The Effects Of Zi-Bu-Pi-Yin Decoction Serum On ER Stress Induced Neuron Apoptosis And Its Mechanisms

Objectives: The endoplasmic reticulum (ER) is an important subcelluar organelle for the synthesis, posttranslational modification, and proper folding of protein. In some conditions, such as accumulation of unfolded protein or disruption of calcium in ER, ER stress is provoked. Normal cells respond to ER stress by activation of the unfolded protein response, to prevent protein misfolding, degrade misfolded protein and facilitate protein proper folding in ER. However, excessive or aberrant ER stress results in cell injury or death. Recent evidence indicates that neurodegenerative disorders in Alzheimer's disease are associated to aberrant ER stress. Further resolution of the molecular relationship between ER stress and neurodegeneration will provide novel insights into the mechanisms of AD pathology, and lead to new therapeutic targets for AD.The previous studies of our group suggested that Zi-Bu-Pi-Yin decoction (ZBPY) has notable neuroprotective effects and functions of against aging. Therefore, further-more exploring the relationship and mechanism between the neuroprotective effects of ZBPY and ER stress induced neuron apoptosis ,will have significance for prevention and cure of AD and other neurodegenerative diseases. In the present study, we set up ER stress model through treatment with tunicamycin (Tm, the inhibitor of N-glycoslytion) on the mouse neuroblastoma Neuro2a cells, Zi-Bu-Pi-Yin Decoction (ZBPY) drug serum acts as intervention. We observe the viability of Neuro2a cells by MTT assay. Applied flow cytometry to observe Neuro2a cells apoptosis and the protective role of ZBPY.At the same time, for the ERS model we also observe the expression of molecular chaperones Glucoseregulated protein78(GRP78), apoptosis promoter CCAAT/EBP homologous protein (CHOP) in protein level by Western blot assay to discuss the effect and mechanism of ZBPY drug serum on neuron death induced by ER stress, and to reveal that the effect of Zi-Bu-Pi-Yin Decoction on the apoptosis of neuron may have relation with ER pathway, to offer a new theory gist to prevent and therapia brain aging and AD; and go further to discuss the drug action and the mechanism of Zi-Bu-Pi-Yin Decoction by serum Pharmacological method.Methods: 1.Neuro2a cells were cultured, when the cells grow over 70-80% treated with Tm which the final concentration is 5μg/ml. Groups of experiment presented here included control, blank serum(10 % ) group, ZBPY drug serum(5%,10%,15% )groups, Tm(5μg/ml) group, blank serum(10 % ) pretreatment group, ZBPY drug serum(5 %,10 %,15%)pretreatment groups. The cells were incubated for 48h, and then the viability was measured by MTT assay.2.Neuro2a cells were cultured, when the cells grow over 70-80% treated with tunicamycin which the final concentration is 5μg/ml. Groups of experiment presented here were the same as above. The cells were incubated for 48h, and then applied flow cytometry to observe Neuro2a cells apoptosis and the protective role of ZBPY.3.Neuro2a cells were cultured, when the cells grow over 70-80% treated with tunicamycin which the final concentration is 5μg/ml. Groups of experiment presented here were the same as above. The cells were incubated for 48h, and then apply Western blot assay to observe the expression of molecular chaperones GRP78, apoptosis promoter CHOP in protein level.Results:1.MTT assay disclosed that there is not significant different among the control, blank serum and ZBPY drug serum groups; but compared with control, the viability of Neuro2a cells of Tm group was reduced, which has significant deviation(P<0.01); compared with Tm group, the viability of 10% blank serum pretreatment has not significant deviation;but compared with Tm group,the viability of ZBPY drug serum pretreatment groups has significant deviation(P<0.01);And there is significant different between 10% blank serum pretreatment group and 10% ZBPY drug serum pretreatment group(P<0.05).2.Flow cytometry analysis disclosed that there is no significant different among the control, blank serum and ZBPY drugserum groups; but compared with control, the viability of Neuro2a cells of Tm group was increased, which has significant deviation(P<0.01); compared with Tm group, the viability of 10% blank serum pretreatment has not significant deviation;but compared with Tm group,the viability of ZBPY drug serum pretreatment groups has significant deviation(P<0.01);And there is significant different between 10% blank serum pretreatment group and 10% ZBPY drug serum pretreatment group(P<0.05).3.The result of Western blot disclosed, after incubated for 24h with Tm, the expression of GRP78 and CHOP protein levels were obviously increase, which compared with control has significant deviation(P<0.05); compared with control, in the blank serum group and ZBPY drug serum groups, the expression of GRP78 and CHOP protein level has no significant deviation; compared with Tm group, the expression of GRP78 and CHOP protein levels of ZBPY drug serum pretreatment groups has significant deviation(P<0.05),but the 10% blank serum group has no significant deviation; there is significant different between 10% blank serum pretreatment group and 10% ZBPY drug serum pretreatment group(P <0.05).Conclusions: 1. ZBPY Decoction can protect the neuron damage induced by ER stress. 2. Zi-Bu-Pi-Yin Decoction inhibited neuron apoptosis induced by ER stress, depended on the concentration.3.The role of Zi-Bu-Pi-Yin Decoction inhibiting the neuron apoptosis induced by ER stress may by down regulating the expression of GRP78 and CHOP, and this may be related to ER stress apoptotic transduction pathway.