基于“肝郁气滞—内质网应激关联”新假说新靶点的老年性痴呆中药干预研究
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摘要
目的
1.证明中药复方多靶点、多环节、多层次的整体性调节方式的作用机理及其化学机制，为进一步研究中药复方治疗阿尔茨海默病.奠定基础。
2.揭示Aβ1-40诱导的AD细胞模型和病证结合动物病理模型在ERS和UPP功能障碍上的分子病理基础和特点。
3.阐明疏肝解郁法对对AD的细胞和动物病理模型ERS及ERAD(UPP)系统的多靶点的调节作用。
4.观察疏肝解郁法对神经细胞ERS模型、AD细胞模型和AD动物模型内质网应激及其内质网相关蛋白降解通路的影响，从基因分子水平探讨其药效学机理。
5.探讨“肝郁气滞-内质网应激关联”假说的科学内涵，为重大疑难疾病老年性痴呆的中医药防治提供新的靶点、开辟新思路。
方法
本课题通过以下几个实验进行研究：
1.实验通过利用SELDI(蛋白芯片质谱仪)对柴胡疏肝散含药血清和安理申含药血清进行检测，使柴胡疏肝散含药血清、安理申含药血清和空白组含药血清之间飞行时间质谱柱状图之间对比。
2.采用高效液相色谱(HPLC)对该复方含药血清、水煎剂和含药血清对NG108-15神经元细胞株干预后进行了细胞内药物成分检测研究。
3.实验用经老化处理的Aβ1-40对NG108-15神经细胞进行诱导，使其成为AD细胞模型，并把未诱导和诱导后的细胞分成6个组，分别用正常兔血清、安理申兔含药血清、高中低浓度柴胡疏肝散含药血清进行干预，干预结束后进行荧光染色和流式细胞仪细胞凋亡测试，另外还对不同组细胞进行ERS、UPP标志性分子的测试。
4.实验用衣霉素（TM）对NG108-15神经细胞进行诱导，使其成为ERS细胞模型，并把未诱导和诱导后的细胞分成6个组，分别用正常兔血清、安理申兔含药血清、高中低浓度柴胡疏肝散含药血清进行干预，干预结束后进行荧光染色和流式细胞仪细胞凋亡测试，另外还对不同组细胞进行ERS、UPP标志性分子的测试。
5.取经Open-field-test试验筛选大鼠，并经Morris水迷宫游泳迅速灵活的大鼠，随机分为6组，即正常对照组、假手术组对照组、模型对照组、西药对照组、柴胡疏肝散高低剂量组，每组l5只。先束缚法建立实验大鼠肝郁模型，接着在建立好的肝郁模型上往实验大鼠的海马注射Aβ1-40创建肝郁型AD病证结合大鼠模型。观测实验各组行为学指标的变化。
结果
1.柴胡疏肝散含药血清有7个特有蛋白表达峰，分别是M2440、M2455、M2602、M5781、M11544、M14285、M14983；安理申组特有的峰是14个，分别是M1562、M2174、M2201、M2216、M2360、M2374、M2681、M2695、M2710、M2909、M3211、M3938、M14063、M15604。
2.柴胡疏肝散各单味中药的成分和柴胡疏肝散水煎剂的成分相比存在着差异；实验利用高效液相色谱法进行分析后，发现在血清和NG108-15细胞中发现很多柴胡疏肝散水煎剂原型成分和移形成分。
3.实验研究中通过MTT实验证明经Aβ1-40对NG108-15神经细胞干预造模后，观察各组NG108-15细胞的活性，再加入柴胡疏肝散含药血清高中低剂量后，细胞的活性明显好于模型组细胞的活性；西药安理申组含药血清和高剂量组柴胡疏肝散含药血清对造模后的细胞的活性都好于其他各组，且两者之间无明显差异。流式细胞仪检测各组细胞的凋亡率，结果证明西药安理申组含药血清和高中低剂量组柴胡疏肝散含药血清对造模后的细胞的活性都明显好于模型组；高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。联合荧光染色也证明高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。DNA琼脂糖电泳以及本实验组既往的研究，观察到柴胡疏肝散含药血清对模型细胞干预处理后，GRP78和CHOP基因显著表达下调，而泛素(ubiquitin，Ub)基因显著表达上调。
4.实验研究中通过MTT实验证明经衣霉素（TM）对NG108-15神经细胞干预造模后，观察各组NG108-15细胞的活性，再加入柴胡疏肝散含药血清高中低剂量后，细胞的活性明显好于模型组细胞的活性；安理申组含药血清和高剂量组柴胡疏肝散含药血清对造模后的细胞的活性都好于其他各组，且两者之间无明显差异。利用流式细胞仪检测各组细胞的凋亡率，结果证明安理申组含药血清和高中低剂量组柴胡疏肝散含药血清对造模后的细胞的活性都明显好于模型组；高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。联合荧光染色也证明高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。DNA琼脂糖电泳以及本实验组既往的研究，观察到柴胡疏肝散含药血清对模型细胞干预处理后，GRP78和CHOP基因显著表达下调，而泛素(ubiquitin，Ub)基因显著表达上调。
5.实验研究结果显示，肝郁造模前、肝郁造模结束时以及老年性痴呆病证结合模型干预治疗使实验各组大鼠的体重发生了变化，存在着差异。模型组大鼠体重和正常组大鼠体重相比，体重在各个阶段都低于正常组，有显著差异（P<0.01）；西药组、中药高剂量组、中药低剂量组和模型组大鼠体重相比，体重在各个阶段都高于模型组，有显著差异（P<0.01）；模型组和假手术组相比，大鼠体重明显低于假手术组，有显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，大鼠体重明显高于中药低剂量组，有显著差异（P<0.01）；西药组和中药高剂量组相比，大鼠体重无明显差异（P>0.05）；肝郁造模前、肝郁造模结束后实验各组大鼠糖水偏好度发生了变化，存在着差异。正常组和模型组糖水偏好度相比，有显著差异（P<0.01）；西药组、中药高剂量组、中药低剂量组和模型组大鼠糖水偏好度相比，有显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，大鼠糖水偏好度相比高于中药低剂量组，有显著差异（P<0.01）；西药组和中药高剂量组相比，大鼠糖水偏好度相比存在差异（P<0.05）；实验各组大鼠在逃避潜伏期相对比中，西药对照组、中药高剂量组、中药低剂量组和模型组相比，存在显著差异（P<0.01）；正常组和模型组相比，存在显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，存在显著差异（P<0.01）；西药组和中药高剂量组相比，未存在差异（P>0.05）；实验各组大鼠120S内找到原平台的记忆相对比中，包括初次找到原平台的时间和穿越原平台的次数，西药对照组、中药高剂量组、中药低剂量组和模型组相比，存在显著差异（P<0.01）；正常组和模型组相比，存在显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，存在显著差异（P<0.01）；西药组和中药高剂量组相比，未存在差异（P>0.05）；Open-field-test水平活动得分，正常组、西药对照组与模型组相比较P<0.01；中药高剂量组和中药低剂量组相比，P<0.01；西药组和中药高剂量组相比，P>0.05；Open-field-test垂直活动得分，正常组、西药对照组与模型组相比较P<0.01；中药高剂量组和中药低剂量组相比，P<0.01；西药组和中药高剂量组相比，P>0.05；柴胡疏肝散水煎剂可以使单胺类脑内神经递质NE、DA和5-HT含量增高。
结论
1.通过以上研究证明柴胡疏肝散含药血清和安理申含药血清飞行质谱图存在着显著差异,说明两者之间对机体的药理作用机理存在差异。
2.柴胡疏肝散含药血清进入NG108-15细胞内部的药物有效成分有多种，符合霰弹靶点理论多种有效成分作用于目标，而且作用的靶点有多个，为进一步探索阿尔茨海默病治疗新靶点提供可能。
3.Aβ1-40成功诱导NG108-15神经细胞成AD细胞模型；衣霉素成功诱导NG108-15神经细胞成ERS细胞模型。
4.柴胡疏肝散含药血清具有改善细胞ERS，激活UPP功能的作用。
5.海马注射Aβ1-40结合束缚法可成功复制病证结合的肝郁型AD动物模型；
6.柴胡疏肝散可明显改善肝郁型AD大鼠模型抑郁症状，增强其学习记忆功能。
7.研究结果表明本理论的实验机理可能与疏肝解郁法可以明显改善内质网应激、增强泛素系统蛋白降解活性有关，其作用涉及多个基因分子靶点。
objective
1．To verify the mechanism of Chinese herbal compounds on themulti-target, multi-link and multi-level holistic regulationpattern，and the difference by comparing with the mechanism onthe single target under western medicine, Aricept(DonepezilHydrochloride Tablets), which is based on the thinking ofproteomics and through the experiments in order to establisha fundamental method for the further research on the effectmechanism of Chinese herbal compounds.
2． To explore the potential chemical mechanism of themulti-target effect on Traditional Chinese Medicine, and tofind new targets for treating Alzheimer's disease.
3． Cellular model of AD and disease-syndrome animalpathological model with molecular pathology were induced by Aβ1-40,and studied based on basic features of the ERS with UPPdysfunction.
4． To study the liver-qi stagnation remising endoplasmicreticulum stress, and the regulatory role of multiple targetson AD cells and pathological animals’ model of ERS and ERAD (UPP) system.
5．To conduct intervention study of endoplasmic reticulumstress from liver-qi stagnation, and to verify the hypothesison “Stagnation of Live-qi associated with the endoplasmicreticulum stress”.
6．To observe liver-qi stagnation model and AD animal modelsof nerve cells ERS model with AD cells endoplasmic reticulumstress endoplasmic reticulum-associated protein degradationpathway, to explore the pharmacodynamic mechanism undermolecular level.
Methods
This thesis includes the following experimental methods:
1．The protein chip mass spectrometer(SELDI)was used to detectthe medicated serum of CHSGS and Aricept.The flight time massspectrum histogram was adopted to make the comparison ofmedicated serum among Chaihu shugan san(CHSGS),Aricept and theblank groups and detect the different proteins so as to verifythe differences in the mechanisms between chinese herbalcompound and western medicine.
2．To do Cells drug ingredients detection research of compoundin containing serum, water decoction and the NG108-15nervecell strain after intervention of containing serum by HighPerformance Liquid Chromatography (HPLC). HPLC was performedto analysis the targets.
3．Experiments by the aging process Aβ1-40on NG108-15nerve cellswere induced, making it a cellular model of AD, and uninducedand induced cells were divided into six groups, respectively,using normal rabbit serum, the Aricept rabbit containing lowconcentrations of serum high school CHSGS intervention serum containing fluorescent staining and flow cytometry apoptosistest after the end of the intervention, in addition to differentgroups of cells ERS, UPP iconic molecular test.
4．The experiment used tunicamycin(TM) was induced in NG108-15cells, the cells become ERS cell model, and the uninduced andinduced cells were divided into six groups. Respectively withnormal rabbit serum, Aricept rabbit serum containing highconcentration, CHSGS containing serum intervention. After theintervention, fluorescence staining and flow cytometryapoptosis test. In addition to the different groups of cellswere ERS with UPP markers testing.
5．For Open-field-test screening test of rats, and the rapidand flexible Morris water maze swimming rats were randomlydivided into six groups, and each group of has fifteen rats,also including the normal control group, sham operation group,model group, western medicine group, CHSGS high dose group.
Results
1.The medicated serum of CHSGS contained seven specific proteinexpression peaks (M2440、M2445、M2602、M5781、M11544、M14285、M14983)； The medicated serum of Donepezil Hydrochloridecontained fourteen specific peaks (M1562、M2174、M2201、M2216、M2360、M2374、M2681、M2695、M2710、M2909、M3211、M3938、M14063、M15604).
2.A variety of effective components were found from CHSGS inhuman serum and NG108-15cells by HPLC，which is in accordancewith the previous hypothesis.
3.The experiments showed that the cell conditions treated byCHSGS on Aβ1-40induced neural cells are better than the sameneural cells but without treated, which was also found in Donepezil Hydrochloride treated neural cells; The apoptosisanalysis by flow cytometry experiments showed that DonepezilHydrochloride and CHSGS treated neural cells are better thancontrols; It also showed that high dose CHSGS treated neuralcells are better than low dose. The experiments by agar gelelectrophoresis showed that GRP78and CHOP gene up regulatedwhile Ub gene down regulated in CHSGS treated neural cells.
4.The MTT experiments showed that CHSGS can improve thetunicamycin (TM) treated NG108-15cells conditions. It alsoshowed that Donepezil Hydrochloride and high dose CHSGS treatedneural cells conditions are better than other controls. Theexperiments by agar gel electrophoresis showed that GRP78andCHOP gene up regulated while Ub gene down regulated in CHSGStreated neural cells.
5． The established model made the rats losing weight incomparison with normal rats, which showed obvious difference(P<0.01). The following medicine feeding experiments showedboth modern medicine and traditional medicine can improve theweight of rats than normal, which showed obvious difference(P<0.01), no difference (P>0.05) between DonepezilHydrochloride and high dose CHSGS. The same phenomenon alsofound in the sugar feeding experiments and memory testing (suchas escapes latency, spatial probe, place navigation,open-field-test and et. al.) experiments. The experiments alsoshowed that CHSGS can increase the expression of NE, DA and5-HTin nerval cells.
Conclusions
1.The study demonstrate that CHSGS containing serum and Ariceptmedicated serum flight spectrum-MS have significant difference,so that differences exist on the pharmacological actionmechanism between two models.
2.The study demonstrate that there are a variety of effectivecompositions of herbs Bupleurum Liver-Coursing powdercontaining serum into NG108-15cells, which is also inconsistent with shotgun target theory of various effectivecomponents acting on the one or multi-targets. The result alsoprovides opportunity for further exploration of newtherapeutic targets in Alzheimer's disease.
3.The study demonstrate that Aβ1-40can induce NG108-15neuralcells to establish AD model; The study also demonstrate thatTM can induce NG108-15neural cells to establish ERS model.
4.The study demonstrate that CHSGS contained serum can improvethe condition of ERS, and activate the function of UPP.
5. The study demonstrates that the hippocampal injection ofAβ1-40can successfully verify the established AD model.
6. The study demonstrates that CHSGS can significantly improvethe condition of AD disease rats and improve their memorialability.
7. The study demonstrates that the mechanism of the theory inthis experiment is that CHSGS can significantly improve thecondition of ERS and increase the expression of Ub enzyme. Thestudy also demonstrates that more than one gene has involvedin this molecular activity.
1.证明中药复方多靶点、多环节、多层次的整体性调节方式的作用机理及其化学机制，为进一步研究中药复方治疗阿尔茨海默病.奠定基础。
2.揭示Aβ1-40诱导的AD细胞模型和病证结合动物病理模型在ERS和UPP功能障碍上的分子病理基础和特点。
3.阐明疏肝解郁法对对AD的细胞和动物病理模型ERS及ERAD(UPP)系统的多靶点的调节作用。
4.观察疏肝解郁法对神经细胞ERS模型、AD细胞模型和AD动物模型内质网应激及其内质网相关蛋白降解通路的影响，从基因分子水平探讨其药效学机理。
5.探讨“肝郁气滞-内质网应激关联”假说的科学内涵，为重大疑难疾病老年性痴呆的中医药防治提供新的靶点、开辟新思路。
方法
本课题通过以下几个实验进行研究：
1.实验通过利用SELDI(蛋白芯片质谱仪)对柴胡疏肝散含药血清和安理申含药血清进行检测，使柴胡疏肝散含药血清、安理申含药血清和空白组含药血清之间飞行时间质谱柱状图之间对比。
2.采用高效液相色谱(HPLC)对该复方含药血清、水煎剂和含药血清对NG108-15神经元细胞株干预后进行了细胞内药物成分检测研究。
3.实验用经老化处理的Aβ1-40对NG108-15神经细胞进行诱导，使其成为AD细胞模型，并把未诱导和诱导后的细胞分成6个组，分别用正常兔血清、安理申兔含药血清、高中低浓度柴胡疏肝散含药血清进行干预，干预结束后进行荧光染色和流式细胞仪细胞凋亡测试，另外还对不同组细胞进行ERS、UPP标志性分子的测试。
4.实验用衣霉素（TM）对NG108-15神经细胞进行诱导，使其成为ERS细胞模型，并把未诱导和诱导后的细胞分成6个组，分别用正常兔血清、安理申兔含药血清、高中低浓度柴胡疏肝散含药血清进行干预，干预结束后进行荧光染色和流式细胞仪细胞凋亡测试，另外还对不同组细胞进行ERS、UPP标志性分子的测试。
5.取经Open-field-test试验筛选大鼠，并经Morris水迷宫游泳迅速灵活的大鼠，随机分为6组，即正常对照组、假手术组对照组、模型对照组、西药对照组、柴胡疏肝散高低剂量组，每组l5只。先束缚法建立实验大鼠肝郁模型，接着在建立好的肝郁模型上往实验大鼠的海马注射Aβ1-40创建肝郁型AD病证结合大鼠模型。观测实验各组行为学指标的变化。
结果
1.柴胡疏肝散含药血清有7个特有蛋白表达峰，分别是M2440、M2455、M2602、M5781、M11544、M14285、M14983；安理申组特有的峰是14个，分别是M1562、M2174、M2201、M2216、M2360、M2374、M2681、M2695、M2710、M2909、M3211、M3938、M14063、M15604。
2.柴胡疏肝散各单味中药的成分和柴胡疏肝散水煎剂的成分相比存在着差异；实验利用高效液相色谱法进行分析后，发现在血清和NG108-15细胞中发现很多柴胡疏肝散水煎剂原型成分和移形成分。
3.实验研究中通过MTT实验证明经Aβ1-40对NG108-15神经细胞干预造模后，观察各组NG108-15细胞的活性，再加入柴胡疏肝散含药血清高中低剂量后，细胞的活性明显好于模型组细胞的活性；西药安理申组含药血清和高剂量组柴胡疏肝散含药血清对造模后的细胞的活性都好于其他各组，且两者之间无明显差异。流式细胞仪检测各组细胞的凋亡率，结果证明西药安理申组含药血清和高中低剂量组柴胡疏肝散含药血清对造模后的细胞的活性都明显好于模型组；高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。联合荧光染色也证明高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。DNA琼脂糖电泳以及本实验组既往的研究，观察到柴胡疏肝散含药血清对模型细胞干预处理后，GRP78和CHOP基因显著表达下调，而泛素(ubiquitin，Ub)基因显著表达上调。
4.实验研究中通过MTT实验证明经衣霉素（TM）对NG108-15神经细胞干预造模后，观察各组NG108-15细胞的活性，再加入柴胡疏肝散含药血清高中低剂量后，细胞的活性明显好于模型组细胞的活性；安理申组含药血清和高剂量组柴胡疏肝散含药血清对造模后的细胞的活性都好于其他各组，且两者之间无明显差异。利用流式细胞仪检测各组细胞的凋亡率，结果证明安理申组含药血清和高中低剂量组柴胡疏肝散含药血清对造模后的细胞的活性都明显好于模型组；高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。联合荧光染色也证明高剂量组柴胡疏肝散含药血清干预保护细胞后，细胞凋亡率明显好于中低剂量柴胡疏肝散含药血清干预保护的细胞。DNA琼脂糖电泳以及本实验组既往的研究，观察到柴胡疏肝散含药血清对模型细胞干预处理后，GRP78和CHOP基因显著表达下调，而泛素(ubiquitin，Ub)基因显著表达上调。
5.实验研究结果显示，肝郁造模前、肝郁造模结束时以及老年性痴呆病证结合模型干预治疗使实验各组大鼠的体重发生了变化，存在着差异。模型组大鼠体重和正常组大鼠体重相比，体重在各个阶段都低于正常组，有显著差异（P<0.01）；西药组、中药高剂量组、中药低剂量组和模型组大鼠体重相比，体重在各个阶段都高于模型组，有显著差异（P<0.01）；模型组和假手术组相比，大鼠体重明显低于假手术组，有显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，大鼠体重明显高于中药低剂量组，有显著差异（P<0.01）；西药组和中药高剂量组相比，大鼠体重无明显差异（P>0.05）；肝郁造模前、肝郁造模结束后实验各组大鼠糖水偏好度发生了变化，存在着差异。正常组和模型组糖水偏好度相比，有显著差异（P<0.01）；西药组、中药高剂量组、中药低剂量组和模型组大鼠糖水偏好度相比，有显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，大鼠糖水偏好度相比高于中药低剂量组，有显著差异（P<0.01）；西药组和中药高剂量组相比，大鼠糖水偏好度相比存在差异（P<0.05）；实验各组大鼠在逃避潜伏期相对比中，西药对照组、中药高剂量组、中药低剂量组和模型组相比，存在显著差异（P<0.01）；正常组和模型组相比，存在显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，存在显著差异（P<0.01）；西药组和中药高剂量组相比，未存在差异（P>0.05）；实验各组大鼠120S内找到原平台的记忆相对比中，包括初次找到原平台的时间和穿越原平台的次数，西药对照组、中药高剂量组、中药低剂量组和模型组相比，存在显著差异（P<0.01）；正常组和模型组相比，存在显著差异（P<0.01）；中药高剂量组和中药低剂量组相比，存在显著差异（P<0.01）；西药组和中药高剂量组相比，未存在差异（P>0.05）；Open-field-test水平活动得分，正常组、西药对照组与模型组相比较P<0.01；中药高剂量组和中药低剂量组相比，P<0.01；西药组和中药高剂量组相比，P>0.05；Open-field-test垂直活动得分，正常组、西药对照组与模型组相比较P<0.01；中药高剂量组和中药低剂量组相比，P<0.01；西药组和中药高剂量组相比，P>0.05；柴胡疏肝散水煎剂可以使单胺类脑内神经递质NE、DA和5-HT含量增高。
结论
1.通过以上研究证明柴胡疏肝散含药血清和安理申含药血清飞行质谱图存在着显著差异,说明两者之间对机体的药理作用机理存在差异。
2.柴胡疏肝散含药血清进入NG108-15细胞内部的药物有效成分有多种，符合霰弹靶点理论多种有效成分作用于目标，而且作用的靶点有多个，为进一步探索阿尔茨海默病治疗新靶点提供可能。
3.Aβ1-40成功诱导NG108-15神经细胞成AD细胞模型；衣霉素成功诱导NG108-15神经细胞成ERS细胞模型。
4.柴胡疏肝散含药血清具有改善细胞ERS，激活UPP功能的作用。
5.海马注射Aβ1-40结合束缚法可成功复制病证结合的肝郁型AD动物模型；
6.柴胡疏肝散可明显改善肝郁型AD大鼠模型抑郁症状，增强其学习记忆功能。
7.研究结果表明本理论的实验机理可能与疏肝解郁法可以明显改善内质网应激、增强泛素系统蛋白降解活性有关，其作用涉及多个基因分子靶点。
objective
1．To verify the mechanism of Chinese herbal compounds on themulti-target, multi-link and multi-level holistic regulationpattern，and the difference by comparing with the mechanism onthe single target under western medicine, Aricept(DonepezilHydrochloride Tablets), which is based on the thinking ofproteomics and through the experiments in order to establisha fundamental method for the further research on the effectmechanism of Chinese herbal compounds.
2． To explore the potential chemical mechanism of themulti-target effect on Traditional Chinese Medicine, and tofind new targets for treating Alzheimer's disease.
3． Cellular model of AD and disease-syndrome animalpathological model with molecular pathology were induced by Aβ1-40,and studied based on basic features of the ERS with UPPdysfunction.
4． To study the liver-qi stagnation remising endoplasmicreticulum stress, and the regulatory role of multiple targetson AD cells and pathological animals’ model of ERS and ERAD (UPP) system.
5．To conduct intervention study of endoplasmic reticulumstress from liver-qi stagnation, and to verify the hypothesison “Stagnation of Live-qi associated with the endoplasmicreticulum stress”.
6．To observe liver-qi stagnation model and AD animal modelsof nerve cells ERS model with AD cells endoplasmic reticulumstress endoplasmic reticulum-associated protein degradationpathway, to explore the pharmacodynamic mechanism undermolecular level.
Methods
This thesis includes the following experimental methods:
1．The protein chip mass spectrometer(SELDI)was used to detectthe medicated serum of CHSGS and Aricept.The flight time massspectrum histogram was adopted to make the comparison ofmedicated serum among Chaihu shugan san(CHSGS),Aricept and theblank groups and detect the different proteins so as to verifythe differences in the mechanisms between chinese herbalcompound and western medicine.
2．To do Cells drug ingredients detection research of compoundin containing serum, water decoction and the NG108-15nervecell strain after intervention of containing serum by HighPerformance Liquid Chromatography (HPLC). HPLC was performedto analysis the targets.
3．Experiments by the aging process Aβ1-40on NG108-15nerve cellswere induced, making it a cellular model of AD, and uninducedand induced cells were divided into six groups, respectively,using normal rabbit serum, the Aricept rabbit containing lowconcentrations of serum high school CHSGS intervention serum containing fluorescent staining and flow cytometry apoptosistest after the end of the intervention, in addition to differentgroups of cells ERS, UPP iconic molecular test.
4．The experiment used tunicamycin(TM) was induced in NG108-15cells, the cells become ERS cell model, and the uninduced andinduced cells were divided into six groups. Respectively withnormal rabbit serum, Aricept rabbit serum containing highconcentration, CHSGS containing serum intervention. After theintervention, fluorescence staining and flow cytometryapoptosis test. In addition to the different groups of cellswere ERS with UPP markers testing.
5．For Open-field-test screening test of rats, and the rapidand flexible Morris water maze swimming rats were randomlydivided into six groups, and each group of has fifteen rats,also including the normal control group, sham operation group,model group, western medicine group, CHSGS high dose group.
Results
1.The medicated serum of CHSGS contained seven specific proteinexpression peaks (M2440、M2445、M2602、M5781、M11544、M14285、M14983)； The medicated serum of Donepezil Hydrochloridecontained fourteen specific peaks (M1562、M2174、M2201、M2216、M2360、M2374、M2681、M2695、M2710、M2909、M3211、M3938、M14063、M15604).
2.A variety of effective components were found from CHSGS inhuman serum and NG108-15cells by HPLC，which is in accordancewith the previous hypothesis.
3.The experiments showed that the cell conditions treated byCHSGS on Aβ1-40induced neural cells are better than the sameneural cells but without treated, which was also found in Donepezil Hydrochloride treated neural cells; The apoptosisanalysis by flow cytometry experiments showed that DonepezilHydrochloride and CHSGS treated neural cells are better thancontrols; It also showed that high dose CHSGS treated neuralcells are better than low dose. The experiments by agar gelelectrophoresis showed that GRP78and CHOP gene up regulatedwhile Ub gene down regulated in CHSGS treated neural cells.
4.The MTT experiments showed that CHSGS can improve thetunicamycin (TM) treated NG108-15cells conditions. It alsoshowed that Donepezil Hydrochloride and high dose CHSGS treatedneural cells conditions are better than other controls. Theexperiments by agar gel electrophoresis showed that GRP78andCHOP gene up regulated while Ub gene down regulated in CHSGStreated neural cells.
5． The established model made the rats losing weight incomparison with normal rats, which showed obvious difference(P<0.01). The following medicine feeding experiments showedboth modern medicine and traditional medicine can improve theweight of rats than normal, which showed obvious difference(P<0.01), no difference (P>0.05) between DonepezilHydrochloride and high dose CHSGS. The same phenomenon alsofound in the sugar feeding experiments and memory testing (suchas escapes latency, spatial probe, place navigation,open-field-test and et. al.) experiments. The experiments alsoshowed that CHSGS can increase the expression of NE, DA and5-HTin nerval cells.
Conclusions
1.The study demonstrate that CHSGS containing serum and Ariceptmedicated serum flight spectrum-MS have significant difference,so that differences exist on the pharmacological actionmechanism between two models.
2.The study demonstrate that there are a variety of effectivecompositions of herbs Bupleurum Liver-Coursing powdercontaining serum into NG108-15cells, which is also inconsistent with shotgun target theory of various effectivecomponents acting on the one or multi-targets. The result alsoprovides opportunity for further exploration of newtherapeutic targets in Alzheimer's disease.
3.The study demonstrate that Aβ1-40can induce NG108-15neuralcells to establish AD model; The study also demonstrate thatTM can induce NG108-15neural cells to establish ERS model.
4.The study demonstrate that CHSGS contained serum can improvethe condition of ERS, and activate the function of UPP.
5. The study demonstrates that the hippocampal injection ofAβ1-40can successfully verify the established AD model.
6. The study demonstrates that CHSGS can significantly improvethe condition of AD disease rats and improve their memorialability.
7. The study demonstrates that the mechanism of the theory inthis experiment is that CHSGS can significantly improve thecondition of ERS and increase the expression of Ub enzyme. Thestudy also demonstrates that more than one gene has involvedin this molecular activity.
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