雌激素延缓衰老及植物雌激素α-ZAL抗Aβ细胞损伤作用的实验研究
摘要
衰老的发生和演变是生物体不可抗拒的自然过程,随着老年医学的飞速发展,有关衰老机制的学说已有上百种,但至今对衰老是如何发生的仍无确切答案。近年研究人员发现:伴随自然衰老过程,活性氧产物生成增多超过机体清除能力,可致细胞及生物大分子受损,其中对核酸的损害主要表现为DNA氧化受损和修复不足,损伤DNA积累激发变异信号诱导性衰老,最终启动衰老过程。减轻DNA损伤,促进损伤修复必然有利于衰老过程的延缓。作为一种内源性激素,雌激素表现出的延缓自然衰老的作用已被流行病学研究证实,但它对衰老过程中细胞DNA的损伤修复是否具有影响,尚未见相关报道。因此,本研究设计了体内、外试验,观察衰老过程中雌激素作用下DNA损伤修复的变化,探讨其可能的延缓衰老作用机制。
伴随衰老进程,神经退行性疾病—阿尔茨海默病(Alzheimer's disease,AD)的发病率呈增高趋势。在AD的发病中,β-淀粉样蛋白(Aβ)的生成、代谢及毒性作用是AD发生的中心环节。Aβ诱导的细胞凋亡是AD神经元丢失的主要原因,因而拮抗Aβ诱导的细胞凋亡是预防AD的重要靶标。雌激素预防AD的作用已得到实验的证实,但植物雌激素-α-玉米赤霉烯醇(α-Zearalanol,α-ZAL)是否具有类似效应,迄今未见报道。本实验设计以雌激素为阳性对照,观察α-ZAL的抗Aβ损伤作用,在细胞水平评价α-ZAL的神经保护作用,为开发新的安全有效的激素替代药物奠定基础。
第一部分雌激素减轻DNA氧化损伤,延缓衰老
一、体外实验
1.应用有限增殖细胞-人胚肺二倍体成纤维细胞(human diploid fibroblasts.HDF)体外培养,发现以无E2血清培养的细胞经50次群体倍增(PD50)后即完全进入衰老状态,而从PD30开始加入E2(10~(-8)、10~(-7)、10~(-6))持续培养,细胞分别经54、57、59次群体倍增后才进入完全衰老状态,表明雌激素具有延长细胞生命周期的作用,且作用表现出一定的剂量依赖关系。
2.在PD50时,10~(-6)M E2培养细胞中β-半乳糖苷酶染色阳性细胞数明显少于无E2培养对照细胞(P＜0.05)。表明雌激素可延缓衰老表型的出现,有确实的抗衰老作用。
3.通过DCFH-DA免疫荧光染色观察到,伴随PD次数的增加,细胞内源性ROS水平升高,而10~(-6)M E2可以减缓该升高过程。表明雌激素可以促进活性氧产物的清除。
4.Western blotting实验结果显示,DNA损伤修复机制调控的重要因子p53和p21WAF1的表达受E2影响。在PD50时,10~(-6)M E2培养细胞中p53和p21WAF1的表达低于对照细胞(P＜0.05),表明雌激素可明显减少DNA损伤及其积累。
5.在PD50时,应用流式细胞术分析细胞周期,发现10~(-6)M E2培养条件下,细胞G1期细胞比例显著低于对照组(P＜0.05);BrdU免疫组织化学染色结果显示,10~(-6)M E2培养细胞中BrdU阳性细胞数明显高于对照细胞(P＜0.05),表明雌激素可以维持DNA复制,延迟细胞周期阻滞的出现。
6.单细胞凝胶电泳实验结果显示,与200μM H_2O_2共孵育2h,细胞DNA明显受损,细胞慧尾拉长,而H_2O_2作用后加入10~(-6)M E2,细胞慧尾长度明显短于单独H_2O_2作用组(P＜0.05),表明雌激素可以明显促进DNA损伤修复。
二、在体实验
1.成年雌性C57小鼠随机分为三组:对照组(Control),假手术加皮下注射生理盐水,腹腔注射芝麻油;模型组(Model),双侧卵巢切除加皮下注射D-半乳糖100mg/kg.d和腹腔注射芝麻油;E2补充组(ERT),双侧卵巢切除加皮下注射D-半乳糖100mg/kg.d和腹腔注射E2 50μg/kg.d,D-半乳糖和E2连续使用8w。
2.MWM测试发现模型组小鼠寻台潜伏期显著长于对照组(P＜0.01),而ERT组小鼠寻台潜伏期短于模型小鼠(P＜0.05),该结果显示雌激素补充可明显改善小鼠空间学习记忆能力。
3.比色法测定脑海马抗氧化酶活力及MDA含量。结果表明,与对照组小鼠相比模型组小鼠MDA含量显著升高(P＜0.01),抗氧化酶SOD及GSH-Px活性显著降低(P＜0.01),ERT组小鼠上述改变部分逆转。
4.组织形态学表明,模型组小鼠海马神经元有明显损伤,表现为海马CA1区神经元尼氏小体蓝色颗粒减少和胞浆空泡样变性,平均积分光密度值显著低于对照组小鼠(P＜0.01),ERT组小鼠海马CA1区尼氏小体增多,平均积分光密度值升高大于模型组小鼠(P＜0.05),表明雌激素可对抗D-半乳糖所致的神经元损伤。
5.免疫组织化学及western blotting结果显示,模型组小鼠海马神经元DNA明显受损,海马CA1区DNA氧化损伤标志物8-oxox-dG阳性细胞数显著高于对照组小鼠(P＜0.01),而DNA损伤碱基切除修复蛋白MTH1及促DNA修复蛋白BDNF的表达均明显降低(P＜0.01);ERT组小鼠CA1区8-oxox-dG阳性细胞数明显减少,MTH1及BDNF的表达明显回升(P＜0.05)。同时,ERT组小鼠应激诱导的促DNA修复蛋白Hsp70的表达亦显著高于模型组(P＜0.01),表明雌激素可通过调节DNA修复机制来减少DNA氧化损伤。
6.末次注射BrdU后24h,模型小鼠海马DG区BrdU阳性细胞数较对照组显著减少(P＜0.01),ERT组小鼠BrdU阳性细胞数明显多于模型组(P＜0.05),表明雌激素可保护小鼠海马区神经干细胞的增殖,有利于衰老细胞更新,延缓组织老化。
7.利用形态学及RT-PCR的方法,观察小鼠子宫的相关变化,发现模型组小鼠子宫显著萎缩,E2补充后子宫重量增加,但子宫内膜癌标志物MN/CA9 mRNA的表达在各组小鼠均未检测到。表明在本实验条件下雌激素使用在保护神经元的同时未使子宫产生异常变化。
本部分研究结果显示,雌激素可通过减少内源性活性氧产物的生成,调控DNA损伤修复蛋白表达,减轻DNA氧化损伤,促进其损伤修复,从而减少由损伤DNA积累所启动的衰老程序而发挥延缓衰老作用。
第二部分植物雌激素α-玉米赤霉烯醇对PC12细胞Aβ_(25-35)损伤的保护作用研究
1.培养PC12细胞,采用MTT方法观察到,5μM Aβ_(25—35)与细胞共孵育24h,活细胞数显著低于对照组(P＜0.01);而将细胞与不同浓度E2或α-ZAL预孵育12h后再加入Aβ_(25—35),活细胞数呈剂量依赖性的升高(P＜0.05),但在相同浓度下α-ZAL的细胞保护效应略低于E2。
2.透射电镜结果显示,5μM Aβ_(25—35)与PC12细胞共孵育24h可导致细胞凋亡。Hoechest33258荧光染色实验结果显示,Aβ_(25—35)作用组凋亡细胞数显著多于对照组(P＜0.01),E2及α-ZAL预孵育均明显减轻Aβ_(25—35)诱导的细胞凋亡(P＜0.05)。
3.生化学检测结果提示,与对照组相比,单独Aβ_(25—35)与PC12细胞共孵育,细胞MDA含量升高(P＜0.01),SOD和GSH-Px活性降低(P＜0.01),而E2及α-ZAL预孵育均能有效对抗Aβ_(25—35)所致的细胞抗氧化酶活性降低及MDA生成增多(P＜0.01),表明E2及α-ZAL可明显对抗Aβ_(25—35)导致的细胞氧化损伤。
4.Western blotting及RT-PCR实验结果显示,Aβ_(25—35)导致PC12细胞抗凋亡蛋白bcl-2表达减少,而促凋亡蛋白bax及caspase-3表达升高,E2及α-ZAL可拮抗上述改变。同时E2及α-ZAL还能诱导抗凋亡基因A20 mRNA的表达,表明E2及α-ZAL可通过调控凋亡相关蛋白及基因的表达而发挥抗细胞凋亡作用。
本部分研究结果显示,E2及α-ZAL均可通过对抗氧化损伤,调控凋亡相关蛋白的表达而发挥抗Aβ_(25-35)诱导的细胞凋亡效应,α-ZAL的细胞保护效应稍弱于E2。
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.
PartⅠ:Effects of estrogen on protect DNA damage and delay aging
Experiment in vitro
1.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~(-6)M 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~(-6)M),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~(-6)M) 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 H_2O_2 for 2h,SCGE assay results showed that the length of comet tail in 10~(-6)M estrogen cultured cell less than the cells which incubated with H_2O_2 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 vivo
1.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.
PartⅡ:Phytoestrogenα-ZAL protects cultured PC12 cells from Aβ_(25-35) insult
1.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.
伴随衰老进程,神经退行性疾病—阿尔茨海默病(Alzheimer's disease,AD)的发病率呈增高趋势。在AD的发病中,β-淀粉样蛋白(Aβ)的生成、代谢及毒性作用是AD发生的中心环节。Aβ诱导的细胞凋亡是AD神经元丢失的主要原因,因而拮抗Aβ诱导的细胞凋亡是预防AD的重要靶标。雌激素预防AD的作用已得到实验的证实,但植物雌激素-α-玉米赤霉烯醇(α-Zearalanol,α-ZAL)是否具有类似效应,迄今未见报道。本实验设计以雌激素为阳性对照,观察α-ZAL的抗Aβ损伤作用,在细胞水平评价α-ZAL的神经保护作用,为开发新的安全有效的激素替代药物奠定基础。
第一部分雌激素减轻DNA氧化损伤,延缓衰老
一、体外实验
1.应用有限增殖细胞-人胚肺二倍体成纤维细胞(human diploid fibroblasts.HDF)体外培养,发现以无E2血清培养的细胞经50次群体倍增(PD50)后即完全进入衰老状态,而从PD30开始加入E2(10~(-8)、10~(-7)、10~(-6))持续培养,细胞分别经54、57、59次群体倍增后才进入完全衰老状态,表明雌激素具有延长细胞生命周期的作用,且作用表现出一定的剂量依赖关系。
2.在PD50时,10~(-6)M E2培养细胞中β-半乳糖苷酶染色阳性细胞数明显少于无E2培养对照细胞(P＜0.05)。表明雌激素可延缓衰老表型的出现,有确实的抗衰老作用。
3.通过DCFH-DA免疫荧光染色观察到,伴随PD次数的增加,细胞内源性ROS水平升高,而10~(-6)M E2可以减缓该升高过程。表明雌激素可以促进活性氧产物的清除。
4.Western blotting实验结果显示,DNA损伤修复机制调控的重要因子p53和p21WAF1的表达受E2影响。在PD50时,10~(-6)M E2培养细胞中p53和p21WAF1的表达低于对照细胞(P＜0.05),表明雌激素可明显减少DNA损伤及其积累。
5.在PD50时,应用流式细胞术分析细胞周期,发现10~(-6)M E2培养条件下,细胞G1期细胞比例显著低于对照组(P＜0.05);BrdU免疫组织化学染色结果显示,10~(-6)M E2培养细胞中BrdU阳性细胞数明显高于对照细胞(P＜0.05),表明雌激素可以维持DNA复制,延迟细胞周期阻滞的出现。
6.单细胞凝胶电泳实验结果显示,与200μM H_2O_2共孵育2h,细胞DNA明显受损,细胞慧尾拉长,而H_2O_2作用后加入10~(-6)M E2,细胞慧尾长度明显短于单独H_2O_2作用组(P＜0.05),表明雌激素可以明显促进DNA损伤修复。
二、在体实验
1.成年雌性C57小鼠随机分为三组:对照组(Control),假手术加皮下注射生理盐水,腹腔注射芝麻油;模型组(Model),双侧卵巢切除加皮下注射D-半乳糖100mg/kg.d和腹腔注射芝麻油;E2补充组(ERT),双侧卵巢切除加皮下注射D-半乳糖100mg/kg.d和腹腔注射E2 50μg/kg.d,D-半乳糖和E2连续使用8w。
2.MWM测试发现模型组小鼠寻台潜伏期显著长于对照组(P＜0.01),而ERT组小鼠寻台潜伏期短于模型小鼠(P＜0.05),该结果显示雌激素补充可明显改善小鼠空间学习记忆能力。
3.比色法测定脑海马抗氧化酶活力及MDA含量。结果表明,与对照组小鼠相比模型组小鼠MDA含量显著升高(P＜0.01),抗氧化酶SOD及GSH-Px活性显著降低(P＜0.01),ERT组小鼠上述改变部分逆转。
4.组织形态学表明,模型组小鼠海马神经元有明显损伤,表现为海马CA1区神经元尼氏小体蓝色颗粒减少和胞浆空泡样变性,平均积分光密度值显著低于对照组小鼠(P＜0.01),ERT组小鼠海马CA1区尼氏小体增多,平均积分光密度值升高大于模型组小鼠(P＜0.05),表明雌激素可对抗D-半乳糖所致的神经元损伤。
5.免疫组织化学及western blotting结果显示,模型组小鼠海马神经元DNA明显受损,海马CA1区DNA氧化损伤标志物8-oxox-dG阳性细胞数显著高于对照组小鼠(P＜0.01),而DNA损伤碱基切除修复蛋白MTH1及促DNA修复蛋白BDNF的表达均明显降低(P＜0.01);ERT组小鼠CA1区8-oxox-dG阳性细胞数明显减少,MTH1及BDNF的表达明显回升(P＜0.05)。同时,ERT组小鼠应激诱导的促DNA修复蛋白Hsp70的表达亦显著高于模型组(P＜0.01),表明雌激素可通过调节DNA修复机制来减少DNA氧化损伤。
6.末次注射BrdU后24h,模型小鼠海马DG区BrdU阳性细胞数较对照组显著减少(P＜0.01),ERT组小鼠BrdU阳性细胞数明显多于模型组(P＜0.05),表明雌激素可保护小鼠海马区神经干细胞的增殖,有利于衰老细胞更新,延缓组织老化。
7.利用形态学及RT-PCR的方法,观察小鼠子宫的相关变化,发现模型组小鼠子宫显著萎缩,E2补充后子宫重量增加,但子宫内膜癌标志物MN/CA9 mRNA的表达在各组小鼠均未检测到。表明在本实验条件下雌激素使用在保护神经元的同时未使子宫产生异常变化。
本部分研究结果显示,雌激素可通过减少内源性活性氧产物的生成,调控DNA损伤修复蛋白表达,减轻DNA氧化损伤,促进其损伤修复,从而减少由损伤DNA积累所启动的衰老程序而发挥延缓衰老作用。
第二部分植物雌激素α-玉米赤霉烯醇对PC12细胞Aβ_(25-35)损伤的保护作用研究
1.培养PC12细胞,采用MTT方法观察到,5μM Aβ_(25—35)与细胞共孵育24h,活细胞数显著低于对照组(P＜0.01);而将细胞与不同浓度E2或α-ZAL预孵育12h后再加入Aβ_(25—35),活细胞数呈剂量依赖性的升高(P＜0.05),但在相同浓度下α-ZAL的细胞保护效应略低于E2。
2.透射电镜结果显示,5μM Aβ_(25—35)与PC12细胞共孵育24h可导致细胞凋亡。Hoechest33258荧光染色实验结果显示,Aβ_(25—35)作用组凋亡细胞数显著多于对照组(P＜0.01),E2及α-ZAL预孵育均明显减轻Aβ_(25—35)诱导的细胞凋亡(P＜0.05)。
3.生化学检测结果提示,与对照组相比,单独Aβ_(25—35)与PC12细胞共孵育,细胞MDA含量升高(P＜0.01),SOD和GSH-Px活性降低(P＜0.01),而E2及α-ZAL预孵育均能有效对抗Aβ_(25—35)所致的细胞抗氧化酶活性降低及MDA生成增多(P＜0.01),表明E2及α-ZAL可明显对抗Aβ_(25—35)导致的细胞氧化损伤。
4.Western blotting及RT-PCR实验结果显示,Aβ_(25—35)导致PC12细胞抗凋亡蛋白bcl-2表达减少,而促凋亡蛋白bax及caspase-3表达升高,E2及α-ZAL可拮抗上述改变。同时E2及α-ZAL还能诱导抗凋亡基因A20 mRNA的表达,表明E2及α-ZAL可通过调控凋亡相关蛋白及基因的表达而发挥抗细胞凋亡作用。
本部分研究结果显示,E2及α-ZAL均可通过对抗氧化损伤,调控凋亡相关蛋白的表达而发挥抗Aβ_(25-35)诱导的细胞凋亡效应,α-ZAL的细胞保护效应稍弱于E2。
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.
PartⅠ:Effects of estrogen on protect DNA damage and delay aging
Experiment in vitro
1.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~(-6)M 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~(-6)M),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~(-6)M) 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 H_2O_2 for 2h,SCGE assay results showed that the length of comet tail in 10~(-6)M estrogen cultured cell less than the cells which incubated with H_2O_2 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 vivo
1.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.
PartⅡ:Phytoestrogenα-ZAL protects cultured PC12 cells from Aβ_(25-35) insult
1.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.
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