| BackgroundWith the rapid growth of aging population, aging and brain aging issues have drawn increasing attention. Aging is a normal physiological phenomenon, if the progress of brain aging evolves too quickly, a pathological degeneration of the nervous system such as Alzheimer’s disease or Parkinson’s disease might occur, resulting in significant social and economic burden. Because of its irreversible character, effective drugs to alleviate the progression of neurodegenerative disease is still unavailable. Therefore, exploring the mechanism of the brain aging and developing effective anti-aging, brain protecting drug is of great significance for the prevention and treatment of neurodegenerative disease and improving the quality of life of older people.D-galactose (D-gal) induced subacute aging model is a currently widely applied aging model in China, which has advantages of short modeling time, stable results, time and money saving. It is widely used in research of mechanisms of aging and anti-aging pharmacological effects. This model is established by continuous subcutaneous injection of large doses of D-galactose, causing glucose metabolism disorder.Flavonoids are ubiquitous in vegetables, fruits, tea and other natural plants. Flavonoids were shown to have free radical scavenging, anti-inflammatory, anti-allergic, anti-viral, anti-proliferation and anti-carcinogenic biological activities [4]. In recent years, the flavonoids’potential function of neuronal regulation, prevention of age-related neurodegenerative diseases have drawn more and more attention. Flavonoids exert a multiplicity of neuroprotective actions within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation and oxidative stress, to regulate a variety of cellular signaling pathways and the potential to promote memory, learning and cognitive functions [5,6]. Extracts of leaves of Diospyros kaki have been used in clinical prevention and treatment of various cardiovascular and cerebrovascular diseases, and flavonoids are the main active ingredients. However, the effect and mechanism of flavonoids extracted from the leaves of Diospyros kaki of delaying brain aging are not very clear. In this study, we applied the widely used D-galactose induced subacute senile model to investigate the possible effect and mechanism.ObjectiveEstablish D-galactose induced subacute aging mouse model, using Morris water maze test, brain oxidative stress detection kit, and inflammation-related indicators detected by western blot, immunohistochemistry and ELISA test, to investigate the cognitive, oxidative stress and inflammation effects of flavonoids extracts from the leaves of Diospyros kaki on aging mice.MethodsForty twelve-week old Kunming male mice were randomly divided into four groups:normal control group (NC group), model group (D-gal group), low-dose flavonoids intervention group (F1 group) and large-dose flavonoids intervention group (F2 group). D-gal group and two intervention groups received injection of D-galactose 100 mg/(kg·d) for 60 consecutive days to build aging model. F1 group and F2 group were fed with flavonoids 40,80 mg/(kg·d) respectively by oral gavage, while NC group and D-gal group were fed with the same volume of normal saline. Morris water maze was used for testing escape latency (EL) and the number of crossings, related kits were used for detecting superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) content in the brain tissue, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were used for the expression of inflammation factors:tumor necrosis factor-alpha (TNF-a) and interleukin-lbeta (IL-1β);the expression of advanced glycation end products (AGEs), receptor for advanced glycation end products (RAGE) and nuclear transcription factor-KB (NF-κB) were detected by western blot。Results1. Morris water maze testCompared with NC group, EL in D-gal group prolonged (P<0.01), the numbers of crossings reduced (P<0.01); inF1 and F2 groups, EL decreased obviously (P<0.05, P<0.01), and the number of crossings improved (P<0.01, P<0.01) compared with D-gal group.2. Oxidative stress detectionCompared with NC group, SOD and GSH-Px activities decreased significantly (P<0.01, P<0.01) in D-gal group, with MDA content increased (P<0.01). In F1 and F2 groups, SOD activity increased (P<0.01, P<0.01), and GSH-Px activity increased (P<0.05, P<0.01), while MDA content decreased (P<0.01, P<0.01).3. The expression of inflammatory factors TNF-α and IL-1β detected by immunohistochemistry.In the cortex and hippocampus of mice brain in D-gal group, TNF-a and IL-1β were stained brown, the number of positive cells was the largest, and the mean optical density increased compared with NC group, the difference was significant (P<0.01, P<0.01); both factors in F1 group were dyed lighter, the number of positive cells decreased, and the mean optical density decreased obviously compared with D-gal group (P<0.01, P<0.01); both TNF-α and IL-1β in F2 group were dyed the lightest, the number of positive cells was the least, and the mean optical density decreased obviously compared with D-gal group (P<0.01,P<0.01).4. The expression of inflammatory factors TNF-a and IL-1β detected by ELISA.The expression of TNF-a and IL-1β in D-gal group increased obviously compared with NC group (P<0.01, P<0.01); the expression of TNF-a in F1 and F2 groups was significantly lower than D-gal group (P<0.01, P<0.01), the expression of IL-1β in F1 and F2 groups significantly decreased in comparison with D-gal group (P<0.01, P<0.01), and changes in F2 group were more obvious (P<0.01).5. The protein expression of AGEs, RAGE and phospho-NF-KB detected by western blot.The expression of AGEs, RAGE and phospho-NF-KB in D-gal group increased obviously compared with NC group (P<0.01, P<0.01, P<0.01); all the above proteins in F1 group were lower than D-gal group (P<0.05, P<0.01, P<0.05); in F2 group all the above proteins significantly decreased (P<0.05, P<0.01, P<0.01).ConclusionFlavonoids extracted from the leaves of Diospyros kaki significantly rescued cognitive decline in subacute aging mice induced by D-galactose. elevated the antioxidant capacity, decreased inflammation in brain tissues. Thus, FLDK shows anti-aging effects. |
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