| Objective: By observing the effect of Jiedu Yizhi Formula on cognitive impairment in Alzheimer’s disease(AD)patients with spleen kidney deficiency and phlegm turbidity obstructing the orifices syndrome,analyzing the improvement of AD cognitive domain by this formula,exploring the correlation between traditional Chinese medicine syndrome and cognitive function,daily living ability,and evaluating the efficacy and safety of Jiedu Yizhi Formula in treating mild to moderate AD.By observing the behavioral indicators of each group of mice,the effect of Jiedu Yizhi Formula on the learning and memory ability of APP/PS1 mice was revealed.By applying pathological and metabolomics related experimental techniques,the improvement of typical pathological characteristics of AD mouse brain tissue by Jiedu Yizhi Formula and its impact on small glial cell activation markers and TREM2 pathway target proteins were clarified.Method:1 Clinical researchCollect AD patients who met the inclusion criteria from the Brain Disease Outpatient Department and Treatment Area of the Third Affiliated Clinical Hospital of Changchun University of Traditional Chinese Medicine from December 2022 to December 2023.Using a randomized controlled method,the enrolled mild and moderate AD patients were randomly divided into a JDYZF group and a Donepezil group.They were treated with modified Jiedu Yizhi Formula and Donepezil Hydrochloride Tablets(trade name: An Lishen),respectively.Both groups were given cognitive and behavioral training and health education for AD,with a course of 24 weeks.Before treatment,12 weeks of treatment,and 24 weeks of treatment,Western medicine scales,dementia traditional Chinese medicine symptom grading and quantification scales,and dementia classification and syndrome elements were conducted.Table measurement to evaluate the effects of drugs on cognitive function and daily living abilities.2 Experimental researchFifty male APP/PS1 transgenic mice were randomly divided into a model control group,a donepezil group,and a low,medium,and high dose group of Jiedu Yizhi Formula.An additional 10 C57BL/6 mice were selected as blank control groups.The Jiedu Yizhi Formula group was orally administered with decoction at concentrations of 1.78g/kg,3.56g/kg,and 7.12g/kg.The Western medicine control group was orally administered with a concentration of 5mg/kg of donepezil solution.The model control group and blank control group were orally administered with an equal volume of physiological saline once a day for 60 consecutive days.Observing the behavior of mice through Morris water maze experiment,observing the pathological changes in the brain of each group of mice through HE staining,Nissl staining,and silver plating staining,detecting the metabolic status of brain tissues in each group of mice through non targeted metabolomics technology,observing the expression of microglial cell markers IBA1,M1 marker CD16,M2 marker CD206 through immunofluorescence technology,and analyzing the expression of blood lipids and serum inflammatory markers such as IL-6,TNF-α,TGF-β,and IL-10 using an enzyme-linked immunosorbent test.Use protein immunoblotting and immunohistochemistry to identify the target proteins of the TREM2 and p38 MAPK pathways.Result:1 Clinical research(1)Statistical analysis was conducted on the basic information and scale scores of 62 enrolled patients,revealing no significant difference in baseline data between the two groups(P>0.05),indicating comparability.The study found that the number of patients with blood lipid abnormalities in their current medical history was the highest(77.42%).Our formula demonstrated the ability to enhance the MMSE,Mo CA,and ADAS-cog scores of AD patients compared to pre-treatment levels,showing significant improvements(P<0.01).Moreover,our prescription led to a significant reduction in scores related to traditional Chinese medicine symptoms like forgetfulness,intellectual decline,and personality quirkiness(P<0.01).Notably,it also improved symptoms such as facial dullness,abnormal behavior,and unclear speech,with statistical significance(P<0.05).Furthermore,our prescription significantly enhanced the traditional Chinese medicine syndrome scores of patients presenting with conditions like spleen deficiency,kidney deficiency,marrow deficiency,phlegm turbidity,and blood stasis(P<0.01).In comparison to the donepezil group,the Jiedu Yizhi Formula did not exhibit a statistically significant improvement in the overall scores of MMSE,Mo CA,and ADAS-cog in AD patients(P>0.05).Yet,it notably enhanced the directional and abstract ability scores within the cognitive domain of AD patients,showing significant improvement(P<0.05).The total effective rate of MMSE score improvement was significantly higher in the Jiedu Yizhi Formula(64.52%)than in the donepezil group(45.16%)(P<0.05).Though no statistically significant difference was observed in the improvement efficiency of Mo CA and ADAS-cog scores between the two groups,the Jiedu Yizhi Formula group showed higher scores compared to the donepezil group.Jiedu Yizhi Formula also demonstrated its ability to enhance the ADL score of AD patients(P<0.05)and significantly improve the HAMD score of patients(P<0.01).It effectively reduced traditional Chinese medicine symptoms of forgetfulness,intellectual decline,facial dullness,peculiar personality,abnormal behavior,and unclear speech in AD patients,with statistically significant differences(P<0.05).Additionally,significant improvements were witnessed in traditional Chinese medicine syndromes like spleen deficiency,kidney deficiency,marrow deficiency,phlegm turbidity,and blood stasis in patients(P<0.01).(2)Correlation analysis found that the spleen deficiency syndrome of 62 patients before treatment had the highest correlation with ADAS-cog and HAMD scores,with correlation coefficient r values of 0.30,both of which were statistically significant(P<0.05),followed by strong correlation between phlegm turbidity and Mo CA and MMSE scores,with correlation coefficient r values of 0.29(P=0.02)and 0.27(P=0.03),both of which were statistically significant(P<0.05).After 24 weeks of treatment with Jiedu Yizhi Formula,an analysis was conducted on the traditional Chinese medicine syndrome and Western medicine scale scores of 31 patients.The spleen deficiency syndrome had a strong positive correlation with ADAS-cog(r=0.46),and the blood stasis syndrome also had a strong positive correlation with ADAS-cog(r=0.58),all of which had significant statistical significance(P<0.01).Correlation analysis was conducted on the difference in score improvement.Phlegm turbidity syndrome had a moderate correlation with ADAS-cog and ADL,with r values of 0.46 and 0.45,respectively,and both had statistical differences(P<0.05);There is a moderate correlation between blood stasis syndrome and ADL(r=0.41,P<0.05).2 Experimental research(1)On the 5th day of the Morris water maze experiment,compared with the model group mice,the escape latency and average swimming distance of mice in the middle and high dose groups of Jiedu Yizhi Formula and the donepezil group were significantly shortened(P<0.01).In the space exploration experiment,compared with the model group mice,the middle and high-dose groups of Jiedu Yizhi Formula and the donepezil group mice crossed the platform area more times,and the first arrival time on the platform was shortened(P<0.05).The residence time and movement distance in the target quadrant were significantly increased(P<0.01).The application of HE and Nissl staining methods confirmed that Jiedu Yizhi Formula can promote the relatively complete morphology and organic arrangement of neurons in the frontal cortex and hippocampus of AD mice,increase the number of Nissl bodies in some areas,and silver staining confirmed that this formula can regulate some neurofibrillary tangles in the hippocampus.Application of immunohistochemistry to confirm that understanding Jiedu Yizhi Formula can reduce Aβ excessive deposition of proteins.(2)Non targeted metabolomics analysis showed that the most identified substances in this study were lipids and lipid molecules,with a total of 380 species.Comparing the differential metabolites between the blank control group and the model control group with those between the detoxification and puzzle formula group and the model control group,it was found that there were significant differences(P<0.05)in 17 endogenous metabolites,including 1-methyluric acid and D-Quinovose.The correlation between 1-methyluric acid and phosphatidylglycerol 34:3 was strong(r=0.77,P<0.05),and the differential metabolites were enriched in caffeine metabolism and glycerophospholipid metabolism pathways(P<0.05).(3)Immunofluorescence experiments showed that compared with the model control group,the Jiedu Yizhi Formula group could reduce the expression levels of M1 markers CD16 and i NOS protein in mouse brain tissue(P<0.05),and increase the expression levels of M2 markers CD206 and Arg-1 protein(P<0.05).The expression of inflammatory factors in the serum of each group of mice was observed using ELISA method.Compared with the model group,the IL-6 content in the blank group was lower(P<0.01),while the IL-6 expression level in the donepezil group and the low-dose group of Jiedu Yizhi Formula slightly decreased(P>0.05);The expression level of IL-6 was significantly reduced in the middle and high dose groups of Jiedu Yizhi Formula(P<0.05).The expression levels of TNF-α in the serum of mice in each group showed that compared with the model group,the expression levels of TNF-α in the middle and high dose groups of Jiedu Yizhi Formula were significantly reduced(P<0.05);There was no significant change in the expression level of TNF-α between the low-dose group of Jiedu Yizhi Formula and the donepezil group(P>0.05).The content of TGF-β in the serum of normal C57 mice is extremely low,while it is higher in APP/PS1 transgenic mice,and it changes with different doses of administration.Compared with the model group,TGF-β in the donepezil group and Jiedu Yizhi Formula are higher,The expression level of the low-dose group was slightly increased(P>0.05);And the TGF-β expression level in the middle dose group of Jiedu Yizhi Formula was increased(P<0.05),and The expression level of TGF-β was found in the high-dose group of Jiedu Yizhi Formula significantly increased(P<0.01).The results of IL-10 expression levels in the serum of each group of mice showed that compared with the model group,there was no difference in IL-10 expression levels between the donepezil group and the low-dose group of Jiedu Yizhi Formula(P>0.05);The expression level of IL-10 in the middle and high dose groups of Jiedu Yizhi Formula increased,and the difference was statistically significant(P<0.05).In the detection of p38 MAPK pathway target proteins in the hippocampal tissues of different groups of mice,it was found that there was no significant difference in p38,ERK,and JNK proteins among each group(P>0.05),while the phosphorylation level of proteins changed significantly.Compared with the model group,the expression level of p-p38 protein in the brain tissue of mice in the middle and high dose groups of Jiedu Yizhi Formula decreased significantly(P<0.05);The expression level of p-p38 protein in brain tissue was slightly reduced in the low-dose Jiedu Yizhi Formula and donepezil groups.The expression levels of p-JNK protein in the brain tissue of mice in the donepezil group and high-dose group of Jiedu Yizhi Formula were significantly reduced compared to the model group(P<0.01).The protein expression levels of p-JNK in the medium dose group of Jiedu Yizhi Formula were also significantly reduced(P<0.05),while the protein expression levels of p-JNK in the low-dose group of Jiedu Yizhi Formula were not significantly different.Compared with the model group,the protein expression level of p-ERK in the brain tissue of mice in the donepezil group and the middle and high dose groups of Jiedu Yizhi Formula decreased significantly(P<0.05);The expression level of p-ERK protein in the brain tissue of the low-dose group of Jiedu Yizhi Formula showed little change(P>0.05).(4)Compared with the blank control group,the expression level of TREM2 protein in the hippocampus of the model group mice was significantly down regulated(P<0.01).Compared with the model group,the expression level of TREM2 protein in the treatment group was upregulated.There was a difference in the upregulation of TREM2 protein expression between the middle and high-dose groups of Jiedu Yizhi Formula and the donepezil group(P<0.05),while the low-dose group of Jiedu Yizhi Formula was slightly upregulated(P>0.05).Compared with the model group,the high-dose group of Jiedu Yizhi Formula showed a significant upregulation of DAP12 protein expression(P<0.01),while the medium dose group of Jiedu Yizhi Formula and the donepezil group showed differences in protein expression upregulation(P<0.05).There was no significant difference in the expression level of Syk protein among different groups(P>0.05),while phosphorylated Syk protein changed.Compared with the normal group,the relative expression level of p-Syk protein in the model group was significantly down regulated(P<0.01).Compared with the model group,the expression level of p-Syk protein in the low-dose groups of donepezil and Jiedu Yizhi Formula did not change significantly(P>0.05),while there was a difference in the upregulation of protein expression between the medium and high-dose groups of Jiedu Yizhi Formula(P<0.05).No Aβ protein expression was detected in normal mice,and abundant Aβ protein deposition was found in the hippocampus and frontal cortex of APP/PS1 mice.Compared with the model group,the down-regulation of Aβ protein expression in the low-dose group of the antidote prescription was not significant(P>0.05),and the down-regulation of protein expression in the middle-dose group of the antidote prescription was differentiated(P<0.05),and the down-regulation of Aβ protein expression in the high dose of the antidote prescription and Donepezil groups was significant(P<0.01).Aβ protein expression was significantly down-regulated(P<0.01).Compared with the model group,the down-regulation of MEK protein expression in the middle-and high-dose groups of Jiedu Yizhi Formula was differentiated(P<0.05),and the down-regulation of MEK protein expression in the donepezil group was significant(P<0.01).Serum lipoprotein assay in mice revealed that compared with the model group of mice,both the medium and high doses of Jiedu Yizhi Formula could reduce the serum TG content of mice with difference(P<0.05),and for the effect on serum CHO and LDL,a significant reduction in the expression level was found only in the high dose group of Jiedu Yizhi Formula(P<0.05),and no significant difference in the lipid content of the rest of the treatment groups was observed(P>0.05).Conclusion:1.Jiedu Yizhi Formula can improve the cognitive domain of AD patients,with the most significant improvement in directional and abstract abilities.This formula can improve symptoms such as forgetfulness and intellectual decline,and significantly alleviate dementia symptoms such as spleen deficiency,kidney deficiency,marrow deficiency,phlegm turbidity,and blood stasis.2.Jiedu Yizhi Formula can improve memory and cognitive dysfunction in APP/PS1 transgenic mice,promote organic arrangement of some neurons in the frontal cortex and hippocampus of AD mice,reduce partial nuclear atrophy,increase the number of Nissl bodies,and reduce neurofibrillary tangles in certain regions.Recall metabolites such as 1-methyluric acid and D-Quinovose,regulate caffeine metabolism and glycerophospholipid metabolism pathways.3.Jiedu Yizhi Formula can regulate the transformation of microglia in the hippocampus of APP/PS1 transgenic mice from M1 type to M2 type,effectively improving neuroinflammation in AD mice.Our formula may exert therapeutic effects on neuroinflammation by activating the TREM2 pathway to regulate metabolic disorders and polarization of microglia. |
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