Intervention Study Of Traditional Chinese Medicine On Alzheimer’s Disease Based On Novel Hypothesis "Stagnation Of Live-Qi Associated With The Endoplasmic Reticulum Stress"

objective1．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.MethodsThis 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.Results1.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. Conclusions1.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.