| Objectives:Alzheimer's disease(AD)is a neurodegenerative disease characterized by insidious onset and progressive impairment of cognitive function,it severely impairs the quality of life and health of patients.At present,the aging population in China is gradually increasing,and the number of AD patients is also increasing year by year,drug therapy can improve the clinical symptoms of AD patients,but cannot completely block the progression of the disease,and the course of medical treatment is long.Previous clinical studies had shown that Huanglian Jiedu Decoction has a therapeutic effect on AD.In terms of experimental studies,Huanglian Jiedu Decoction can inhibit the characteristic pathological changes of AD and down-regulate the expression of inflammatory factors in AD model mice.This research adopted the single-center,random,positive control drug,non-inferiority design,the positive control drug is donepezil,the effect of Huanglian Jiedu Decoction on AD was comprehensively evaluated from the aspects of holistic cognitive function,the ability of daily living,quality of life,and syndrome of traditional Chinese medicine(TCM),the time-effect character of Huanglian Jiedu Decoction was analyzed by observing the curative effect at the different time points and the efficacy maintenance in the different period time.The clinical significance of TCM syndromes in AD patients was illustrated from the view of modern medicine by analyzing the correlation between TCM syndromes and holistic cognitive function,the ability of daily living,and quality of life,So as to provide a research basis for TCM diagnosis and treatment of AD and application of Huanglian Jiedu Decoction in AD treatment.In this study,the effects of Huanglian Jiedu Decoction on AD model mice were observed from the many levels of the ability of learning and memory,A? and relevant inflammatory cytokines,histopathology,activation of microglia,NLRP3 inflammasome associated protein and gene expression,and the possible mechanism of Huanglian Jiedu Decoction in the intervention of AD was explored.Methods:1.Clinical observation:50 cases of AD patients meeting the inclusion criteria were enrolled in the study.The study included the screening of AD patients,baseline evaluation,randomization,treatment phase(12 weeks),and follow-up phase(12 weeks).After screening,the AD patients were randomly divided into the experimental group and the control group,the baseline evaluations were performed for both groups to assess comparability.In the treatment stage,the control group was treated with donepezil,and the experimental group was treated with Huanglian Jiedu Decoction.During the follow-up phase,Huanglian Jiedu Decoction and donepezil were discontinued.In terms of efficacy evaluation indexes,the primary observation indexes were MMSE and ADAS-COG scales to evaluate holistic cognitive function,ADL scales to evaluate activities of daily living,QOL-AD scales to evaluate the quality of life,and PES-D/11 scales to evaluate the distribution and degree of TCM syndrome elements.The main observation indexes of the two groups at 4th,8th,12th,16th,20th,24th weeks were compared with those before treatment,the differences between the main observation indexes of the two groups at the same time point were analyzed.The differential ratios of the main observation indexes in the two groups were analyzed from the 12th week to the 16th week,from the 16th to the 20th week,from the 20th week to the 24th week,then the difference of maintenance efficacy between the two groups was compared.By analyzing the correlation between PES-D/11 scores and MMSE,ADAS-COG,ADL,QOL-AD scores,the correlation between TCM syndromes and holistic cognitive function,the ability of daily living activities,quality of life was explored.The differences of secondary outcome indexes between the two groups were compared in the 12th week,and the effects of Huanglian Jiedu Decoction and donepezil on TCM syndrome were observed,the differences in the efficacy of the two groups were analyzed.Finally,the safety and compliance of the two groups were evaluated.2.Experimental study:the 9-month-old Tau/APP/SP1 transgenic AD model mice were randomly divided into the model group,donepezil group,Huanglian Jiedu Decoction high-dose group,Huanglian Jiedu Decoction medium-dose group,Huanglian Jiedu Decoction low-dose group.The model group was given sodium carboxymethyl cellulose solution,the donepezil group was given donepezil hydrochloride tablet solution,and the Huanglian Jiedu decoction groups were given 865mg·kg-1·d-1,433mg·kg-1·d-1,and 216mg·kg-1·d-1 solution of different concentrations of Huanglian Jiedu Decoction extract,respectively,for 3 months by intragastric administration.In experiment 1,the Morris water maze test was used to evaluate the learning ability and memory ability of mice in the five groups.In experiment 2,the expression levels of A? 1-40,A? 1-42,IL-1?,IL-18 in hippocampal tissues of mice in the five groups were detected by ELISA.In Experiment 3,HE staining was used to observe the changes of histomorphometry in the hippocampal CA1 region of each group.The immunohistochemical method was used to observe the morphology and number of activated microglia cells in the hippocampal CA1 region of each group.In Experiment 4,the protein expression levels of NLRP3,ASC,Pro-caspase-1,Caspase-1 p20 in the hippocampal tissues of each group were detected by western blot,and the mRNA expression levels of NLRP3,ASC,Caspase-1 in the hippocampal tissues of each group were detected by real-time quantitative PCR.Results:1.Clinical observation(1)Recruiting patients50 patients with AD were included,of which 8 cases with shedding and 42 patients completed the study,including 21 cases in the experimental group and 21 cases in the control group.(2)Baseline dataThere were no significant differences between the experimental group and the control group in terms of general information,dementia evaluation-related scale score,TCM syndrome element distribution and syndrome degree,and neuroimaging score before treatment(P>0.05).(3)Comparison of curative effect indexes at each time pointIn the MMSE scale score,the score of the experimental group and control group in the 4th,8th,20th,24th weeks has no significant difference from that before treatment(P>0.05),the score of the two groups in the 12th,16th weeks was increased respectively compared with that before treatment(P<0.05).In the 4th,8th,12th,16th,20th,24th week,the score of the experimental group has no significant difference from that of the control group(P>0.05).In the ADAS-Cog scale score,the score of the experimental group and control group in the 4th,8th,20th,24th weeks has no significant difference from that before treatment(P>0.05),the score of the two groups in the 12th,16th weeks was decreased respectively compared with that before treatment(P<0.05).In the 4th,8th,12th,16th,20th,24th week,the score of the experimental group has no significant difference from that of the control group(P>0.05).In the ADL scale score,the score of the experimental group in the 4th,8th,20th,24th weeks has no significant difference from that before treatment(P>0.05),the score of the control group in the 4th,8th,16th,20th,24th weeks has no significant difference from that before treatment(P>0.05),the score of the experimental group in the 12th,16th weeks was decreased compared with that before treatment(P<0.05),the score of the control group in the 12th weeks was decreased compared with that before treatment(P<0.05).In the 4th,8th,12th,16th,20th,24th week,the score of the experimental group has no significant difference from that of the control group(P>0.05).In the QOL-AD scale score,the score of the experimental group in the 4th,20th,24th weeks has no significant difference from that before the treatment(P>0.05),the score of the control group in the 4th,8th,20th,24th weeks has no significant difference from that before the treatment(P>0.05),the score of the experimental group in the 8th,12th,16th weeks was increased compared with that before treatment(P<0.05),the score of the control group in the 12th,16th weeks was increased compared with that before treatment(P<0.05).In the 4th,8th,12th,16th,20th,24th week,the score of the experimental group has no significant difference from that of the control group(P>0.05).The results of repetitive measurement deviation analysis showed that time factors affected MMSE,ADAS-COG,ADL,QOL-AD scores in the 2 groups of patients.MMSE and QOL-AD scores of the two groups have no interaction effect between time and group,ADAS-Cog and ADL scores of the two groups have interaction effects between time and group.In terms of MMSE,ADAS-COG,ADL,QOL-AD scores,there were significant differences between scores at each time point as an intra-subject difference factor(P<0.05),while there was no significant difference between scores of the two treatments as an inter-subject difference factor(P>0.05).The MMSE and QOL-AD scores increased with the prolongation of treatment time in the two groups and both reached the highest level at the 12th week of enrollment.ADAS-COG and ADL scores decreased with the extension of treatment time in the two groups,and both reached the lowest level at the 12th week of enrollment.After the treatment,the MMSE and QOL-AD scores of the two groups decreased,while the ADAS-COG and ADL scores increased.(4)The maintenance effect of efficacyFrom the 12th week to the 16th week of enrollment,there were significant differences in the differential ratio of ADL and QOL-AD scores between the experimental group and the control group(P<0.05),indicating that the maintenance efficacy of the experimental group was better than that of the control group,while there was no significant difference in the differential ratio of MMSE and ADAS-cog scores between the two groups at the same period time(P>0.05).There was no significant difference in the differential ratio of MMSE,ADAS-COG,ADL,QOL-AD scores between the experimental group and the control group from the 16th to the 20th week,and from the 20th to the 24th week(P>0.05).(5)The correlation between TCM syndrome element and cognitive function,ability of daily living,and quality of lifeThe result of Spearman's correlation analysis showed that there was a negative correlation between MMSE score and the scores of medullary decrease,exuberant toxin(r=-0.31,P=0.04;r=-0.33,P=0.03).There was a positive correlation between the scores of kidney deficiency,exuberant toxin,and the score of ADAS-COG(r=0.39,P=0.01;r=0.31,P=0.04).There was a positive correlation between the scores of medullary decrease,exuberant toxin,and the score of ADL score(r=0.35,P=0.02;r=0.32,P=0.04).The scores of kidney deficiency,phlegm turbidity,exuberant toxin were negatively correlated with the score of QOL-AD(r=-0.35,P=0.03;r=-0.36,P=0.02;r=-0.32,P=0.04).(6)The influence of Huanglian Jiedu Decoction on TCM syndrome elementsthe scores of Yang excess,fire-toxicity,phlegm turbidity of the experimental group after the treatment(the 12th week)were lower than that before treatment(P<0.05),the scores of kidney deficiency,qi deficiency,blood deficiency,medullary decrease,blood stasis,Yang excess,phlegm turbidity,fire-toxicity of the control group after treatment(the 12th week)had no significant change with that before treatment(P>0.05).After the treatment(the 12th week),the scores of Yang excess and fire-toxicity in the experimental group were lower than those in the control group(P<0.05),while there was no statistical difference in the scores of kidney deficiency,Qi deficiency,blood deficiency,medullary decrease,phlegm turbidity,and blood stasis between the two groups(P>0.05).(7)Safety observationThere was no abnormality in safety-related laboratory indices of patients in both two groups during enrollment,treatment,and follow-up.In terms of adverse reactions,there was 1 case of nausea in the control group and 2 cases of abdominal distension in the experimental group,both of which were mild.There was no significant difference in the incidence of adverse reactions between the two groups(P>0.05).(8)Compliance observationThe drug compliance rate of the experimental group was 85.71%(18/21),and that of the control group was 80.95%(17/21).There was no statistical difference in the drug compliance rate between the two groups(P>0.05).2.Experimental study(1)Effect of Huanglian Jiedu Decoction on the ability of learning and memory of the AD model miceIn the first 4 days of training and the fifth day of orientation navigation tests,the escaping latencies and the total swimming distances of the five groups were gradually shortened.The results of orientation navigation tests in the 5th day showed that the escaping latencies of the donepezil group,the HLJDT high-dose group,the HLJDT middle-dose group were significantly reduced compared with the model group(P<0.01),the escaping latencies of the HLJDT low-dose group also reduced compared with the model group(P<0.05),the total swimming distance of the donepezil group,the HLJDT high-dose,middle-dose,and low-dose group were less than that of the model group(P<0.05).The results of spatial probe tests showed that the times across platform and the time percent during platform quadrant of the donepezil group,HLJDT high-dose group,and HLJDT medium-dose group were significantly higher than that of the model group(P<0.01),and the time of the first arrival at the original platform of the donepezil group,HLJDT high-dose group,and HLJDT medium-dose group was significantly lower than that of the model group(P<0.01).Compared with the model group,the times across platform and the time percent during platform quadrant of the HLJDT low-dose group was increased(P<0.05),and the time of the first arrival at the original platform of the HLJDT low-dose group was decreased(P<0.05).(2)Effects of Huanglian Jiedu Decoction on the expression of A? and related inflammatory factors in the hippocampus of the AD model miceIn respect of the expressions of A ? 1-40 and A ? 1-42 in the mice hippocampus,compared with the model group,the expression of soluble and insoluble A ? 1-40,soluble and insoluble A ? 1-42,total A ? 1-40,total A ? 1-42 in the HLJDT high-dose,medium-dose,and low-dose group were significantly lower(P<0.01),the expression of insoluble A ? 1-40 in the donepezil group reduced(P<0.05),the expression of soluble and insoluble A ? 1-42,soluble A ? 1-40,total A? 1-40,total A ? 1-42 in the donepezil's group significantly reduced(P<0.01);Compared with the donepezil group,the expressions of soluble and insoluble A ? 1-40,soluble and insoluble A ? 1-42,and total A ? 1-42 in the HLJDT high-dose group were decreased(P<0.05),and the expressions of total A ? 1-40 in the HLJDT high-dose group were significantly decreased(P<0.01).The expressions of soluble and insoluble A ? 1-40,soluble and insoluble A ? 1-42,total A ? 1-40,total A ? 1-42 in the HLJDT medium-dose group were decreased than that of the donepezil group(P<0.05).Compared with the HLJDT low-dose group,the expressions of soluble A ? 1-40 and total A ? 1-40 in the HLJDT high-dose group were decreased(P<0.05,P<0.01),and the expressions of soluble A ? 1-40 and total A ? 1-40 in the HLJDT medium-dose group were decreased(P<0.05).In terms of the expression of IL-1? in the mice hippocampus,the expression of the HLJDT high-dose,medium-dose,and low-dose groups and the donepezil groups were lower than that of the model group(P<0.05).In terms of the expression of IL-18 in the mice hippocampus,the HLJDT large-dose group,HLJDT medium-dose group,and HLJDT small-dose group and donepezil group were significantly lower than the model group(P<0.01),the expression of the HLJDT high-dose group and the HLJDT medium-dose group were lower than that of the model group(P<0.05),the expression of the HLJDT high-dose group and the HLJDT medium-dose group were lower than that of the HLJDT low-dose group(P<0.05).(3)Effects of Huanglian Jiedu Decoction on the hippocampal histomorphology and the activation state of microglia in AD model miceThe HE staining results of the hippocampal CA1 region showed that the cell hierarchy of the model group was reduced,the tissue structure was loose,the arrangement was disordered,the contour was not clear,the nerve cells were swollen,and some cells had vacuolar degeneration or shrinkage necrosis.Compared with the model group,the morphological manifestations of hippocampal CA1 in the donepezil group,the HLJDT high-dose group,the HLJDT medium-dose group,and the HLJDT low-dose group were improved,the pyramidal cells were arranged neatly,the tissue structure was clearer,and there were fewer cells with vacuolar degeneration or shrinkage necrosis.In terms of the total number of neurons in the hippocampal CA1 region,the HLJDT high-dose group and the HLJDT medium-dose group were significantly increased compared with the model group(P<0.01),and the donepezil group and the HLJDT low-dose group were increased compared with the model group(P<0.05).The immunohistochemical staining results of the hippocampal CA1 region showed that the microglial cells in the model group were active and amoeba-like.The donepezil group,and the HLJDT high-dose,medium-dose,and low-dose groups had smaller microglia,lighter staining,longer spines of nerve cells,and fewer activated microglia than the model group.In terms of the numbers of activated microglia,the HLJDT high-dose group and the HLJDT medium-dose group were significantly decreased compared with the model group(P<0.01),the donepezil group and the HLJDT low-dose group were decreased compared with the model group(P<0.05),the HLJDT high-dose group and the HLJDT medium-dose group were decreased compared with the donepezil group(P<0.05).(4)Effect of Huanglian Jiedu Decoction on the expression of NLRP3 inflammasome relevant protein and mRNA in the hippocampusThe results of Western blot showed that the protein expressions of NALRP3,ASC,Caspase-1 p20 of the donepezil group and HLJDT high-dose,medium-dose,and low-dose groups were significantly lower than that of the model group(P<0.01).The protein expressions of NALRP3,ASC,Caspase-1 p20 of the donepezil group,HLJDT high-dose group,and HLJDT medium-dose group were lower than that of the HLJDT low-dose group(P<0.05).There was no significant difference in the protein expression of Pro-caspase-1 among the five groups(P>0.05).The results of Real-time quantitative PCR showed that the mRNA expressions of NLRP3,ASC,Caspase-1 of the donepezil group and HLJDT high-dose,medium-dose,and low-dose groups were significantly lower than that of the model group(P<0.01).The mRNA expressions of NLRP3,ASC,Caspase-1 were not significantly different among other groups(P>0.05).Conclusions:1.Clinical studies suggest that Huanglian Jiedu Decoction can improve the AD patients' holistic cognitive function and activity of daily living,quality of life.Huanglian Jiedu Decoction have an earlier effect on improving the quality of life than donepezil and have the better maintenance efficacy on improving the ability of daily living and the quality of life than donepezil.In AD patients,kidney deficiency,medullary decrease,exuberant toxin were correlated with holistic cognitive function.Medullary decrease and exuberant toxin were correlated with the activity of daily living.Exuberant toxin and phlegm turbidity were correlated with quality of life.Huanglian Jiedu Decoction can effectively reduce the symptomatic severity of Yang excess,fire-toxicity,phlegm turbidity in AD patients,and its improvement effects on Yang excess syndrome and fire-toxicity syndrome were better than donepezil.Huanglian Jiedu Decoction showed good performance in drug safety and patient compliance.2.Experimental studies have shown that Huanglian Jiedu Decoction can improve learning and memory ability of Tau/APP/PS1 transgenic mice,can reduce the expressions of hippocampal soluble and insoluble A ? 1-40,total A ? 1-40,soluble and insoluble A ?1-42,total A ? 1-42 and IL-18,IL-1?,can reduce the pathological damage of hippocampus,can inhibit the activation state of hippocampal microglia,can down-regulate the protein expression of NLRP3,ASC,Caspase-1 p20 and the mRNA expression of NLRP3,ASC,Caspase-1 in hippocampus tissue.In terms of the effect difference of the different-dose Huanglian Jiedu Decoction,the high-dose group and the medium-dose group have the better inhibition effect on the expression of soluble A ? 1-40,total A ? 1-40,IL-18 and the better down-regulated effect on the protein expression of NLRP3,ASC,Caspase-1 p20 in the hippocampus than those of the low-dose group.The results above suggest that the improvement in learning and memory ability of Tau/APP/PS1 transgenic mice by Huanglian Jiedu Decoction may be related to its inhibiting the expression of NLRP3 inflammasome relevant proteins and genes,regulating the activation state of microglia cells,and inhibiting neuroinflammation and protecting neurons. |
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