| Background: Alzheimer’s disease is a chronic neurodegenerative disease. Pathophysiology of Alzheimer’s disease shows the accumulation of amyloid plaques outside neurons and Tau protein forms neurofibrillary tangles inside cells. Alzheimer’s disease is characterized by the loss of neuros and synapses. The cause for Alzheimer’s disease is still unknown, but among all the hypotheses,amyloid hypothesis is the most convincing one. The hypothesis postulates that extracellular amyloid beta deposits are the fundamental cause of the disease. And it developed with recent research that the non-plaque Aβ oligomers, these toxic oligomers are the primary cause of the disease. The accumulation of Aβ oligomers in Alzheimer’s disease brains forms senile plaques. And the recruit of these oligomers out of the neurons induces the disrupted ion homeostasis and leads to apoptosis. The intrinsic pathway of apoptosis is mitochondrial dysfunction, as the dysfunction is activated, mitochondrial membrane potential is dissipated,and the permeability of the mitochondrial membrane is increased, and the release of reactive oxygen species is amplified. The expression of some characteristic proteins related to apoptosis will be altered. Which means the expression of pro-apoptosis proteins will be upregulated,whereas the anti-apoptosis proteins will downregulate their expression. Aβoligomers are derived from the proteolysis of amyloid precursor protein. This protein is cleaved by beta secretase and gamma secretase. And the Aβ oligomers exist in two types,Aβ1-42 and Aβ1-40. Aβ25-35 will be utilized in this experiment since this existence is the efficient fragment from Aβ1-42, and also possesses cytotoxicity. Kai Xin San (KXS) was first recorded in Beiji Qianjin Medicine Compound which was written by Simiao Sun back in Tang dynasty.As the development of nowadays pharmacological research, KXS is a fundamental drug for depression. The components of KXS are polygalae radix, ginseng radix et rhizoma, poria and acori tatarinowii rhizoma,the components are specialized to cure Alzheimer’s disease in high frequency, which suggests that KXS may be an effective drug to cure Alzheimer’s disease.And in Chinese medicine theory, all these four components ingratiate the mechanism of Alzheimer’s disease. Traditional research of Chinese medicine in vitro employs direct drug delivery methods, which means the drug carrier is the medicine itself. However, the composition of Chinese medicine is always complex and unpredictable, and it is hard to describe and simulate the fact on the medical metabolism in vivo by this traditional method.Another method of study was proposed hence the situation. which is serum pharmacology.The essential of serum pharmacology is the medicated serum of drug is the carrier in the experiment, drug is administered orally to rats, and after some days administration, collect serum from rats, and apply to cells as drug. Serum pharmacology compensates for the insufficiency of traditional research strategies, and is widely used in Chinese medicine pharmacology study. Based on the background as mentioned, we aim to investigate the the protective effect of medicated of Kai Xin San (KXS) on Aβ25-35 induced injury in human neuroblastoma SH-SY5Y cells and probable mechanism. And try to ascertain if the mechanism includes the protection of mitochondria.Objective: To investigate the protective effect of medicated of Kai Xin San (KXS) on Aβ25-35 induced injury in human neuroblastoma SH-SY5Y cells and its probable mechanisms.Methods: Healthy adults SD rats were divided into four groups randomly, orally administered KXS (in three doses,0.58g/Kg, 1.16g/Kg, 2.32g/Kg) and distilled water equally, twice a day for 7 days, Blood was taken from abdominal aorta, centrifuged and inactivated,KXS-medicated serum was prepared, 1h after KXS administration at 8th day. Effective components of KXS-medicated serum were detected by UPLC-MS technique to postulate primarily. SH-SY5Y cells were cultured with AP25-35(10μmol/L) for 24h in the presence and absence of KXS-medicated serum. to conclude the protective effect of medicated of Kai Xin San (KXS) on AβP25-35 induced injury in SH-SY5Y cells and its probable mechanisms. The effective components were tested by HPLC and mass spectrometer. The Cell viability was measured by MTT method. Cell apoptosis, ROS level and mitochondrial membrane potential were detected by flow cytometry and took photos by laser scanning confocal microscope. The expressions of Bcl-2, BAX, p-Akt, Aktl/2/3 and Caspase-3 were inspected by Western Blot,Results: Based on the result of primary mass spectrogram, at least ten effective components in KXS-medicated serum including sibiricose A5, sibiricose A6, tenuifoliside B, tenuifoliside C, tenuifoliside D, β-asarone, ginsenoside Rbl, ginsenoside Rhl, poricoic acid B, poricoic acid DM and poricoic D were examined and speculated. The concentration of AP25-35 is 10μmol/L and the rat serum of the ratio in cell culture fluid was 10% after the filter of experimental conditions. Compared with the blank serum control group, Aβ25-35 significantly inhibited cell viability,increased the cell apoptosis and ROS level and decreased the mitochondrial membrane potential. The addition of KXS-medicated serum prior to the treatment of Aβ25-35 could significantly enhance the cell survival rate, attenuate the production of ROS in cells, reduce the cell apoptosis, and increase the mitochondrial membrane potential.Compared with the blank serum control group, Aβ25-35 conspicuously upregulated the expression of protein BAX and Caspase-3, downregulated the expression of protein Bcl-2,and the ratio of expression of p-Akt was increased .The addition of KXS-medicated serum prior to the treatment of Aβ25-35 could significantly upregulated the expression of protein Bcl-2, the ratio of expression of p-Aktl was decreased, and downregulated the expression of protein BAX and Caspase-3.Conclusions: Medicated serum of KXS has a protective effect against the Aβ25-35-induced injury in SH-SY5Y cells, which is associated with the inhibition of apoptbsis and protection of mitochondria. |
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