The Protection Of Decoction "Gengnianchun" To PC12 Cells And Hippocampal Cells From Aβ_25-35 Insult

Perimenopausal syndrome (PMS), as a common kind of disease in aged women, is due to the decline of ovary function and decrease of estrogen level. It's due to the deficit of kidney's function in Chinese traditional medicine. Its typical manifestations are hectic fever, perspiration, insomnia, menstrual disorder, atrophy of genital tract, the change of mental status and even dementia. Alzheimer's disease (AD) is an age-related neurodegenerative disease characterized by the progressive degeneration and loss of neurons in the brain, which has been correlated with the appearance of neurofibrillary tangles and senile plaques. AD is also one type of dementia. Amyloid beta protein (Aβ) is the major component of senile plaques and considered to have a causal role in the development and progress of AD and this hydrophobic polypeptide is proteolytically produced from amyloid precursor protein.The principle of GNC decoction is nourishing the Kidney and the Liver and clearing Heart-fire. It is verified that this decoction is clinically effective, especially improving the memory. Our previous study has found that GNC appears to good effects in controlling the neuro-endocrino-immune network by up-regulating the expression of estrogen receptor, enhancing the OVX rats'manifestation in Morris water maze test, and GNC might increase the RNA and protein expression of ERβin hippocampus, and regulate the cholinergic neuron function so as to enhance the learning and memory ability.In this study, we observed the protective effects of medicated rat serum on Aβ25-35-induced PC12 cells and hippocampal cells'insult and tried to supply some evidences for its possible mechanism.PartⅠThe protection of decoction "Gengnianchun" to PC12 cells from Aβ25-35 insultObjective:To investigate the effect of medicated rat serum containing decoction "Gengnianchun" (GNC) and its protection to pheochromocytoma (PC12) cells from direct amyloidβ(Aβ)25-35 insulting and to find its possible mechanism. Methods:Medicated rat serum was prepared by intragastric administrating of GNC in a dose of 10 times of human for ovariectomized SD rats. CCK-8 assay was used to evaluate the effect of medicated rat serum on PC12 cells' viability with concentration of 5％,10% and 20% and to investigate the cell viability with concentration of 5μM,10μM,20μM,50μM and detect the cytotoxicity of Aβ25-35 to PC 12 cells to set a cell model of Alzheimer's disease. Five groups were set as normal group, MRS group, NGF group, NRS group, model group. The protection of medicated rat serum to PC 12 cells from direct Aβ25-35 insult by using CCK-8 assay to evaluate the cell viability, Annexin V-FITC/PI flow cytometry assay to detect cell apoptosis rate and western blotting assay to analyze the expression of Bcl-2, Bax and active caspase-3 protein.Results:After culturing for 24 h and 48 h with a same concentration, MRS group had higher cell viability than NRS group (p<0.01). Culturing with MRS, the group with higher concentration and longer culturing time had higher cell viability (p<0.01) and the 20% MRS group had the highest cell viability.Both culturing for 24 h and 48 h with Aβ25-35, the cell viability was decreased significantly as comparing with that of the normal group as control (P<0.01) and in a concentration and time-dependent manner.Comparing with the model group, the cell viability of MRS group was increased significantly (P<0.01) and MRS had the similar effect as NGF.Comparing with the model group, the cell apoptosis rate of MRS group was decreased significantly (P<0.01) and MRS had the similar effect as NGF.The ratio of Bax/Bcl-2 of MRS group was decreased as comparing with that of model group (P<0.05). The result of the expression of active caspase-3 was consistent with the ratio of Bax/Bcl-2.Conclusions:The MRS group had higher cell viability than the NRS group at the same concentration. Aβ25-35 was neurotoxicity in a manner of concentration and time-dependent. The pretreatment with medicated serum reduced the apoptosis rate in PC12 cells with Aβ25-35 insult compared with the model group and MRS had nearly the same effect as the positive control NGF, and may through the pathway regulating the expression of Bcl-2, Bax and active caspase-3 protein. PartⅡThe protection of decoction "Gengnianchun" to hippocampal cells from Aβ25-35 insultObjective:To investigate the effect of medicated rat serum containing decoction "Gengnianchun" (GNC) and its protection to primary hippocampal cells from direct Aβ25-35 insult and to find its possible mechanism.Methods:The primary hippocampal cells were cultured with fetal rats from pregnant SD rats (p16 d-18 d). The cells were treated as same as the PC12 cells and LDH release assay was used to detect the cell viability and western blotting assay was used to analyze the expression of Bcl-2, Bax and active caspase-3 protein.Results:Comparing with the normal group, the model group had more releasing of LDH. While comparing with the model group, The MRS group had lower level of LDH release (P<0.01) and MRS had nearly the same effect as NGF.The Bax/Bcl-2 ratio was higher in the model group than the MRS group. The ratio of Bax/Bcl-2 in the MRS group was decreased as comparing with the model group (P<0.01). The result of the expression of active caspase-3 was consistent with the ratio of Bax/Bcl-2.Conclusions:MRS accelerated the cytoactivity of hippocampal cells and may through the pathway regulating the expression of Bcl-2, Bax and active caspase-3 consistent with the PC 12 cells.