Studies Of Estrogen Delays Senescence And Phytoestrogen α-ZAL Protects Cells Against Aβ Toxicity

At present,there is no explicit theory to explain the mechanism underlying aging, which is a spontaneous and irreversible process.During the last years,scientists had found that accompanying with the aging process,when the production exceeds the capacity of detoxification,reactive oxygen species(ROS) can cause DNA damage in cells and organism.Extensive observations suggest that the oxidative damage of DNA accumulation can induce stress or aberrant signaling inducing senescence which play a central role in the pathogenesis of aging.As a result,reduce the damage of DNA will be benefit to delay aging.As an endogenous hormone,the effects on extending lifespan of estrogen have been demostrated in epidemiologic survey.However,the effect on DNA damage and repair has not been reported yet.In present study,the effects of estrogen on DNA damage and repair was investigaed to find the possible mechanism in vivo and in vitro experiments.Alzheimer's disease(AD) is the most common form of dementia,the incidence of which increases with aging.The formation,metabolism and toxicity of Aβplay an important role in AD pathophysiology.Inhibiting the neuronal apoptosis induced by Aβwhich greatly contributed to neuronal loss is the potential strategy of AD treatment.The neuroprotective of estrogen have been approved,nevertheless,there is rare research in the relationship betweenα-ZAL and Aβ.The present study was designed to evaluate the neuroprotective ofα-ZAL against Aβinduced cells damage in cultured PC12 cells when used E2 as positive control. Partâ… :Effects of estrogen on protect DNA damage and delay agingExperiment in vitro1.Human diploid fibroblasts(HDF) reaching senescence in PD50 which was cultured in estrogen deficiency medium.Cells respectively proliferated and reached senescence in PD54,PD57 and PD59 incubated with estrogen at 10^-8,10^-7 or 10^-6M concentration.These results suggested that estrogen could extend lifespan in dose dependent manner.2.In senescence phase(PD50),while cells cultured in medium supplemented with estrogen(10^-6M),the number ofβ-gal positive cell was less than the control which was cultured in estrogen deficiency medium(P＜0.05).The result suggested that senescence phaenotype could be delayed by estrogen treatment.3.DCFH-DA result showed that ROS significantly increased accompanying with the PDs prgress.Estrogen(10^-6M) may weaken the process.It suggested that estrogen may facilitate the clearance of ROS.4.Western blotting results showed that the expression levels of p53 and p21 which played an important role in DNA repair has been regulated by estrogen.Compared with the cells cultured in estrogen deficiency medium,there was a significant decrease of p53 and p21 protein in PD50(P＜0.05).It suggested that DNA damage and accumulate was slightly when cells was incubated with estrogen.5.In senescence phase(PD50),cell cycle analysis with FACS showed that cell cycle arrest was delayed by estrogen,the number of cell in G1 phase was less than the control which was cultured in estrogen deficiency medium(P＜0.05),meanwhile the numer of BrdU positive cell high than the control(P＜0.05),shows that estrogen may be attributed to DNA replication.6.After treat with 200μM H₂O₂ for 2h,SCGE assay results showed that the length of comet tail in 10^-6M estrogen cultured cell less than the cells which incubated with H₂O₂ without estrogen,it means that estrogen treatment facilitate DNA repair which DNA damage induced by ROS increase may benefit to decrease damage DNA accumulation.Experiment in vivo1.Mice were randomly divided into three goups.Mice with sham operation and s.c. saline and i.p.sesame oil were used as the control group;mice with ovariectomy and s.c.D-galactose(100mg/kg.d) and i.p.sesame oil were used as the model group; mice with ovariectomy and s.c.D-galactose(100mg/kg.d) and i.p.E2(50μg/kg.d) were used as the ERT goup.Mice were injected daily D-galactose and E2 for 8w.2.Morris water maze the result showed that the escape latencies of model mice increased compared to Control mice(P＜0.01),estrogen treatment decreased the escape latencies compared to model mice(P＜0.05).3.Compared to control mice,D-gal treatment significantly elevated MDA level (P＜0.01) and reduced the activities of SOD and GSH-Px(P＜0.05)in hippocampus. Estrogen treatment can partly reversed the changes.4.Histomorphological observation showed the neurons in CA1 region of hippocampal in model mice were obviously damaged,which were manifested by severely decrease of Nissl body and cytoplasm vacuolar degeneration,the values of optical density decreased compared to control mice(P＜0.01).Estrogen administration could significantly increase the Nissl body in DG,the values of optical density increased compared to model mice(P＜0.05).It means that estrogen protect neurons against the neurotoxicity of D-gal.5.Immunohistochemistry and western blotting results showed 8-oxo-dG,a biomarker of DNA oxidative damage,increased and the expression of MTH1,an oxidized purine nucleoside tiphosphatase which play an important role in base excision repair mechanism,decreased and BDNF,a protein may facilitate DNA repair,decreased in hippocampus CA1 region in model mice compared with control mice(P＜0.05). Estrogen treatment obviously rivalized these changes induced by D-gal. Simultaneously,the levels of Hsp70,a protein promote DNA repair which expression induced by oxidative stress were elevated in estrogen supplementary group compared with model group in hippocampus(P＜0.05).6.BrdU immunohistochemistry method was used to detect the neural stem cells proliferation.The number of BrdU positive cells in DG region of hippocampus was markedly decreased in model mice compared with control(P＜0.01) and it was returned in ERT mice,P＜0.05 compared to model mice.Results showed estrogen protect neural stem cells proliferation,which suggested that estrogen treatment may maintain senescence cell renew to delay organism aging. 7.We assessed the effect of estrogen in uterus by the histomorphological observation and RT-PCR.The results indicated that canceration phaenotype MN/CA9mRNA was not been found in our experiment,although tha weight of uteru in ERT mice than in model mice.It means that estrogen protect neurons and have not seriously side effcct on uteru.In summary,the present research demonstrated that estrogen may delay aging, which maybe related to reduce the production of ROS,attenuate DNA oxidative damage,promote DNA repair and decrease the damaged DNA accumulation.Partâ…¡:Phytoestrogenα-ZAL protects cultured PC12 cells from Aβ_25-35 insult1.Culture PC12 cells,the number of living cells were obviously decreased while cells incubation with 5μM Aβ_25-35 for 24h compare with no-Aβ_25-35 treated cells by using MTT assays.Incubating PC12 cells with E2 orα-ZAL at different concentrations 12h before Aβ_25-35 addition can increase the number of living cells in an dose-dependent way,the number of living cells markly increased compared with Aβ_25-35-treated cells(P＜0.05),but the protective effect ofα-ZAL was weaker than that of E2 at the same concentration.2.Results of electronmicroscopic observation indicated that incubation with Aβ_25-35 for 24h induced apoptosis.Immunofluorescent staining analysis with Hoechest33258 shown the number of positive cells increased after Aβ_25-35-treated compared with Control(P＜0.01),E2 andα-ZAL could decreased the percentages of apoptotic cells(P＜0.05).3.Results of biochemical analysis showed that Aβ_25-35 treatment significantly elevated MDA level(P＜0.01) and reduced the activities of SOD and GSH-Px(P＜0.01) compared with Control,E2 andα-ZAL enhanced the activities of SOD,GSH-Px and reduced the production of MDA,P＜0.01 compared with Aβ_25-35-treated cell.The results showed that E2 andα-ZAL supplements protect cells from oxidative insults.4.Western blotting and RT-PCR results showed that Aβ_25-35 could decrease the expression levels of bcl-2,up-regulate bax and caspase-3 expression level compared with control,E2 andα-ZAL could against these changes.Simultaneously,E2 and α-ZAL induce the expression of A20 mRNA,an anti-apoptosis gene may contribute to inhibit apoptosis.In summary,the present results demonstrated thatα-ZAL could effectively protect cell from apoptosis induced by Aβ_25-35 by maintaining the function of oxidation reduction system and regulating the expression of apoptosis-related gene,just like as E2,but the effect ofα-ZAL was weaker than that of E2.