| As a representative neurodegenerative disorder,the Alzheimer's Disease(AD)is frequently seen in senior populations.There are large amounts of AD patients worldwide as well as in China.AD is rapidly prevailing,with its medical treatment demanding a tremendous expenditure.It has therefore become a huge burden for modern society and economics.Nevertheless,current therapies can only,to some extent,attenuate the relevant symptoms instead of suspending the pathological development through dealing with irreversible neurodegeneration.By contrast,Traditional Chinese Medicine(TCM)is pharmacologically featured as acting through multiple targets and pathways.It has demonstrated an advantageous and unique role in AD therapy.To study the pathophysiology of AD and develop efficient therapies is not only of critical value for medical science but also of clinical significance.This dissertation is generally composed of 3 parts including literature review,clinical investigation,and experimental research.Part 1:literature reviewBased on those literatures relevant in the field of TCM and modern medicine,we characterize AD from the aspects of its epidemiology,pathophysiology,conventional therapies,and TCM philosophy on AD,respectively.Part 2:clinical investigationPurpose:In order to identify the merits of formula Di Huang Yin Zi in treating mild AD with sound evidences,we seek to evaluate its efficacy in comparison with that of Aricept(Donepezil Hydrochloride).Both TCM and modern medicine rating scales are designed.Methods:50 patients with mild AD were included and randomly distributed into TCM cohort and Aricept cohort,respectively.Patients in TCM cohort were administrated with modified formula Di Huang Yin Zi and provided with health education,while patients in Aricept cohort were administrated with Aricept and provided with health education.The therapies lasted for 3 months.Ratings were carried out before and after therapies,respectively.The therapeutic efficacy of formula Di Huang Yin Zi on mild AD was assessed.Results:1.On the therapeutic efficacy of TCM symptoms:According to the TCM rating scale for AD,the total scores,subscores for primary and secondary symptoms are consistently reduced in TCM cohort in comparison with those in Aricept cohort.The reductions are significant(P<0.01).Among primary symptoms,subscores for amnesia and skilled maneuver are comparatively less and the reductions are also significant(P<0.01).Among secondary symptoms,subscores for sour waist and knees,cold limbs,fatigue and sleepiness,vertigo and tinnitus,fecal incontinence are all comparatively less and the reductions are statistically significant(P<0.05).2.MMSE The MMSE total score,subscores for calculating skill,memory,and language are all increased in TCM cohort in comparison with those in Aricept cohort.These increases are considerably significant(P<0.01).3.MoCA The MoCA total score,subscores for visuospatial and executive functions,attention,language,and delayed memory are all increased in TCM cohort in comparison with those in Aricept cohort.These increases are considerably significant(P<0.01).The subscore for abstract thinking is significantly increased as well(P<0.05).4.ADL In terms of ADL score,no significant difference is determined between TCM and Aricept cohorts(P>0.05).Conclusions:1.The TCM rating scale for AD can be used to assess the severity of TCM symptoms in AD patients.A higher score according to it should be associated with more TCM symptoms or indicate that TCM symptoms are more severe in patients.The formula Di Huang Yin Zi can significantly improve those TCM symptoms in patients with mild AD.In particular,the improvements turn out to be prominent regarding amnesia,skilled maneuver,sour waist and knees,cold limbs,fatigue and sleepiness,vertigo and tinnitus,and fecal incontinence.2.The formula Di Huang Yin Zi can significantly improve advanced intelligence of patients.According to MMSE,the improvements are particularly prominent in calculating skill,memory,and language,respectively.According to MoCA,significant improvements by this formula are seen in items including visuospatial and executive functions,attention,language,abstract thought,and delayed memory.Part 3:experimental researchExperiment 1:The improvement of learning and memory ability in AD rat models by formula Di Huang Yin ZiPurpose:We observe behavioral metrics in rats and seek to figure out the impact of formula Di Huang YIn Zi on learning and memory ability in AD rats.Methods:120 male rats were randomly distributed into sham group,AD group,Aricept group,high-dose TCM group,moderate-dose TCM group,and low-dose TCM group.Animals in all groups except the sham group were injected with A?1-42 via lateral ventricles to establish AD model.Rats in Aricept and TCM groups were administrated with relevant agents via intragastric gavage,while those in sham and AD groups were administrated with isovolumetric saline.The administrations were delivered once per day and were consecutively carried out for 30 days.The behaviors of rats were characterized in the context of Morris water maze test,step-down test,step-through test,and Y-maze test,respectively.Results:1.Morris water maze test During the place navigation,3-day administration with the formula Di Huang Yin Zi can significantly shorten the escape latency and mean distance of AD rats in comparison with those AD rats without any treatment(P<0.01).During the spatial probe,3-day administration with the formula Di Huang Yin Zi can significantly upregulate the counts of AD rats traversing the platform and the time of their spending in the target quadrant(P<0.01).The time of their staying in the opposite quadrant was correspondingly reduced(?<0.01).2.Step-down test In comparison with those AD rats without any treatment,the latency to step down in AD rats were significantly increased by the formula Di Huang Yin Zi at high,moderate,and low doses(P<0.01).The counts of error were correspondingly decreased(P<0.05).3.Step-through test In comparison with those AD rats without any treatment,the latency in AD rats was significantly increased by the formula Di Huang Yin Zi(P<0.01).The counts of error were correspondingly decreased(P<0.01).4.Y-maze test In comparison with those AD rats without any treatment,the spontaneous alternation in AD rats was significantly increased by the formula Di Huang YIn Zi(P<0.01).Conclusions:1.The outcomes from Morris water maze test indicate that formula Di Huang Yin Zi can significantly improve the spatial leanrning and memory ability of AD rats.2.The formula Di Huang Yin Zi can significantly increase the latency of AD rats during step-down and step-through tests.It can also reduce the count of errors.It indicates that this formula should function in improving memory.3.According to Y-maze test,the formula Di Huang Yin Zi can significantly improve working memory of AD rats.Experiment 2:The impact of formula Di Huang Yin Zi on energetic metabolism,mitochondrial structure and function in vivo and in vitroPurpose:The following metrics are detected in rat brain tissues of all groups,including mitochondrial swelling,mitochondrial membrane potential,activities of respiratory complex I,?,?,and ?,amounts of ATP,ADP,AMP,EC,and PCr,activities of key enzymes involved in tricarboxylic acid cycle(PDH,KGDH),the level of MDA,activities of SOD and GSH-PX,expression of CREB and PGC-la.These studies facilitate our understanding about the impact of formula Di Huang Yin Zi on energetic metabolism,mitochondrial structure and function inside AD rat brain cells.Discussions were made about the mechanisms underlying the impact of formula Di Huang Yin Zi on mitochondrial function in AD rat brain.Methods:After those behavioral tests mentioned above,we randomly selected 8 rats from each group.The mitochondrial swelling was detected according to A520 absorption.Assay kits were introduced to detect the mitochondrial membrane potential,activities of respiratory chain complex ?,?,?,and ?,activities of key enzymes(PDH,KGDH)involved in tricarboxylic acid cycle,level of MDA,and activities of SOD and GSH-PX,respectively.ATP,ADP,AMP,PCr were detected by means of high performance liquid chromatography.The expression of CREB and PGC-la were determined through Western blot.Results:1.The impact of formula Di Huang Yin Zi on energy charge and the amount of energy-rich phosphates inside AD rat brain.In comparison with those rats without any treatment,the formula Di Huang Yin Zi can significantly increase the amount of ATP,ADP,PCr,and EC(P<0.01),whilst it considerably reduces the amount of AMP(P<0.01).2.The impact of formula Di Huang Yin Zi on mitochondrial swelling in AD rat brain.In comparison with those rats without any treatment,the formula Di Huang lin Zi can significantly upregulate mitochondrial A520 absorption in AD rats(P<0.01).3.The impact of formula Di Huang Yin Zi on mitochondrial membrane potential in AD rat brain tissue.In comparison with those rats without any treatment,the formula Di Huang Yin Zi can significantly upregulate mitochondrial membrane potential in AD rats(P<0.01).4.The impact of formula Di Huang Yin Zi on the activity of mitochondrial respiratory chain complex ?,?,?,and ? in AD rat brain tissue.In comparison with those rats without any treatment,the formula Di Huang Yin Zi can significantly upregulate the activity of respiratory chain complex ?,?,and ? in AD rats(P<0.01).Nevertheless,the activity of complex ? is not affected(P>0.05).5.The impact of formula Di Huang Yin Zi on the activity of those key enzymes involved in mitochondrial tricarboxylic acid cycle.In comparison with those rats without any treatment,formula Di Huang Yin Zi can significantly increase the acitivities of PDH and KGDH in rat brain tissue(P<0.01).6.The impact of formula Di Huang Yin Zi on the level of MDA and acitivities of SOD and GSH-PX in AD rat brain tissue.In comparison with those rats without any treatment,formula Di Huang Yin Zi can considerably reduce the level of MDA in rat brain tissue(P<0.01),whilst it can significantly upregulate the activities of SOD and GSH-PX CP<0.01).7.The impact of formula Di Huang Yin Zi on the expression of CREB and PGC-la in AD rats.In comparison with those rats without any treatment,formula Di Huang Yin Zi can significantly upregulate the expression of phosphorylated CREB and PGC-1?(P<0.01).Conclusions:1.The formula Di Huang Yin Zi can significantly improve the dysfunction of energy metabolism in AD rat brain tissue,increase energy charge of brain tissue as well as the amount of energy-rich phosphates.The energy supply inside the brain is correspondingly enhanced.2.The formula Di Huang lin Zi plays a protective role against mitochondrial injuries regarding its structure and function in AD rat brain tissue.Through maintaining mitochondrial membrane integrity,it can increase activity of mitochondrial respiratory enzyme complex and activity of key enzymes involved in tricarboxylic acid cycle including pyruvate dehydrogenase and a-ketoglutarate dehydrogenase.Therefore,the mitochondrial structure is protected and its function is maintained.3.The formula Di Huang Yin Zi can reduce the oxidative stress and lipid peroxidation resulted from mitochondrial injuries in AD rat brain tissue.Meanwhile,it can upregulate antioxidative capacity of the body and,in turn,mitigate the additional mitochondrial injuries induced by oxidative free radicals.4.The formula Di Huang Yin Zi can facilitate CREB phosphorylation and upregulate the expression of PGC-1?,leading to an increased biosynthesis of mitochondria.5.Finally,mediated by the activation of CREB/PGC-1? pathway,the formula Di Huang Yin Zi can facilitate the mitochondrial biosynthesis,increase structral integrity of mitochondria,and improve its function.Consequently,the energy metabolism of AD rat brain tissue is improved,resulting in a better energy supply. |
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