| Objective:Alzheimer's disease(AD)is a chronic,insidiously progressive,age-related degenerative brain disorder that irreversibly impairs cognitive function,often accompanied by mental and behavioral abnormalities and impairment of daily living.With the extension of human life expectancy,its prevalence rate increases year by year,seriously threatening the physical health and quality of life of the elderly,and bringing heavy economic and psychological burden to society and families.Modern studies have shown that AD is caused by multiple factors,while existing drugs can only improve the clinical symptoms of AD patients by acting on a single target,but cannot reverse the progression of the disease or completely cure it.Moreover,with the extension of medication time,many adverse drug reactions gradually become prominent.Traditional Chinese medicine is often characterized by multi-pathway and multi-target action,which is more consistent with the pathological mechanism of AD.Therefore,it is one of the important approaches to study the prevention and treatment of AD to give full play to the advantages of Traditional Chinese medicine and actively explore effective prescription for the treatment of AD.Neuroinflammation,as an important pathological feature and pathogenic factor of AD,can lead to neuronal apoptosis and vascular injury,and has the characteristics of body deterioration and brain and marrow injury.Its toxic effect is consistent with the internal toxicity category of AD " damaged brain collaterals by toxin " theory.Therefore,huanglian Jiedu Decoction and its related prescriptions are widely used in the study of AD.This team for many years has been committed to the basis of huanglian jiedu decoction to treat old age dementia and clinical research,basic research found that huanglian jiedu decoction can obviously improve the APP/PS1 double transgenic AD mice learning memory ability,can reduce model mouse within A beta precursor protein gene expression and age spots,reduce mouse brain tissue and serum level of IL-6,IL-1 ?;Clinical studies have found that Huanglian Jiedu Decoction can effectively improve the cognitive and daily living ability of senile dementia patients,and can effectively improve the symptoms of Yangkang,Huodu and Tanzhuo syndrome of senile dementia patients.However,there are some problems in the preliminary studies,and the underlying molecular mechanism of the anti-inflammatory effect of Huanglian Jiedu Decoction has not been further explored in the basic studies.Clinical research huanglian Jiedu Decoction alone in the treatment of AD is often limited by its bitter cold properties.Therefore,based on the previous research results,this project is divided into basic and clinical research.In the basic research,AD model mice with Tau/APP/PS1 transgenes that are more consistent with the pathological characteristics of AD are selected as research objects to study the effects of Huanglian Jidu Decoction on their learning and memory ability,histopathological changes and inflammatory factors.The changes of TLR2/MAPK signal pathway related molecules in huanglian Jiedu Decoction intervention were further studied to explore the potential molecular mechanism of huanglian Jiedu Decoction intervention on AD.Clinical study was conducted to explore the improvement effect of the observation group(Huanglian Jiedu Decoction combined with Xixin Decoction and Donepezil)compared with the control group(Donepezil)on the overall cognitive function,daily living ability and TCM syndrome score of AD patients with mild to moderate Tanzhuomengqiao syndrome,as well as the effect on serum inflammatory indicators.Methods:1.Experimental study:In this study,8-week-old SPF Tau/APP/PS1 three transgenic AD model mice were bred in a cage according to male: female = 1:2.Seventy-five nine-month-old male mice identified as Tau/APP/PS1 genotype after breeding were selected as research objects and randomly divided into five groups: Model group,Donepezil group,Huanglian Jiedu Decoction(HLJDT)low-dose group(HLJDT-L),Huanglian Jiedu Decoction(HLJDT)medium-dose group(HLJDT-M)and Huanglian Jiedudu Decoction high-dose group(HLJDT-H).Each group was given different interventions: Model group was given 0.9% normal saline solution,donepezil group was given donepezil hydrochloride aqueous solution,low-dose,medium-dose and high-dose huanglian Jiudu decoction groups were given different concentrations of Huanglian Jiudu decoction extract aqueous solution 975mg/kg/d,1950mg/kg/d and 3900mg/kg/d,respectively.All the mice in the five groups were given intragastric administration for 3 months continuously.Morris water maze test was used to evaluate the learning and memory ability of mice in each group.HE staining was used to observe the pathological changes of hippocampal CA1 region in each group.The expression levels of TNF-?,IL-6,IL-4 and IL-10 in hippocampal tissues of each group were detected by ELISA.The expression levels of CD86 and CD206 m RNA in hippocampus were detected by real-time fluorescence quantitative PCR.The protein expression levels of TLR2,My D88,P38,P-P38,ERK,P-ERK,JNK and P-JNK in hippocampal tissues of each group were detected by Western blot.2.Clinical research:This research into the group of the total,discharge standard of 58 patients with AD,AD patients were randomly divided into control group and observation group,29 cases in each group,with more than the control group given Donepezil therapy,observation group in the control group on the basis of huanglian jiedu decoction is used to joint Xixin Decoction granules treatment,two groups of patients for treatment of 24 weeks.MMSE scale and ADAS-Cog scale were used to evaluate the changes of overall cognitive function of patients in the two groups,and ADL scale was used to evaluate the changes of daily living ability of patients in the two groups.SDSD scale was used to evaluate the changes of TCM syndrome scores in the two groups.The TCM curative effect of the two groups was judged by the reduction rate of syndrome score.The changes of serum inflammatory factors TNF-? and IL-6 in 2 groups were detected by ELISA.The baseline information of gender,age,education level and disease course before treatment was compared between the two groups to evaluate whether the two groups were comparable.The changes of overall cognitive function,daily living ability,TCM syndrome score and inflammatory factors were compared between the two groups before and after treatment,and the differences of TCM efficacy between the two groups after treatment were compared.The efficacy of different intervention drugs and the differences between the two groups were evaluated.Finally,the safety level of the two groups was evaluated.Results:1.Experimental research(1)The effect of Huanglian Jiedu Decoction on the learning and memory ability of Tau/APP/PS1 AD model miceDuring the 5-day positioning navigation experiment,the escape latency of mice in each group showed a decreasing trend and was gradually shortened,suggesting that the learning ability of searching for hidden platform in water was gradually improved through training of mice in the 5 groups.The results at the end of the positioning navigation experiment(day 5)showed that compared with the model group,the latent period of escape in donepezil group and HLJDT low-dose,medium-dose and high-dose groups was significantly reduced(P < 0.01),and the latent period of escape in HLJDT low-dose group was longer than that in Donepezil group,with statistical difference(P < 0.01).There was no statistical difference in escape latency between the high-dose HLJDT group and the medium-dose HLJDT group(P > 0.05).The results of space exploration experiment showed that compared with the model group,the number of platform crossing,residence time in target quadrant and swimming distance in target quadrant of donepezil group and HLJDT low,medium and high dose groups were significantly increased(P < 0.01),and compared with donepezil group,The number of crossing platforms,residence time in the target quadrant and swimming distance in the target quadrant decreased in the low-dose HLJDT group(P < 0.05),while there were no significant differences in the number of crossing platforms,residence time in the target quadrant and swimming distance in the target quadrant between the high-dose HLJDT group and the medium-dose HLJDT group(P > 0.05).(2)The effect of Huanglian Jiedu Decoction on pathological morphology of hippocampus in Tau/APP/PS1 transgenic AD model miceHE staining was used to observe the histopathological morphology of hippocampal CA1 region in each group.The results showed that the number of neurons in hippocampal CA1 region of mice in model group was significantly reduced,the hierarchical arrangement of neurons was disorder,the cell morphology was abnormal,and the nucleus pyknosis accompanied by staining was deepened.Compared with model group,the number of neurons in hippocampal CA1 region of donepezil group,HLJDT low-dose group,HLJDT medium-dose group and HLJDT high-dose group was significantly increased(P < 0.01),and the arrangement of neurons was more regular,the cell morphology was significantly recovered,and the nuclear pyknosis phenomenon was significantly improved.Compared with donepezil group,neurons in hippocampal CA1 region of HLJDT low-medium-high dose group had orderly arrangement and no significant difference in nuclear pyknosis.The number of neurons in hippocampal CA1 region of HLJDT medium-high dose group was significantly increased(P < 0.05).There was no statistical difference in the number of neurons in hippocampal CA1 region of HLJDT group(P > 0.05).(3)The effect of Huanglian Jiedu Decoction on the expression of inflammatory factors in hippocampus of Tau/APP/PS1 transgenic AD miceThe expression level of TNF-? in hippocampus of all groups showed that compared with model group,the expression level of TNF-? in donepezil group and HLJDT low-medium-high-dose group was significantly decreased(P < 0.01).Compared with donepezil group,the expression level of TNF-? in HLJDT low-dose group had no significant difference(P > 0.05),while the expression level of TNF-? in HLJDT medium-dose high-dose group was significantly decreased(P < 0.05,P < 0.01).The expression level of TNF-? was significantly decreased with the increase of HLJDT dose(P < 0.05).The expression level of IL-6 in hippocampus of each group showed that,compared with model group,the expression level of IL-6 in donepezil group and HLJDT low-medium-high-dose group was significantly decreased(P < 0.01);Compared with donepezil group,there was no significant difference in the expression level of IL-6 in the low-dose HLJDT group(P > 0.05),but the expression level of IL-6 in the medium-dose HLJDT group was significantly decreased(all P < 0.01).The expression level of IL-6 decreased significantly with the increase of HLJDT dose(P < 0.01).The expression level of IL-4 in hippocampus of each group showed that,compared with model group,the expression level of IL-4 in donepezil group and HLJDT low-medium-high-dose group was significantly increased(P < 0.01);Compared with donepezil group,the expression level of IL-4 in HLJDT low-dose group had no significant difference(P > 0.05),while the expression level of IL-4 in HLJDT medium-dose group was significantly increased(all P < 0.01).The expression level of IL-4 increased significantly with the increase of HLJDT dose(P < 0.01).The expression level of IL-10 in hippocampus of each group showed that compared with model group,the expression level of IL-10 in donepezil group and HLJDT low-medium-high-dose group was significantly increased(P < 0.01).Compared with donepezil group,there was no significant difference in the expression level of IL-10 in the low-dose HLJDT group(P > 0.05),but the expression level of IL-10 in the medium-dose HLJDT group was significantly increased(all P < 0.01).The expression level of IL-10 was significantly increased with the increase of HLJDT dose(P < 0.01).(4)Effects of Huanglian Jiedu Decoction on mrna expression levels of CD86 and CD206 in hippocampus of Tau/APP/PS1 AD model miceThe expression levels of CD86 and CD206 m RNA in hippocampus of each group were significantly decreased(P < 0.05),and significantly increased(P < 0.05)compared with model group.Compared with donepezil group,there was no significant difference in the expression levels of CD86 and CD206 m RNA in low and medium dose HLJDT group(P > 0.05),but the expression level of CD86 mrna in high dose HLJDT group was significantly decreased(all P < 0.01),and the expression level of CD206 m RNA was significantly increased (all P < 0.05).(5)Effects of Huanglian Jiudu Decoction on protein expression levels of TLR2,My D88,P38,P-P38,ERK,P-ERK,JNK and P-JNK in hippocampus of Tau/APP/PS1 transgenic AD miceThe protein expression levels of TLR2,My D88,P38,P-P38,ERK,P-ERK,JNK and P-JNK in the hippocampus of each group of mice showed that,compared with the model group,The protein expression levels of TLR2,My D88,P-P38,P-ERK and P-JNK in HLJDT low,medium and high dose groups were significantly decreased(P < 0.01),but the protein expression levels of P38,ERK and JNK were not significantly different(P > 0.05),and the protein expression levels of donepezil group were not significantly different(P > 0.05).The expression of TLR2,My D88,P-P38,P-ERK and P-JNK in the hippocampus of mice was significantly decreased with the increase of HLJDT dose(P < 0.01),while there was no significant difference in the expression of P38,ERK and JNK(P > 0.05).2.Clinical study(1)Completion of case recruitmentIn this study,a total of 58 cases of mild to moderate AD with Tanzhuomengqiao syndrome were included,including 6 cases in the control group and 4 cases in the observation group.Altogether,10 cases were shed.Finally,48 cases were completed in the 6-month study,including 23 cases in the control group and 25 cases in the observation group,with a total shedding rate of 17.2%.(2)Baseline dataBefore treatment,there were no significant differences between control group and observation group in general information including gender,age,education level,course of disease,severity of dementia,cognitive correlation scale score,daily living ability scale score,TCM syndrome score,serum TNF-? and IL-6 levels(P > 0.05).(3)Comparison of efficacy indexes between the two groups before and after treatment and 24 weeks after treatmentThe MMSE scores of the two groups were statistically different after 24 weeks of treatment compared with before treatment(P < 0.05).Compared with the control group,MMSE score of observation group was significantly different after 24 weeks of treatment(P < 0.05).After 24 weeks of treatment,there was a statistical difference in The ADAS-Cog score between the two groups compared with that before treatment(P < 0.05).Compared with the control group,the ADAS-cog score of the observation group was statistically different after 24 weeks of treatment(P < 0.05).After 24 weeks of treatment,there was statistical difference in ADL score between the two groups compared with before treatment(P < 0.05).Compared with the control group,the ADL score of the observation group was statistically different after 24 weeks of treatment(P < 0.05).Compared with before treatment,there was no statistical difference in TCM syndrome score after 24 weeks of treatment in the control group(P > 0.05),while there was statistical difference in TCM syndrome score after 24 weeks of treatment in the observation group(P < 0.01).Compared with the control group,the TCM syndrome scores in the observation group were statistically different after 24 weeks of treatment(P < 0.01).After treatment,the total effective rate was 52.17% in the control group and 88.00% in the observation group,and there was a statistical difference between the two groups in the efficacy of TCM syndrome(P < 0.05).Compared with the levels of serum inflammatory factors,the levels of serum TNF-? and IL-6 were statistically different between the two groups after 24 weeks of treatment(P < 0.05).Compared with the control group,the levels of TNF-? and IL-6 in the observation group were statistically different after 24 weeks of treatment(P < 0.05).(4)Safety observationBefore and after treatment,the vital signs of patients in both groups were stable,liver and kidney function,blood glucose,hematuria,fecal routine and electrocardiogram showed no obvious abnormalities.Some adverse reactions occurred during the study,including 2 cases in the control group,which were relieved significantly after symptomatic treatment.There were 3 cases in the observation group with short duration of symptoms and good tolerance,and the related symptoms disappeared without treatment.The incidence of adverse reactions was 9% in the control group and 12% in the observation group,and there was no significant difference in the incidence of adverse reactions between the two groups(P > 0.05).The safety of the two groups was further compared,and there was no statistically significant difference between the two groups(P > 0.05).Conclusion:1.Experimental studies show that:Huanglian Jiedu decoction can be effectively improved Tau/APP/PS1 transgenic AD mice showed learning and memory ability,improved pathological morphology of hippocampal tissue and protected neurons,improved neuroinflammation of hippocampal tissue,regulated polarization of microglia in hippocampal tissue and inhibited expression of TLR2,My D88,P-P38,P-ERK and P-JNK proteins in hippocampal tissue.Based on all the results,we speculated that Huanglian Jiudu Decoction could regulate microglia polarization,possibly by inhibiting the expression of TLR2,My D88,P-P38,P-ERK and P-JNK proteins in mouse hippocampus,and down-regulating the expression of TNF-? and IL-6 downstream pro-inflammatory factors,thereby protecting neurons.Improved the learning and memory function of Tau/APP/PS1 transgenic mice.In addition,different doses of Huanglian Jiedu Decoction have different differences in the observation of different indicators,but in general,medium and high doses are better than low doses.From the point of view of safety,medium doses of Huanglian Jiedu Decoction may be the ideal safe and effective dose in this study,and the dose of Huanglian Jiedu Decoction needs to be further explored in the future.2.Clinical studies showed that:Huanglian Jiedu Decoction combined with Xixin Decoction and Donepezil could effectively improve the overall cognitive function,daily living ability and TCM syndrome score of AD patients,which were all better than the control group treated with Donepezil alone.In the control group treated with donepezil alone,TCM syndrome score did not change significantly before and after treatment,indicating that TCM assisted western medicine treatment can make up for the deficiency of ineffective improvement of TCM syndrome by western medicine.In addition,huanglian Jiedu Decoction combined with Xixin Decoction and Donepezil can significantly reduce the expression of serum inflammatory factors in Patients with AD compared with donepezil,indicating that Traditional Chinese medicine may play an anti-inflammatory role. |
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