| Objective:Alzheimer’s disease(AD)is a degenerative brain disease that occurs silently and gradually reduces the memory and cognitive ability of the affected person.The disease is characterized by memory loss,temperament changes,disorganized logic,slowed movements,and even complete loss of self-care ability.As China enters an aging society,the incidence of AD continues to rise,and with the disappearance of the demographic dividend among young adults,the social problems caused by AD will become increasingly prominent.Currently,known drugs for AD,such as cholinesterase inhibitors,NMDA receptor antagonists,Aβinhibitors,and Tau protein-targeting drugs,have very limited efficacy on the disease.The development of new drugs has frequently hit a wall,and even some western drugs have to be withdrawn from the stage of history because of the serious adverse reactions or toxic side effects found in the process of clinical use.Some TCM prescriptions have shown good effectiveness against AD.The advantage of TCM is that it treats both the symptoms and the root of the disease,and has few toxic side effects.Therefore,it is imperative to explore and validate Chinese medicine prescriptions.Shenghui Tang is from Chen Shiduo’s "Discernment Record" of Qing Dynasty and is said to be "the holy medicine for forgetfulness" because of its effect of benefiting qi and nourishing blood,as well as transporting the heart and kidney.Professor Zhu Weifeng,our supervisor,has made elaborate cuts and clever combinations based on the ancient formula to form modified Shenghui Decoction.The formula is a combination of both the symptoms and the root cause and treats both deficiency and reality.In this study,the therapeutic effects of modified Shenghui Decoction were evaluated from two aspects:clinical experiment and animal experiment.In the clinical trial,the efficacy of Gao Wei Sheng Hui Tang was evaluated by a randomized controlled experimental method,and patients’ cognitive function,daily living ability,and changes in Chinese medicine symptoms after taking the medicine.The clinical experiment was conducted to investigate the efficacy of Jia Wei Sheng Hui Tang and to provide a basis for the treatment of AD in TCM.The animal experiments were conducted to investigate the effects of the therapeutic intervention of Jia Wei Sheng Hui Tang on AD in terms of behavioral changes in AD mice,histomorphological changes in the hippocampus of mice,deposition of Aβ1-42 in hippocampal tissue,and effects on PI3K/Akt/mTOR signaling pathway.Methods:Clinical trial:76 patients with mild and moderate AD were included in this study and randomly divided into Chinese and Western medicine groups.16 cases were shed,and 60 cases were finally completed in the study,30 cases in each group.The western medicine group was treated with donepezil hydrochloride,and the Chinese medicine group was treated with modified Shenghui Decoction.The treatment duration was 12 weeks in both groups.The efficacy of the medication was evaluated in terms of the patient’s overall cognitive function(assessed by MMSE scale and ADAS-Cog scale scores),changes in Chinese medical evidence(assessed by PES-D/11 scale),and ability to perform activities of daily living(evaluated by ADL scale),respectively.The safety of medication administration and patient compliance were also evaluated.Animal experiments:Experiment 1:Effects on the learning and memory ability of modified Shenghui Decoction on AD model mice。Thirty 6-month-old female APP/PS1 mice were randomly divided into a model group,Western medicine group,and Chinese medicine group,each group included 10 mice.Ten female wild-type C57BL/6J mice of the same age were used as the normal group.The Western medicine group was given Donepezil hydrochloride by gavage,the Chinese medicine group was given Shenghui Tang by gavage,and the normal and model groups were given saline by gavage.The normal group and the model group were given saline gavage.The gavage continued for 30 days.Morris water maze experiment(localization navigation experiment and spatial exploration experiment)was used to observe the effect of Shenghui Tang on the learning and memory ability of AD animals.Experiment 2:Observation of histopathological changes and Aβ1-42 deposition in the hippocampus of APP/PS1 mice.After the mice finished the water maze experiment,the hippocampal tissue was stripped from the whole brain,paraffin-embedded,and sectioned.HE staining and immunohistochemistry of Aβ1-42 were completed following the experimental steps required for HE staining and immunohistochemistry.Microscopic examination and image analysis were performed.And evaluate the integrated optical density value of Aβ1-42 in each group.Experiment 3:The effect of Shenghui Tang with added flavor on PI3K/AKT/mTOR pathway in AD mice.Fresh hippocampal tissues from each group of mice were examined for PI3K,AKT,p-AKT,mTOR,and p-mTOR protein expression according to the western blot assay.Results:Clinical experiment:general data of age,duration of illness,gender,education level,occupation,and concomitant diseases of subjects in both groups before the start of treatment were not found to be statistically significant(P>0.05)and comparable between the 2 groups.The differences were not statistically significant(P>0.05)and were comparable between the two groups in terms of MMSE,ADAS-Cog,ADL scale,and degree of Chinese medical evidence before treatment.There was no statistically significant difference between the two groups in the group comparison of MMSE score,ADAS-Cog score,and ADL score performed before and after treatment(P>0.05).After treatment,MMSE scores improved in both groups compared to pre-treatment.The MMSE scores were statistically different in the Western medicine group after treatment compared with those before treatment(P<0.05);the difference was significant in the Chinese medicine group after treatment compared with those before treatment(P<0.01).After treatment,the ADAS-Cog scores decreased in both groups compared with those before treatment,and the difference was significant(P<0.01).After treatment,ADL scores improved in both groups compared with those before treatment,with a significant difference(P<0.01).There was no statistically significant difference between the two groups for each type of evidence before treatment(P>0.05).In the inter-group comparison between the two groups,after treatment,there was a statistically significant difference in the changes in the types of symptoms of kidney deficiency,qi deficiency,marrow reduction,toxicity,and blood stasis in the Chinese medicine group compared with the Western medicine group(P<0.05);there was a statistically significant difference in the changes of spleen deficiency,hyperactivity,and phlegm(P<0.01);there was no statistically significant difference in the symptoms of blood deficiency(P>0.05).In the intra-group comparison between the two groups before and after treatment,there was a statistically significant difference in the changes in the symptoms of pith reduction in the Western medicine group(P<0.05),but no statistically significant difference in the changes in the other types of symptoms(P>0.05);there was a statistically significant difference in the changes in kidney deficiency,spleen deficiency,qi deficiency,yang hyperactivity,toxicity and blood stasis in the Chinese medicine group(P<0.05),and there was a statistically significant difference in the changes in pith reduction and phlegm turbidity(P<0.01).(P<0.01),and there was no statistical difference in the changes in blood deficiency(P>0.05).There was no statistical difference between the two groups in terms of safety and compliance(P>0.05).Animal experiments:Positioning navigation experiment.On the 1st day,in the escape latency time,the escape latency time of mice in the model group was higher than that of the normal group,and the difference was statistically significant(P<0.05).There was no statistical difference between the escape latency time of the western medicine group and the Chinese medicine group and the normal group and the model group(P>0.05),and no statistical difference was seen between the western medicine group and the Chinese medicine group(P>0.05).In terms of swimming distance,the mice in the model group had higher swimming distances than the normal group,and the difference was statistically significant(P<0.01),and the western medicine group and the Chinese medicine group had higher swimming distances than the normal group(P<0.05).There was no statistically significant difference in the swimming distance between the model group,the western medicine group,and the Chinese medicine group(P>0.05).On the 5th day,the escape latency time of the mice in the model group,western medicine group,and Chinese medicine group was higher than that of the normal group,and the difference was statistically significant(P<0.01);the escape latency time of the western medicine group was lower than that of the model group,and the difference was statistically significant(P<0.05);the escape latency time of the Chinese medicine group was lower than that of the model group,and the difference was statistically significant(P<0.01);there was no statistical difference between the western medicine group and the Chinese medicine group.There was no statistical difference between the western medicine group and the Chinese medicine group(P>0.05).In terms of swimming distance,the mice in the model group,the western medicine group,and the Chinese medicine group had higher swimming distances than the normal group,and the difference was significant(P<0.01),and the swimming distance in the western medicine group and the Chinese medicine group was lower than that in the model group,and the difference was statistically significant(P<0.05),and the swimming distance between the western medicine group and the Chinese medicine group was not statistically different(P>0.05).Spatial exploration experiment.For the comparison of the number of platform crossing,the mice in the model group crossed the platform less than the normal group,and the difference was significant(P<0.01),the mice in the western medicine group and the Chinese medicine group crossed the platform less than the normal group,and the difference was statistically significant(P<0.05),the mice in the model group crossed the platform less than the western medicine group and the Chinese medicine group,and the difference was statistically significant(P<0.05),and the number of platform crossing between the western medicine group and the Chinese medicine group There was no statistical difference(P>0.05).For the comparison of the time to reach the original platform for the first time,the time taken by the mice in the model group,western medicine group,and Chinese medicine group was higher than that in the normal group,and the difference was significant(P<0.01),and the time taken by the mice in the western medicine group and Chinese medicine group was lower than that in the model group,and the difference was statistically significant(P<0.05),and the time taken between the western medicine group and Chinese medicine group was not statistically different(P>0.05).For the comparison of the target quadrant time ratio,the model group,western medicine group,and Chinese medicine group were lower than the normal group,and the difference was significant(P<0.01),the western medicine group and Chinese medicine group were higher than the model group,and the difference was significant(P<0.01),and there was no statistical difference between the western medicine group and Chinese medicine group(P>0.05).Hippocampal region HE staining.Normal group:the number of neurons in the hippocampal region was normal,with a neat and tight arrangement and full and uniform morphology.The nuclei were purple,the pyramidal cells were regular,the dendrites were neatly arranged in strips,and the number was normal.In the model group,the number of neurons in the hippocampus was significantly reduced,the arrangement was disorganized,the nuclei were fixed and darkly stained,the number of pyramidal cells was significantly reduced,the arrangement was disorganized,and there was vacuolar degeneration and necrosis of fixation.In the Chinese medicine group,the number of neurons and pyramidal cells in the hippocampal region was less than that in the normal group,but more than that in the model group.The arrangement was neat and the morphology was more regular.Occasional vacuolar degeneration was seen in the pyramidal cells.The cell consolidation and necrosis were better than those in the model group.Western medicine group:the situation was similar to that of the Chinese medicine group.Immunohistochemistry of Aβ1-42 in the hippocampal region.The integral optical density results showed that the expression of Aβ1-42 in the hippocampus of mice in the model group was higher than that in the normal group,and the difference was statistically significant and obvious(P<0.01);the deposition of Aβ1-42 in the western medicine group and the Chinese medicine group was lower than that in the model group but higher than that in the normal group,and the difference was statistically significant and obvious(P<0.01);the difference was not statistically significant when comparing the deposition of the two groups in the western medicine group and the Chinese medicine group(P>0.05).Western blot.Compared with the normal group,the hippocampal PI3K,AKT,p-AKT,mTOR,and p-mTOR protein expressions were significantly higher in the model group compared with the normal group(P<0.01);the hippocampal AKT,mTOR,and p-mTOR protein expressions were higher in the Chinese medicine group and the western medicine group than the normal group(P<0.01),but lower than the model group(P<0.01),and the differences were significant.In terms of PI3K protein expression,PI3K expression in the western medicine group was higher than that in the normal group(P<0.05),which was statistically different,but lower than that in the model group(P<0.01),which was significant.The expression of PI3K in the Chinese medicine group was higher than that in the normal group(P<0.01),but lower than that in the model group(P<0.01),with significant differences.In terms of p-AKT protein expression,p-AKT expression in the Chinese medicine group was higher than that in the normal group(P<0.05),which was statistically different,but lower than that in the model group(P<0.01),which was significant.The p-AKT expression in the Western medicine group was higher than that in the normal group(P<0.01),but lower than that in the model group(P<0.01),with significant differences.There was no statistically significant difference in the comparison of PI3K,AKT,p-AKT,mTOR,and p-mTOR protein expression results between the Chinese medicine group and the western medicine group(P>0.05).Conclusions:1.This study showed that the addition of Shenghui Tang improved the scores of MMSE and ADL and decreased the scores of ADAS-Cog in patients with mild to moderate AD,and improved language ability,concentration ability,orientation,recall,practical ability,and activities of daily living in AD patients.As for the effect on TCM symptoms,the western drug Donepezil only improved the symptoms of medullary decompensation,but not the rest of the common symptoms of dementia,such as kidney deficiency,spleen deficiency,blood deficiency,qi deficiency,hyperactivity,toxicity,phlegm,and blood stasis.Except for the improvement of blood deficiency,the other 8 common symptoms of dementia were all improved,especially the improvement of medullary hypoplasia and phlegm.The majority of patients had good compliance with modified Shenghui Decoction.Moreover,the formula is clinically safe,has good clinical use prospects,and is worth promoting.2.APP/PS1 model mice have obvious memory and spatial orientation impairment.Both modified Shenghui Decoction and donepezil can improve the memory and spatial orientation ability of APP/PS1 mice,and the effects are similar.Both modified Shenghui Decoction and donepezil could inhibit the pathological changes caused by AD to a certain extent,inhibit the aggregation of Aβ1-42 in the hippocampal region of AD mice,and play a role in protecting the nerves in the hippocampal region.The levels of PI3K,AKT,p-AKT,mTOR,and p-mTOR protein were significantly increased in the model group,indicating that PI3K/Akt/mTOR is closely related to the pathogenesis of AD.The expression of PI3K,AKT,p-AKT,mTOR,and p-mTOR proteins can be reduced and the PI3K/AKT/mTOR signaling pathway can be inhibited by administering modified Shenghui Decoction. |
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