant decrease in levels of cytochrome P450 E1 (CYP2E1) and the lipid peroxidation product malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were observed in the 20(R)-Rg3-treated group. These effects were associated with a significant increase of AGEs. More importantly, 20(R)-Rg3 effectively attenuated D-galactose induced apoptosis in liver and kidney via restoring the upstream PI3K/AKT signaling pathway. Taken together, our study suggests that 20(R)-Rg3 may be a novel and promising anti-oxidative therapeutic agent to prevent aging-related injuries in liver and kidney.

An imbalance between oxidants and antioxidants in favor of the oxidants, potentially leading to damage, is termed as ‘oxidative stress’. Some studies indicate that reactive oxygen species (ROS) may increase in resistance of the aqueous humor drainage or induce the rearrangement of human trabecular meshwork and result in glaucoma finally. An eight-week d-galactose injection was used to mimic high oxidative stress rats. Behavior tests, fundus images, anterior chamber depth, anterior chamber diameter (ACDia) and pupil diameter (PD) were investigated. Intraocular pressure (IOP) measurement and serum were taken for antioxidant capability of superoxide dismutase (SOD) and the amount of nitric oxide (NO). After perfusion, Müller’s cells and astrocyte, microglial and inducible Nitric Oxide Synthase (iNOS) were labeled. The activities of SOD were decreased after induction. The spatial memory ability and muscular endurance were impaired through Morris water maze (MWM) and weight-loaded forced swimming test. These results demonstrated that oxidative stress was exacerbated by d-galactose. The IOP was gradually increased in the third week after d-galactose induction. Immunohistochemical stains showed that the density of Müller’s cells, astrocytes and microglia increased in d-galactose group. Microglia mostly became active. These data indicate that oxidative stress may result in the impairment of behavior, increase of Müller’s cells, astrocyte and microglia which is induced by d-galactose. The correlation between oxidative stress and elevation of IOP should be further studied. These may involve in the pathologies of aging or other neurodegenerative disease.