| BackgroundAlzheimer’s disease (AD) is the most common type of senile dementia. Its clinical symptoms include progressive cognitive dysfunction, difficulty in remembering recent events, and behavioral issues.According to recent studies, mitochondrial dysfunction is a major sign during the early period of AD and damage to the mitochondrion is implicated in the development of the disease. Known as the powerhouse of cells, mitochondria produce adenosine triphosphate (ATP) in the presence of oxygen through oxidative phosphorylation; ATP releases energy that could be utilized by human body. Lots of research suggested that AD patients were distinguished by low glucose metabolism that was prior to the appearance of clinical symptoms, and the mitochondrial enzyme activity in the respiratory chain and oxidative phosphorylation was altered. In fact, altered energy supply in mitochondria happens preceding the pathology of AD. Therefore, it is of great importance to study the early change of mitochondria.In this research Shenzhi Jiannao Formula, a compound of Chinese herbal monomers was employed. In experimental animal models, this compound proved to be effective in protecting the neural cells in hippocampal CA1 region from damage. The choline acetyltransferase expression was stimulated and the cholinesterase expression was inhibited, thus enhancing the level of acetyl choline; neural cell death was slowed down due to restricted production of free radicals and thereupon the learning ability and memory function of the animals were improved. In a cellular culture model simulating Alzheimer’s disease induced by the β-amyloid (A(3), Shenzhi Jiannao Formula was effective in inhibiting the release of lactic dehydrogenase, restricting the production of free radicals and apoptin, blocking programmed death of neurons, and maintaining their cell morphology and survival rate. It demonstrated that Shenzhi Jiannao Formula could resist the toxic effect of Aβ on neural cells. Considering that anoxia could lead to mitochondrial damage which is the major cause of sporadic Alzheimer’s disease, protecting mitochondria from hypoxic damage is a potential treatment method. Focusing on various stages of mitochondrial damage, this research gave full play to the advantage of Chinese herbal compounds in multiple-target therapy and attempted to explore how Shenzhi Jiannao Formula function to protect mitochondria.PurposeTo explore Shenzhi Jiannao Formula’s protection of mitochondria at the molecular and cellular level in the cellular culture model, to provide an experimental basis for the application of this formula in clinical treatment of AD, and also to pave way for further development of Chinese herbal compounds.MethodsA neural cell culture model with AD was established using human neuroblastoma cell line (SH-SY5Y). MTT assays were conducted on the three ingredients (ginsenoside, timosaponin, Paeoniflorin) of Shenzhi Jiannao Formula respectively to determine when their concentration would be effective and non-toxic for normal SH-SY5Y cells. According to orthogonal experimental design, the three ingredients were mixed up in different concentrations and applied to SH-SY5Y cells with AD and normal SH-SY5Y cells. The most effectively matched group was selected by comparison of cell death rates in SH-S Y5 Y cell culture model with AD and normal SH-S Y5 Y cells. Mitochondria isolation kits were used to fractionate and keep mitochondria. Tests on the activity of mitochondrial enzymes, including mitochondrial respiratory chain complex I and IV, were performed. Tests on mitochondrial function were performed, including the oxidative damage of mtDNA and the expression of APP and Aβ.ResultsThe maximal nontoxic concentration of the three ingredients were 12.5ug/mlwith ginsenoside,6.25ug/ml with timosaponin, and 3.1252ug/ml with paeoniflorin. In the application of orthogonal experimental design, the mix-up of the three was optimized when ginsenoside was at 6.25ug/ml, timosaponin 6.25ug/ml, paeoniflorin 3.1252ug/ml. The cell death rate and mitochondrial activity in SH-SY5Y cell culture model with AD were higher and stonger than in normal SH-SY5Y cells; after being treated by Shenzhi Jiannao Formula, its cell death rate was brought down significantly.SH-SY5 Y cells with AD had more serious mtDNA damage and higher level of Aβ than the normal SH-SY5Y cells; after being treated by Shenzhi Jiannao Formula, its Aβ level was brought down significantly.ConclusionsThe ingredients of Shenzhi Jiannao Formula could protect cells from anoxia-induced death, help to maintain mitochondrial activity, improve damaged mitochondrial DNA and reduce A(3 accumulation. This research suggested that this Chinese herbal compound could protect hypoxic neurons via the mitochondrial mechanism and its clinical application should be considered based on the concrete experimental data. |
PDF Full Text Request