Study On Neuroprotective Effects Of Ginsenoside Rg1 In Postmenopause Women Alzheimer's Disease Cell Model And Its Mechanism

BackgroundAlzheimer's disease (AD) is a neurodegenerative disorders correlated with age. AD which first reported by German doctor Alosis Alzeimer's is the most common cause of dementia.It's reported that AD affect one of every ten people over age 65 and nearly 35% over age 85 in USA. This translates into more than 4.5 million AD patients in the US, a number expected to reach 9 million by 2030 and 16 million by 2050 as the US population ages. AD has been the fourth cause of old people's death next to cardiovascular diseases,cancer,stroke. The societal impact of AD is considerable, in the US alone, the economic burden of AD is estimated to be in excess of＄110B annually. China is a big population country, the aging population is more serious. There are more than 10 percent sixty years or older population, so the number of dementia patients will be increasing significantly, especially in the older old.Recent studies showed that except for aging, heredity and environment, hormone especially estrogen plays an important role in pathogenesy of AD. Various epidemiological studies showed that women suffer from AD at rate of 1.6-3 times more then men. One hypothesis used to explain the gender bias of a higher risk of AD in women is that women are generally living longer than men. Indeed, it has been calculated that women older than 50 years of age comprise a major portion of the total population of any country in the modern western world. Estrogen shows profound protective effects in vivo and vitro experiments. Several studies indicate that estrogen replacement therapy (ERT) increases performance in some memory/cognition tests in AD patients. However, we have to recognize the side-effects of ERT. It probably increases proliferative and oncogenic activities on non-neuronal cells such as cells in breast and endometrium.Accordingly, growing attention has been paid to Phytoestrogens for its potential therapeutic use in a range of hormone dependent diseases, and has been proven to possess neuroprotective effects. Panax ginseng (Araliaceae) is an important Chinese herbal medicine has been used widely for thousands of years in China, and recently being widely studied in the west. Ginsenosides were reported to be the most abundant, active compounds attributing to the pharmacological actions of ginseng. As one of nearly forty different ginsenosides has been identified from ginseng, ginsenoside Rg1 has been attracting considerable attention for its effects on central nervous system. However, the molecular mechanisms underlying the neuroprotective effect of Ginsenoside Rgl have not been fully studied. In our experiments, we designed to explore the underlying mechanisms of the neuroprotective effect of Ginsenoside Rgl.ObjectiveThis study was performed to assess the neuroprotective effect of Ginsenoside Rgl as well as the underlying mechanisms.MethodsPart 1 Primary neuronal cell cultures and establishment of Aβ25-35-insulted primary culture neuronal cell modelPrimary cultured neurons were prepared from new birth Wistar rats within 24 hours. After removal of meanings, the brains were treated with 0.125% trypsin for 15 min at 37℃. The reaction was stopped using DMEM/F12 medium supplemented with 10% fetal bovine serum. The pellet was dissociated by repeated passage through a series of fire-polished constricted Pasteur pipettes. The cultures were placed in a humidified atmosphere of 95% air and 5% CO2 at 37℃and routinely observed under phase-contrast inverted microscope. Cells were used for experiments after 7 days in vitro.Aβ25-35 was dissolved in sterile Sodium Chloride and diluted in DMEM/F12 medium. This stock was stored at -20℃and incubated for 7 days at 37℃prior to use. Various concentrations of Aβ25-35 (Oμmol/l,2.5μmol/l,5μmol/l,7.5μmol/l,10μmol/1) were added into culture medium for 24hu 48h and 72h to determine the suitable concentration of the model. Cell viability was assessed by MTT method and LDH release assessment.Part 2 Ginsenoside Rgl inhibits the cytotoxicity insulted by AP25-35 and its mechanismNeuroprotective effects of Ginsenoside Rgl inhibiting the cytotoxicity of AP25.35 were detected by the MTT and measurement of LDH release; neuroprotective effects of Ginsenoside Rgl protecting apoptosis induced by Aβ25-35 were detected by AnnexinV/PI staining; Bcl-2,Bax,caspase3,ERαand ERβmRNA expression were detected by real-time PCR; Bcl-2,Bax,active caspase3,ERαand ERβprotein expression were detected by Western blot analysis.ResultsPart 1 Primary neuronal cell cultures and establishment of Aβ25-35-insulted primary culture neuronal cell model1. Establishment of Aβ25-35-insulted primary culture neuronal cell model The results showed the toxicity that Aβ25-35 induced to neuronal cells were in a dose-dependent and time- dependent manner. Both MTT and LDH release results showed, the cytotoxicity of Aβ25-35 came to the biggest in 5μmol/l and 72h groups. Accordingly, we eventually choose 72h treatment of 5μM Aβ25-35 for further experiments.2. the change of cellar morphology Observation of the Model group showed that the number of the neurons decreased, the cells were edematus, nucleus membranes crimpled or ruptured, chromatin aggregated, chromatin aggregated, the number of neuritis decreased, neuritis became shorter even disappeared. The changes were less prominent in ginsenoside Rgl and E2 groups. Part 2 Ginsenoside Rgl inhibits the cytotoxicity insulted by Aβ25-35 and its mechanism1. Ginsenoside Rgl inhibit the cytotoxicity of Aβ25-35 After treated with Aβ25-35, the cell viability was decreased remarkably and LDH release was increased compared with Control group (P<0.01). The cell viability was decreased and LDH release was inhibited in ginsenoside Rgl and E2 groups compared to Model group (P<0.01 or P <0.05). The cell viability of E2 group was higher than ginsenoside Rgl groups, however there was no significant difference between them (P>0.05).2. Ginsenoside Rgl protects apoptosis induced by Aβ25-35 The early apoptotic cells that had bound AnnexinV-FITC would show green staining in the plasma membrane. The late apoptotic cells that had lost membrane integrity would show red staining (PI) throughout the nucleus and a halo of green staining in the plasma membrane. Dead cells would show only red staining (PI) throughout the nucleus. The numbers of early apoptotic cells of Model group were larger than Control group (P<0.01). Various concentrations of ginsenoside Rgl and E2 significantly reduced the numbers of early apoptotic cells compared with Model group (P<0.05).3. Analysis of Bcl-2,Bax,caspase3,ERa and ERβmRNA expression by real-time PCR The expression of Bcl-2 mRNA in Model group was higher significantly than that in Control group (P<0.01). The expression of Bcl-2 mRNA in various concentrations of ginsenoside Rgl and E2 groups increased statistically compared to that in Model group (P<0.01 or P<0.05), and the expression of Bax mRNA decreased (P<0.01 or P<0.05). It resulted in an up-regulation of Bcl-2/Bax ratio (P<0.01 or P 0.05). The expression of caspase3 mRNA in ginsenoside Rg110-7 M,ginsenoside Rgl 10-6M and E2 groups decreased statistically compared to that in Model group (p<0.01 or P<0.05), There was no significant difference between ginsenoside Rgl 10-8M group and Model group(P>0.05). The expression of ERa and ERβmRNA in Model group decreased significantly compared to that in Control group (P<0.01 or P<0.05). The expression of ERβmRNA in various concentrations of ginsenoside Rgl and E2 groups increased statistically compared to that in Model group (P<0.01 or P<0.05). The expression of ERa mRNA in various concentrations of ginsenoside Rgl and E2 groups increased compared to that in Model group, but there was no significant difference between them (P>0.05).4. Bcl-2,Bax,active caspase3,ERαand ERβprotein expression detection by Western blot analysis Levels of Bcl-2 protein were up-regulated after treated with ginsenoside Rgl and E2 (P<0.01 or P<0.05), and levels of Bax protein were down-regulated (P<0.05). It resulted in an up-regulation of Bcl-2/Bax ratio (P<0.01). Ginsenoside Rgl down-regulated the expression of active caspase3 protein similar to E2 group (P<0.01 or P<0.05). There are ERa and ERβprotein expression in primary cultured neuronal cells. The expression of ERa and ERβprotein in Model group decreased significantly compared to that in Control group (P<0.01). There was no significant difference between ginsenoside Rg1,E2 groups and Model group of ERa protein expression (P>0.05). The expression of ERβprotein in various concentrations of ginsenoside Rgl and E2 groups increased statistically compared to that in Model group (P<0.01 or P<0.05).Conclusion1. Both ginsenoside Rgl and E2 can inhibit the cytotoxicity of Aβ25-35. Ginsenoside Rgl shows neuroprotective effects probably because it can protect apoptosis induced by Aβ25-35.2. The mechanism of protecting apoptosis induced by Aβ25-35 for ginsenoside Rgl and E2 may be associated with increasing the ratio of Bcl-2/Bax which down-regulation the activation of caspase 3 and finally inhibit neuron cells apoptosis.3. Both ERa and ERβwere expressed in primary cultured neuronal cells.4. ERs play an important part in AD. Ginsenoside Rgl shows estrogenic-neuroprotective effects. The estrogenic-neuroprotective effects of ginsenoside Rgl at least partly via ERP-dependent pathway, not ERa-dependent pathway.