Mechanism Of PC12 Cells Injury Induced By Aβ And Neuroprotective Effect Of Donepezil

Alzheimer's disease(AD), the most common form of dementia in adults is a neurodegerative disorder characterized by selective neuronal loss and the presence of two different types of fibril deposits: senile plaques(SP) and neurofibrillary tangles(NFT). The epidemic study showed that the incidence of AD is around 5%-10% which brought a great burden to the society. Till now its etiological factors are still not very clear. Scientist supposed that there were some hypotheses about it, such as A β cascade themry choline hypothesis and gene theory. Among those hypotheses, A β cascade theory was more and more recommended. The core of senile plaques is mainly composed of an anyloid P -peptide(A P). A P is hydrolyzed by β, r -site APP-cleaving enzyme from cell membrane. Some studies showed that A P could affect the equilibration of Ca2+ and oxidative stress, inducing DNA break and neurofibrillary tangles, so that caused neural cells apoptosis and necrosis. More and more scientists paid close attention to Aβ and several experiments speculated that it brought toxic effect to neural cells although its idiographic nosogenesis was unclear.Donepezil is a new reversible acetylcholinesterase inhibitor which is used to treat mild-moderate AD in clinical trials recent years. Its mechanism is complicated and has many varieties, for example,inhibiting cholinesterase, relieving damage of free radical improvingcerebral blood flow etc., however whether it could reduce cellapoptosis induced by Aβ is not a final conclusion.AIM: Study the mechanism of PC 12 cells injury induced by Aβ andneuroprotective effect of donepezil.METHODS: Our experiment used cell culture MTT assaysmeasuring lactate dehydrogenase(LDH) release rate immunocytochemical stain TUNEL and transmission electronmicroscope etc. to observe the neurotoxicity and damage type of PC 12cells induced by A β and the neuroprotective mechanism of donepezilon it.In the first part of our study, By using cell culture MTT assays measuring lactate dehydrogenase(LDH) release rate TUNEL and transmission electron microscope, we observed the neurotoxity of different concentration of Aβ 25-35 and judged what kind of damage type they are.In the second part of our study, by using immunocytochemical stain, we observed the different expession of Bcl-2> Caspase-3 among different groups. And by using flow cytometer Annexin梀 method, we observed their condition of apoptosis and the protective effect of donepezil in PC 12 cells.RESULTS: In the first part of our study, MTT assays showed that cell viability decreased under Aβ 25-3 5 treatment(5umol/L, 10umol/L, 20umol/L). The results of TUNEL showed that after Aβ treatment the apoptotic index increased(7.9% 15.6% 27.8%) and proliferation activity decreased(t =13.89> P <0.001). LDH assays showed that LDH increased under Aβ 25-35 treatment increased(2100+150U/L, 2700 +150U/L, 3600+150U/L). Electron microscope showed aftertreatment with A P 20umol/L, apoptosis and necrosis occurred both with the rate of them was 1:2. However apoptosis was the main type of injury when the concentration of Aβ was 10umol/L.In the second part of our study, under donepezil pretreatement, the apoptotic cell rate and LDH release rate decreased (1950+ 150U/L, 2400+150U/L)compared with former Aβ injury group (2850 + 150U/L)and the proliferation activity increased. Immunocytochemical method showed that under donepezil pretreatement, the expression of Bcl-2 increased(67% 60%) while the expression of Caspase-3 decreased(12%, 27%). However there was no significant difference between Aβ injury group and donepezil lag treatment group. CONCLUSIONS: Conclusions in the study were drawn as following:1. Aβ could induce PC 12 cells apoptosis and necrosis while donepezil could protect PC 12 cells against them both.2. Apoptosis was the leading role within A β 5 10umol/L, while necrosis and apoptosis both happened...