【作者】 王文君；

【导师】 李大金；

【作者基本信息】 复旦大学 ， 妇产科学， 2009， 博士

【摘要】 妇女进入围绝经期以后,卵巢功能减退,出现了血管舒缩症状、神经精神症状,以及骨质疏松、学习记忆力减退,甚至发生阿尔茨海默病(Alzheimer’s disease,AD)。对于AD的治疗尚处于对症阶段,雌激素和中医药在AD防治中的作用正在引起重视。雌激素替代治疗同时增加了子宫内膜癌、乳腺癌、心脏疾患和中风等的发病风险,故受到限制。中医中药对由雌激素下降引起的围绝经期综合征有较好的疗效,因此中医中药对于雌激素下降有关的学习记忆能力减退甚至AD可能有良性调节作用。我院根据中医理论及多年临床实践,以滋肾柔肝清心泻火为治则制成以补肾为主更年春方(GNC),对更年期综合征疗效良好,尤其可以改善大脑功能、增强记忆能力。既往研究发现,GNC可通过提高雌激素受体(ER)含量对神经内分泌免疫网络起良性调节作用;GNC可以提高大鼠切除卵巢后Morris水迷宫成绩;GNC可提高海马中ERβmRNA和蛋白的表达、调节海马胆碱能指标并可能由此改善学习和记忆能力;而且GNC不导致子宫内膜增生,提示中药GNC对改善学习和记忆能力有潜在的应用前景。本研究拟通过体外实验研究,解析GNC复方及主要单体成分对神经细胞损伤的保护作用及其分子机制。第一部分中药GNC对PC12细胞的保护作用目的探讨、分析中药GNC含药血清和主要单体及其复合物对PC12细胞作用的有效浓度及时间。方法3月龄SD雌性大鼠去势后随机分组,分别以中药复方GNC颗粒制剂或生理盐水灌服,制备GNC含药血清及正常大鼠血清。以GNC含药血清、正常大鼠血清、F-12培养基(含5%FBS+15%HS,作为空白对照)、GNC复方中主要单体成分芍药苷(Pae)、黄连素(Ber)、知母皂苷A-Ⅲ(Tim)、淫羊霍苷(Ica)及其复合物处理PC12细胞。MTT法检测细胞增殖活力。摸索中药含药血清作用的有效浓度及时间;摸索四种中药单体及复合物(Ica+Tim+Pae+Ber)作用的有效浓度及时间。结果MTT法显示:不同浓度GNC含药血清培养PC12细胞24h、48h、72h后,均表现在20%浓度对PC12细胞活力的促进作用最强,随着培养PC12细胞的GNC含药血清浓度下降,细胞活力呈逐渐减弱趋势;与正常大鼠血清、空白对照比较,20%GNC含药血清培养PC12细胞24、48、72小时后细胞活力均明显增强。与空白对照比较,各中药单体及单体复合液培养PC12细胞24小时后,细胞活力无明显差异,但Ber在浓度为1umol/L和中药单体复合物在浓度梯度3(含Ber 1umol/L、Tim 1umol/L、Pae 1g/L、Ica 1ng/ml)培养细胞时活力似有升高趋势。结论在本研究比较的各不同浓度GNC血清中,20%GNC血清为GNC作用PC12细胞最适有效剂量;处理24小时可作为GNC含药血清作用PC12细胞有效时间。第二部分Aβ25-35导致PC12细胞损伤目的探讨并分析Aβ25-35导致PC12细胞损伤的有效时间和剂量,以建立阿尔茨海默病体外细胞模型,为后续解析GNC复方及主要单体成分保护神经细胞免受损伤的作用及其分子机制奠定基础。方法应用不同浓度Aβ25-35处理PC12细胞24h、48h,MTT法测定细胞活力,倒置显微镜观察细胞形态。结果终浓度分别为5、10、20、及40μmol/L的Aβ25-35培养PC12细胞24h、48h后,MTT法显示的细胞活力均有明显下降(P＜0.01);且随着Aβ25-35浓度增加或培养时间延长,细胞活力有明显减弱趋势。倒置相差显微镜观察:对照组细胞密集,细胞间连接紧密,细胞有立体感;在Aβ25-35作用下,有活力细胞数目减少,细胞间连接较松,胞浆较暗淡,细胞碎片较多,贴壁细胞欠透明,部分发生皱缩,胞浆中有较多颗粒。结论Aβ25-35有明显剂量、时间依赖性地抑制PC12细胞活力作用;采用20umol/1的Aβ处理PC12细胞24小时可制备AD体外模型。第三部分中药GNC方对Aβ诱导PC12细胞损伤的保护作用及其细胞内信号通路目的探讨GNC方是否能明确改善神经细胞凋亡?GNC复方哪些成分是保护神经细胞免受损伤的主要成分?GNC方有无提高ER亚型、起到类似SERM的作用?GNC方是否能激活MAPK信号转导途径?方法设置7个实验组:(1)对照组;(2)Aβ(模型)组;(3)正常大鼠血清组;(4)GNC含药血清组;(5)Ber组;(6)中药单体复合物组;(7)NGF(阳性对照)组。其中(1)组细胞以常规培养基(F-12培养基:含5%FBS+15%HS)与其它各组同步培养;(2)组与(3)-(7)组同步加入Aβ25-35(20umol/L),不加其它药;(3)-(7)组在细胞传代进人对数生长期后(24h左右)分别加药,其中(3)-(6)组加药的浓度根据第一部分摸索的最适浓度加上,(7)组加入终浓度为10 mg/L的NGF。(3)-(7)组分别于加药2小时后加Aβ25-35(20umol/L),继续分别培养24小时,MTT检测细胞活力,流式细胞仪检测细胞凋亡率,Western-blotting检测PC12细胞caspase-3、bax和bcl-2、p-ERK1/2、ER亚型蛋白表达。结果MTT法检测细胞活力显示:与Aβ处理的模型组比较,GNC含药血清组、Ber组、中药单体复合物组、NGF组的细胞活力均明显增强(P＜0.01);中药单体复合物组及Ber组细胞活力均明显低于GNC含药血清组(P＜0.05和P＜0.01);中药单体复合物组细胞活力明显高于Ber组比较,(P＜0.05)。Annexin V-FITC/PI双标记FCM分析PC12细胞早期凋亡显示:与Aβ模型组比较,GNC含药血清组、Ber组、中药单体复合物组、NGF组的PC12细胞早期凋亡率均明显下降(P＜0.01);中药单体复合物组及Ber组的PC12细胞早期凋亡率均明显高于GNC含药血清组(P＜0.01);中药单体复合物组FCM测得PC12细胞早期凋亡率明显低于Ber组(P＜0.01)。Western-blotting结果显示,GNC含药血清、NGF处理的PC12细胞凋亡抑制基因bcl-2表达水平明显高于促凋亡基因bax;中药单体复合物对PC12细胞抑凋亡基因bcl-2表达略高于促凋亡基因bax。各组caspase-3的表达量在Aβ组最高,正常大鼠血清组较Aβ组稍低,Ber组、中药单体复合物组其次,NGF组、GNC含药血清组明显减低,NGF组最低。Western-blotting检测结果还显示:各组均未能测出ERα蛋白条带,提示ERα不参与对PC12细胞的保护作用。ERβ表达水平高低依次为:GNC含药血清组、中药单体复合物组、NGF组、Ber组、正常大鼠血清组、Aβ组。Western-blotting检测结果还发现:各组中p-ERK1/2含量高低依次为:GNC含药血清组、NGF组、中药单体复合物组、Ber组、正常大鼠血清组、Aβ组。结论1.GNC含药血清、Ber、中药单体复合物均能抑制Aβ致PC12细胞凋亡,对Aβ致PC12细胞的损伤有保护作用,其中GNC含药血清作用最强,与NGF作用相仿。2.PC12细胞表达ERβ蛋白,但不表达ERα蛋白。GNC复方能明显提高PC12细胞中ERp表达。3.GNC复方能激活神经细胞MAPK信号通路,提高PC12细胞p-ERK1/2的表达。第四部分GNC方通过ERβ保护PC12细胞免于损伤及其信号转导机制目的探讨GNC复方是否通过ERK途径介导抗凋亡作用;以及细胞内信号转导途径?方法实验设置:(1)对照组;(2)Aβ组;(3)MAPK阻断+GNC含药血清组;(4)MAPK阻断+中药单体复合物组;(5)ER拮抗+GNC含药血清组;(6)ER拮抗+中药单体复合物组;(7)GNC含药血清组;(8)中药单体复合物组。分别培养24小时后,AnnexinV-FITC/PI双染色流式细胞仪(FCM)检测PC12细胞早期凋亡;Western-blotting检测各组PC12细胞caspase-3、bax和bcl-2蛋白表达。分别培养10分钟后,Western-blotting检测PC12细胞p-ERK1/2表达水平,分析ER介导的细胞内信号转导途径。结果与GNC含药血清组比较,MAPK阻断+中药含药血清组、ER拮抗+中药含药血清组的PC12细胞早期凋亡率均明显增加(P＜0.01);与中药单体复合物组比较,MAPK阻断+中药单体复合物组、ER拮抗+中药单体复合物组的PC12细胞早期凋亡率均明显增加(P＜0.01),提示中药含药血清及中药单体复合物均可通过ER及MAPK途径抑制PC12细胞凋亡。Western-blotting检测结果可以看出:中药含药血清组、中药单体复合物组PC12细胞抑凋亡基因bcl-2含量明显高于促凋亡的bax含量,MAPK阻断+中药含药血清组、ER拮抗+中药含药血清组、MAPK阻断+中药单体复合物组、ER拮抗+中药单体复合物组的PC12细胞中抑凋亡基因bcl-2含量明显低于促凋亡的bax含量;且此4组中caspase-3的表达量也较中药含药血清组、中药单体复合物组明显增加,说明中药含药血清及中药单体抑制Aβ致PC12细胞凋亡的作用至少通过ER和MAPK途径调节bax、bcl-2的表达。分析中药提高ER含量与信号转导途径间的关系发现:含药血清组及中药单体复合物组PC12细胞有一定量的p-ERK表达,含药血清组p-ERK较中药单体复合物组略多,加入ER拮抗剂后两组PC12细胞均未测出p-ERK表达,提示中药GNC提高p-ERK的表达至少有ER的参与。结论1.GNC含药血清及中药单体在抑制Aβ致PC12细胞凋亡的作用过程中涉及ER及MAPK信号途径。中药含药血清及中药单体抑制Aβ致PC12细胞凋亡的作用通过ER和MAPK途径调节bax、bcl-2的表达。2.ER介导中药GNC激活MAPK信号通路。更多还原

【Abstract】 The perimenopausal syndrome, such as vasomotion, mental and psychological symptoms as well as osteoporosis, degeneration of study and memory ability, even Alzheimer’s disease occur with ovary aging in menopausal period.The therapy of AD is just at the symptomatic, and the estrogen and traditional Chinese medicine (TCM) get more and more attention. For the increasing risk of endometrial carcinoma, breast cancer and stroke companying estrogen replacement therapy, HRT is limited. A lot of clinical and experimental studies have shown that TCM is effective in treatment of perimenopausal syndrome resulting from estrogen decreasing without apparent side effects, which gives a new therapeutics for AD. According to TCM theory, tonifing Kidney is appropriate to improve the learning and memory in menopause.The principle of GNC decoction is nourishing the Kidney and the Liver and clearing Heart-fire. It is verified that this decoction is clinically effective, especially improving the memory. Our previous study has found that GNC appears to good effects in controlling the neuro-endocrino-immune network by up-regulating the expression of estrogen receptor, enhancing the OVX rats’ manifestation in Morris water maze test, and GNC might increase the RNA and protein expression of ERβin hippocampus, and regulate the cholinergic neuron function so as to enhance the learning and memory ability.We are to investigate the roles of GNC decoction and its monomers in neuroprotection and try to find the mechanism in the present study.Part I Protective Effects of Gengnianchun Decoction on PC12CellsObjective To observe and compare the protective effects of Gengnianchun (GNC) Decoction and its main monomers on PC12 cells.Methods The serum of the rats was got after perfusion of GNC granula to OVX SD rats. PC 12 cells were treated with the serum or main monomers i.e. Paeoniflorin(Pae), Timosaponin A-III(Tim), Berberine Hydrochloride (Ber) and Icariine (Ica) that are the main substances in GNC decoction, respectively, and then analyzed by MTT assay for cell viability to find the proper culturing concentration and duration.Results Comparing with serum from normal rats and control group, the serum with GNC Decoction reinforced PC12 cells activity, with the strongest effect in 20% concentration of the serum at 24 h, 48 h, 72 h. Culturing with herb monomers show little difference except the Ber at the concentration of 1umol/L and mixed fluid of herb monomers at the concentration of degree 3 ( including Ber1umol/L、Tim1umol/L、Pae 1g/L、Ica 1ng/ml) which seems increased but can’t make sure. Therefore, we choose the herbage containing serum and the herb monomer and mixed ones to culture the target cells.Conclusions GNC Decoction might exert a potential neuroprotective effect. The serum with GNC Decoction was found to be stronger than the main monomers.Part II PC12 Cells injury induced by AP25-35Objective To establish the AD model in vitro by Ap25-35-induced PC12 cells injury.Methods PC12 cells were incubated by different concentration of Aβ25-35 for 24 hours, 48 hours, respectively, and the cell livability was determined by MTT assay. Meanwhile, cellular morphological change was observed by phase contrast microscope.Results After PC12 cells were treated by different concentrations of 5,10,20,40μmol/ L of Aβ25-35 for 24 hours and 48 hours, the cell viability decreased, and the injury cells were more in number and nuclei contrasted, which shows obviously dose and time-dependent. Therefore, the AD model in vitro has been built successfully.Conclusions A|325-35 can inhibit the viability and promote its apoptosis of PC12 cells in a dose and time-dependent manner, and the PC12 cell injury may be as AD model.Part III Protective effects of Gengnianchun Decoction on PC12 cells injury induced by Aβ25-35 and its signal transductionObjective To investigate whether GNC can ameliorate the apoptosis of neuron and what are the effective ingredients. To elucidate which signaling transduction pathway mediates its anti-apoptosis action.Methods The Ap25-35-induced PC12 cell injury were cultured with GNC-containing serum and its monomers such as Paeoniflorin(Pae), Timosaponin A-III(Tim), Berberine Hydrochloride (Ber) and Icariine (Ica) that are the main substances in GNC decoction. The early apoptosis was determined by AnnexinV-FITC / PI flow cytometry. The protein expression of caspase-3, bax, bcl-2, p-ERK1/2 and ER subtypes were measured by western blotting.Results It has been found by MTT assay that the GNC-containing serum protects PC12 cells from the Aβ25-35-induced injury that is similar with NGF and that the monomers from GNC also protects the cells from injury (P<0.05), which suggests that GNC-containing serum, Ber, and herb monomer mixture all improve the cell viability of PC12 cells.It has been found by FCM that the GNC-containing serum and monomers of GNC inhibits the cell apoptosis induced by Aβtreatment, which suggests that the GNC-containing serum, Ber, herb monomer mixture all protects from the Aβ-induced apoptosis of PC12 cells.From the result of Western-blotting we can find there is much more expression of bcl-2 than bax in GNC-containing serum treatment compared to the Aβ-induced P12 cell injury. The expression of caspase-3 is decreased significantly in the treatment with the GNC-containing serum in comparison with the Aβ-induced P12 cell injury, which suggests that the GNC-containing serum protects P12 cell from the apoptosis induced by Aβthat is superior to the monomers of GNC.Also it can be found from the result of Western-blotting that there is no ERαband expressed in PC 12 cells. However, the GNC-containing serum increases significantly expression of ERβin the injured P12 cells induced by Aβ, suggesting the herb can elevate the expression of ERβin PC 12 cell.Western-blotting also shows that the expression of p-ERK1 / 2 is increased in the injury cells treated with GNC-containing serum, suggesting the herb can increase the expression of p-ERK1 / 2 in the Aβ-induced injured P12 cells.Conclusions The serum containing GNC, and its monomers can inhibit P12 cell apoptosis induced by Aβand protect cell from damage, which may be via ERβand MAPK pathway. Part IV Protective effects of Gengnianchun Decoction on Aβ-induced PC12 cells injury by ERβand its signal transductionObjective To probe into which signaling transduction pathway is involved in GNC’s anti-apoptosis.Methods The early apoptosis was determined by AnnexinV-FITC / PI flow cytometry. The protein expression of caspase-3, bax, bcl-2, p-ERK1/2 and ER subtypes were measured by western blotting. The protein expression was measured again after pre-treating respectively by ER antagonist ICI182, 780 and blocker of MAPK.Results Compared with the GNC+Aβ, MAPK blocker, ER antagonist abolishes p-ERK increase and the PC12 cell early stage apoptosis protection induced by GNC and its monomer mixture (P<0.01), and inhibits bcl-2, ERβexpression, and enhances bax, caspase3 expression in the cells, which suggests that ERβand MARK pathway is involved in the protection of GNC from P12 cell apoptosis.Conclusions Up-regulation of ER and MAPK pathway and down-regulation of apoptosis signal pathway are involved in the protection of GNC from PC12 cell injury induced by Aβ. The up-regulation MAPK pathway by GNC may be via ERβ-mediated.更多还原