Study On The Protective Effects Of Estrogen In The Impairment Of PC12 Cells Induced By Aβ

Introduction: Alzheimer disease (AD), as one of the most frequent dementia, is a brain degenerative disease suffered at gerontics and preseniums, accompanied with chronic progressing inreversible failures of memories, cognitive disorders and personality changes. The etiopathogenisis of AD is not clear, and we are short of effective policies in its treatments. The incidence of AD increases quickly with the world population ageing. Under the heavy influences on patient's physical and mental health and the quality of their lives, AD has been the fourth cause of death in the developed countries. It has become so troublesome a problem that more and more attentions have been paid to it.As far as we know, the amyloid β protein (Aβ) hypothesis has a cardinal position in the pathogenisis of AD . Aβ is an important component of senile plaques (SP), which is a diagnostic pathological change of AD. Aβ has different kinds of states. It is reported that the fibriform aggregate state of Aβ has neural toxic effects in different ways, which play an unlear role in the onset of AD. Little has been known about the effects of soluble Aβ on the neurons, so further research should be done in this field.Estrogens have wide and important effects on the central nervous system. The low level of estrogen in the post-menopause has been considered to take part in the onset of AD. However, there still have some disputes of the estrogen replacements in the therapies of AD. It is necessary to do some study on this point.The changing fluidity of membranes has great effects on the functions of the cells, which is also a sensitivity index to early damage of the cells. Some papers have reported the effects of Aβ on the fluidity of membranes, however, the effects of estrogen have not been reported yet.The cholinergic hypothesis is another important pathogenetic hypothesis for AD. Previous researches focused on the morphological changes of cholinergic neuron or the biochemistry changes of choline acetyl transferase (ChaT) and cholinesterase (AchE). Now, some researchers have much interesting in the change of nicotinic acetylcholine receptor (nAchR). There has one paper reported by the foreign researchers on the the relationship between Aβ and nAchR, however, the influences of estrogen to the expression of nAchR have never been reported yet.For the further study of the pathogenisis of AD, especially the functions of the soluble Aβ to the onset of AD; the validation of estrogens, whether it could protect the neuron, and the possible mechanism of estrogens, Four parts have been studied as follows.Part One Effects of estrogen on the damage of Aβ 25-35 to PC12 cellsObjective: To investigate the changes of viability and membrane permeability for PC12 cells acted by soluble Aβ 25-35 and the influences of 17β -estradiol (17βE₂) to the changes.Methods: 1. cell culture: PC12 cells were grown in DMEM medium containing 5% fetal bovine serum, 5% horse serum, 100ü/ml penicillin, 100μg /ml streptomycin in a 5% CO₂ humidified incubator at 37℃.The medium were replaced every 2-3 days,and PC12 cells were passaged every 4-6 days. 2. Grouping: PC12 cells were put in three groups, i.e. blank group, Aβ group (treated with 5μM soluble Aβ25-35), and 17βE₂ intervention group (treated with 5μM soluble Aβ 25-35 plus 17β-E₂). For 17βE₂ intervention group, PC12 cells were divided into five subgroups according to concentration gradient (0.1 nmol·L-1, 1 nmol·L-1, 10 nmol·L-1, 100 nmol·L-1, 1000 nmol·L-1). 3. Cell viability detection by MTT method: PC12 cells suspension at exponential phase were seeded randomly in 96 well cell culture cluster at 2×10⁴ cells/ml density (200μl each well). They were given different treatment according grouping criteria and grown continuingly for 0.5h. 6h, 24h, 48h, 72h respectively after adherence, then optical density(OD₅₇₀) values were detected by MTT method. Within each group six samples were set. 4. Degree of injury detection by LDH: Leakage concentrations of LDH in culture fluid supernatant were detected by LDH kit. 5. Statistical method: Measurement data was expressed in mean±SEM (X ± S), group comparisons used one-way ANOVA followed by Studen-Newman-Keuls (SNK-q) test, and SPSS10.0 statistics software was used.Results: 1. Effect of soluble Aβ 25-35 on MTT reduction: By contrast to blank group, OD₅₇₀ for A β group decreased significantly (P<0.01) at every time point and dynamically in a time-dependent manner. 2. The influence of 17βE₂ to the change of MTT reduction induced by Aβ 25-35: MTT reduction for 10^-10M 17βE2 intervention group had no significant difference contrast to blank group (P>0.05), but for 10^-9-10^-6M 17βE2 intervention groups, MTT reduction increased significantly (P<0.01) at every time point except for 0.5h in a dose-dependent manner. 3. Effect of soluble Aβ 25-35 on leakage concentrations of LDH: By contrast to blank group, Leakage concentrations of LDH for Aβ group increased significantly (P<0.01) at every time point and dynamically in a time-dependent manner. 4. The influence of 17βE₂ to the change of leakage concentrations of LDH induced by Aβ 25-35: Leakage concentrations of LDH for 10^-10M 17βE2 intervention group had no significant difference contrast to blank group (P>0.05), but for 10^-9 -10^-6M 17βE2 intervention groups, leakage concentrations of LDH decreased significantly (P<0.01 or P<0.05) at every time point in a dose-dependent manner.Conclusion: Ultra- physiologic dose Aβ , even in soluble form, does toxic actions early and time-dependently to neurons with their viability cut down and membrane integrity damaged. PC12 cell injured by soluble Aβ 25-35 can be used as cell model that represents earlier pathological lesion of AD. Estrogen has anti- Aβ and neuroprotective effect in a dose-dependent manner.Part Two Effect of estrogen on the change of Membrane fluidity for PC12 cells induced by Aβ 25-35Objective: To investigate the change of membrane fluidity for PC12 cells acted by soluble Aβ 25-35 and the influences of 17βE2 to the change.Methods: 1. cell culture: As part one. 2. Grouping: As part one. 3. PC12 cells membrane fluidity detected by fluorescence polarization method: PC12 cells suspension at exponential phase were seeded randomly in 6 well cell cultre cluster at 2×10⁵cells/ml density (2ml each well). They were given different treatment according grouping criteria and grown continuingly for 0.5h, 6h, 24h, 48h, 72h respectively after adherence, then washed and collected with Hanks solution, loaded with l,6-diphenyl-l,3,5-hexatriene (DPH) fluorescent probe (final concentration 2×10^-6mol/L, away from light, incubation for 30 min). At last, fluorescence intensitis (I_VV, I_VH, I_HH, I_HV) were measured respectively by fluorospectrophotometer (condition: excitation wave 362nm, emission wave 432nm, excitation slit 10nm, emission slit 10nm, 25±0.5℃), fluorescence polarization (P) and microviscosity (η) were calculated. Within each group four samples were set. 4. Statistical method: As part one. Results: 1. Effect of soluble Aβ 25-35 on PC12 cells membrane fluidity: By contrast to blank group, P and η for Aβ group increased significantly (P<0.01) at every time point and dynamically in a time-dependent manner. 2. The influence of 17βE₂ to the change of membrane fluidity induced by Aβ 25-35: P and η for 10^-10M 17βE2 intervention group had no significant difference contrast to blank group (P>0.05), but for 10^-9-10^-6 M 17βE2 intervention groups, P and η decreased significantly (P<0.01 or P<0.05) at every time point in a dose-dependent manner.Conclusion: 5μM soluble Aβ 25-35 leads to decreased PC12 cells membrane fluidity (i.e. increased fluorescence polarization and microviscosity), which appears more vulnerable and also time-dependent. It provides new evidence for Aβ membrane toxicology. Estrogen can improve neuron membrane fluidity in a dose-dependent manner, which has never been reported and adds new clue to mechanism of estrogen.Part Three Effect of estrogen on the change of free radical metabolism in PC12 cells induced by Aβ 25-35Objective: To investigate the change of free radical metabolism in PC12 cells acted by soluble Aβ 25-35 and the influences of 17βE2 to the change.Methods: 1. cell culture: As part one. 2. Grouping: As part one. 3. Cytoplasm MDA level andGSH-P_X activity detection: PC12 cells suspension at exponential phase were seeded randomly in 6 wellcell cultre cluster at 5×10⁵cells/ml density (2ml each well). They were given different treatmentaccording grouping criteria and grown continuingly for 24h after adherence, then washed with PBS,lysed by specific lysate into cytoplasm fluid. Cytoplasm MDA levels and GSH-P_X activitis weredetected using TBA and DTNB methods according kit instructions respectively. Within each group fivesamples were set. 4. Statistical method: As part one.Results: By contrast to blank group, cytoplasm MDA level for Aβ group increased significantly(P<0.01) and GSH-P_X activity decreased significantly (P<0.01) respectively after 24hr. But for 10^-10-10^-6M 17βE2 intervention groups, cytoplasm MDA level decreased significantly (P<0.01 or P<0.05) and GSH-P_X activity increased significantly (P<0.01 or P<0.05) respectively in a dose-dependent manner.Conclusion: 5μM soluble Aβ 25-35 makes PC12 cells generate more free radical, damages free radical-clearing system ability, and as a result results in oxidative stress, which probably is the cause of cell damage and decreasad membrane fluidity found above. Estrogen can activate antioxidase activity, enhance free radical-clearing ability and lower free radica level in a dose-dependent manner, which probably is the root of its neuroprotective effect.Part Four Effect of estrogen on the change of nAchR expression in PC12 cells induced by Aβ 25-35Objective: To investigate the change of nAchRα 7 expression in PC12 cells acted by soluble Aβ 25-35 and the influences of 17βE₂ to the change.Methods: 1. cell culture: As part one. 2. Grouping: PC12 cells were put in three groups, i.e. blank group, Aβ group (treated with 5μM soluble Aβ 25-35), and 17βE₂ intervention group (treated with 5μM soluble Aβ 25-35 plus 17β-E₂). For 17βE₂ intervention group, PC12 cells were divided into four subgroups according to concentration gradient (1 nmol·L-1, 10 nmol·L-1, 100 nmol·L-1. 1000 nmol·L-1). 3. The observation of nAchRα 7 in PC12 cell by indirect immunofluorescence (IF) staining: Coverslips precoated with polylysine were put into 24 well cell cultre cluster, and PC12 cells suspension at exponential phase were seeded in the wells at 2×10⁴ cells/ml density (1ml each well). They were given different treatment according grouping criteria and grown continuingly for 72h after adherence, then were fix, permeabilized, blocked, reacted with primary antibody and secondary antibody, mounted in turn, at last were observed and shot. 4. The detection of nAchRα 7 protein expression level by Western-blot analysis: PC12 cells suspension at exponential phase were seeded in 6cm dishes precoated with polylysine at 2×10⁵ cells/ml density (4ml each well). They were given different treatment according grouping criteria and grown continuingly for 72h after adherence. Membrane proteins were extracted by using lysis buffer and ultracentrifugation, and then quantitated by BCA method, denatured in SDS-loading buffer, submitted to electrophoresis on 12% SDS-polyacrylamide gel (PAGE)( 40μl per lane), blotted onto PVDF membrane in graphite box in turn. The PVDF membranes were blocked (in 5% skimmed milk powder for 2hr), incubated with primary antibody (goat polyclonal anti-α 7 antibody, Santa Cruz Biotechnology Inc, 1:500, 4℃, overnight) and horseradish peroxidase (HRP)-conjugated secondary antibody (rabbit anti-goat, 1:5000, 1hr) in turn , illuminated using enhanced chemiluminescence (ECL) kit, at last were exposed to films, bands on which were quantified with Koda gel imaging system . Experiment above was carried out repeattedly for four times. 5. Statistical method: Western-blot gray scale results were used to semiquantitative analysis. method as part one.Results: 1. Indirect IF staining: nAchRα 7 in PC12 cells appeared to be a ring in green fluorescence surrounding cell surface , of which there was difference between different groups, fluorescence intensity for blank group was stronggest, that for A β group weakened obviously, on contrast to A β group, fluorescence intensity for 17βE₂ intervention group (10^-9-10^-6M) brightened to some degree. 2. Western-blot analysis: By contrast to blank group, nAchRα 7 protein expression level in Aβgroup decreased significantly ( 65%, P<0.01); The levels in 17βE₂ intervention group were up-regulated significantly (P<0.01) in a dose-dependent manner,when compared with Aβ group.Conclusion: For the first time, nAchRα 7 is observed to distribute on PC12 cell surface using Indirect IF staining. Aβ makes the fluorescence intensity for nAchR α 7 weaken ,which can be reversed by estrogen . Combined with Western-blot analysis, it is confirmed that Aβ inhibit the expression of nAchRα 7, while estrogen up-regulate the expression in a dose-dependent manner.