Study On The Mechanism Of Calming Nerves And Invigorating Intelligence Therapy For AD Based On P38 MAPK Signal Pathway

Objective:Discuss the connotation of"spirit"and"intelligence"from the theory of traditional Chinese medicine,and explore the relationship between spirit,soul,mind,intelligence and thinking in combination with the theory of five viscera,and on this basis,further explore the internal theoretical basis of the treatment of AD with the method of calming the nerves and improving intelligence.The effect of Shenghui granule on p38 signal pathway of scopolamine AD model rats was observed through animal experiments.In combination with serum pharmacology,the effect of Shenghui Granule containing serum on p38 signal pathway of APPsw-shsy-5y cells was further observed.The serum pharmacochemistry,network pharmacology and molecular docking technology were used to explore the material basis of efficacy of Shenghui Granule and its mechanism of action in treating AD.Molecular dynamics simulation was used to observe the action mechanism of active ingredients on key action targets.Method:1.Explore the connotation of"spirit"and"intelligence"in traditional Chinese medicine,the relationship between"spirit","intelligence"and the five viscera gods,and the relationship between sleep and dementia by consulting relevant literature.To further explore the internal theoretical basis of the treatment of AD with the method of calming the nerves and strengthening the intelligence and the theoretical basis of the treatment of AD with Shenghui Granule.2.Experiment 1:40 male SD rats were randomly divided into four groups（10 in each group）,namely blank group,model group,Shenghui granule group,and donepezil group.The administration treatment was as follows:blank group（ultrapure water gavage）,model group（ultrapure water gavage）,Shenghui granule group（Shenghui granule 4.86g·kg-1·d-1,gavage）and donepezil group（donepezil 1 mg·kg-1·d-1,gavage）.The experiment lasted for 28 days.In the first 18 days,rats in each group were treated by gavage.In addition to gavage,behavioral experiments were also conducted on the 19th to28th days.Rats were treated by intraperitoneal injection 30 minutes before the test.In addition to the blank group,the other groups were intraperitoneally injected with scopolamine hydrobromide injection 5 ml·kg-1·d-1 every day.The blank group was intraperitoneally injected with the same dose of normal saline.The cognitive ability and anxiety state of rats in each group were evaluated by water maze and open field test.HE staining was used to observe the pathological damage degree of EC and CA1 in rat brain tissue.The activity of neurons in EC and CA1 regions was observed by c-fos immunofluorescence staining.Use WB to detect p-p38,p38,Tau,p-Tau,Bcl-2 and Bax proteins.3.Experiment 2:First of all,the AD pathological neural cell model of APPsw-SHSY-5Y cell line was constructed,and then the drug-containing serum of Shenghui Granule was prepared.The most suitable culture concentration of drug-containing serum was selected through CCK8 experiment.Observe the expression of APP and p-p38 in APPsw-SHSY-5Y cells by immunofluorescence and WB,and detect extracellular A by ELISAβ_1-42level.Use U46619 to activate p38 and observe whether the medicated serum of Shenghui Granule can reduce the activation of p38 pathway.4.Experiment 3:Prepare the medicated serum of Shenghui Granule,use the triple four-stage liquid chromatography-mass spectrometry,Waters Acquisition UPLC BEH C18column,and Agilent Mass Hunter Workstation analysis software to compare the medicated serum,blank serum,and drug monomer mass spectra of Shenghui Granule,and analyze and identify the blood components of Shenghui Granule.Use Pub Chem,Swiss Target Prediction and Pharmapper databases to obtain blood component targets,and use Alzheimer’s disease as the keyword to collect AD disease targets from Gene Cards and OMIM.Venny was used to analyze the intersection between the target of blood component of Shenghui granules and AD disease target to obtain the possible target of Shenghui granules.Use Metascape to conduct KEGG analysis on the intersection target and observe whether it is enriched into MAPK pathway.Use molecular docking technology to dock key targets and analyze molecular-protein interactions.5.Experiment 4:Use molecular dynamics simulation to simulate the key targets for 30ns,and use RMSD,protein and small molecule distance analysis to observe the stability of the complex.Packmol was used to model protein and small molecules at a ratio of 1:8,and molecular dynamics simulation was used again to observe the movement of protein and small molecules within 50 ns.After the simulation,the distance analysis between protein and small molecule was used to observe the proximity of small molecule and binding site,and the movement track was sampled and observed.The ligand receptor binding energy was calculated using MM-GBSA method.Result:1.Theoretical research:Traditional Chinese medicine"gods"are divided into broad gods and narrow gods.The broad sense of God encompasses the external manifestations of the entire human body’s life activities,while the narrow sense of God refers to high-level spiritual activities such as human spirit,emotions,consciousness,and thinking."Essence"is the primary material foundation of"God","blood"is an important source of"God",and the essence of water and grain is the material guarantee of"God".The five zang organs contain five gods,which are closely related to human spiritual,emotional,and willpower activities.God,soul,soul,will,will,thought,consideration,and intelligence are the manifestations of the gradually advanced and complex functional activities of various organs,reflecting the unique observation and thinking of traditional Chinese medicine on the physiological,psychological,and spiritual states of the human body.Human thinking and decision-making activities are jointly influenced by the five viscera and six viscera,and the process includes multiple stages such as sensation,cognition,thinking,decision-making,and action.The five viscera are closely related to dementia and insomnia.There is a certain theoretical basis for regulating the five zang organs to calm the five spirits and treating dementia by calming the five spirits.2.Experimental study:Experiment 1:（1）Water maze behavioral experiment results:after 5 days of training,the escape latency of all groups gradually decreased.The escape latency of rats in blank group,Shenghui granule group and Donepezil group was 21.7±6.3 s,37.9±9.8 s and 31.1±8.8 s respectively.The escape latency of model group was 52.4±9.5 s,which was significantly higher than that of other groups.The number of times of platform crossing showed that compared with the blank group（4.4±0.8）,the number of times of platform crossing in the model group（1.1±0.6）was significantly reduced（P<0.01）;Compared with the model group,Shenghui Granule group（3.1±1.1）and Donepezil group（3.5±0.8）were higher than the model group（P<0.01,P<0.01）.The result of residence time ratio in the target quadrant showed that the percentage of residence time in the target area in the model group was（34.0±4.3%）,which was significantly lower than that in the blank group（24.1±3.4%）（P<0.01）;The percentage of swimming time in the target area of Shenghui Granule group（31.0±4.3%）and Donepezil group（28.8±3.1%）was higher than that of the model group（P<0.05,P>0.05）.（2）The results of open-field experiment:the time of rats staying in the central area showed that compared with the blank group（4.5±2.0 s）,the time of rats staying in the model group（1.2±0.7 s）was significantly reduced（P<0.01）;Compared with the model group,the rats in Shenghui Granule group（3.2±0.9 s）and Donepezil group（3.4±1.0 s）spent significantly longer in the central area（P<0.05,P<0.05）.The number of rats entering the central area showed that compared with the blank group（3.3±0.9）,the number of rats entering the central area in the model group（0.7±0.6）was significantly reduced（P<0.01）;Compared with the model group,the number of rats entering the central area in Shenghui Granule group（2.2±0.6）and Donepezil group（1.7±0.8）increased significantly（P<0.01,P>0.05）.The results of total exercise distance of rats showed that compared with the blank group（3467.2±692.4 cm）,the total exercise distance of rats in the model group（652.3±272.9 cm）was significantly reduced（P<0.01）;Compared with the model group,the total exercise distance of rats in Shenghui Granule group（2464.2±699.7 cm）and Donepezil group（2701.0±585.8 cm）were significantly increased（P<0.01,P<0.01）.（3）HE staining results:The neurons in the EC area and the CA1 area of the hippocampus of the blank group were arranged in order,while the neurons in the model group could observe obvious eosinophilic cytoplasm,nuclear pyknosis,neuronal swelling,pyramidal cell contraction and vacuolation.Compared with the model group,the number of eosinophilic stained neurons in the Shenghui Granule group and the Donepezil group after treatment was less,showing slight neurotoxicity,and nuclear pyknosis and vacuolation were alleviated.（4）C-fos fluorescence staining results:compared with the blank group,the c-fos fluorescence intensity in the hippocampus and EC region of the model group decreased.Compared with the model group,the c-Fos fluorescence intensity of Shenghui granule group and donepezil group increased.（5）WB detected the expression of p38MAPK signal pathway related protein in the hippocampus:the expression of p38 MAPK in the hippocampus of rats in the model group was significantly higher than that in the blank group(P;Compared with the blank group,the expression of Tau protein in the hippocampus of the model group was significantly higher than that of the blank group(P;Compared with the model group,the expression of Tau protein in Shenghui Granule group and Donepezil group decreased（P<0.05,P<0.05）.Compared with the blank group,the expression of Bax protein in the hippocampus of the model group was significantly higher than that of the blank group(P;Compared with the model group,the expression of Tau protein in Shenghui Granule group and Donepezil group decreased（P<0.01,P<0.01）;Compared with the blank group,the expression of Bcl-2 protein in the hippocampus of the model group was significantly lower than that of the blank group（P<0.01）;Compared with the model group,the expression of Bcl-2 protein in Shenghui Granule group and Donepezil group increased（P<0.05,P<0.01）.Experiment 2:（1）The results of CCK8 showed that the optimal concentration of the drug-containing serum culture of Shenghui granules was 10%.（2）APP immunofluorescence and WB results:Compared with empty plasmid group,APP fluorescence expression in APPsw group increased;Compared with the APPsw group,the fluorescence intensity of APP in the blank serum group was not significantly different,while the fluorescence intensity of the drug-containing serum group was significantly reduced.The results of WB test were consistent with those of immunofluorescence staining.Compared with the empty plasmid group,the expression of APP protein in APPsw group increased（P<0.05）.Compared with the APPsw group,there was no significant difference in the blank serum group（P>0.05）,while the expression of APP protein in the drug-containing serum group was significantly lower（P<0.05）.（2）Results of p-p38 immunofluorescence and WB:Compared with empty plasmid group,the expression of p-p38 fluorescence in APPsw group was increased;Compared with the APPsw group,the fluorescence intensity of p-p38 in the blank serum group was not significantly different,while the fluorescence intensity of the drug-containing serum group was significantly reduced;WB test results showed that there was no significant difference in the expression of p38 in each group;Compared with empty plasmid group,the expression of p-p38 protein in APPsw group was increased（P<0.05）;Compared with the APPsw group,there was no significant difference in the blank serum group（P>0.05）,while the expression of p-p38 protein in the drug-containing serum group was significantly lower（P<0.05）.（3）ELISA results:compared with empty plasmid group,APPsw group AβThe level of 1-42 increased significantly（P<0.01）;Compared with APPsw group,blank serum group AβThere was no significant difference in the level of 1-42（P>0.05）,but the level of serum containing drugs was significantly lower（P<0.05）.（4）p38 pathway activation test results:compared with APPsw cell group,the expression of p-p38,APP and Bax protein in APPsw+drug-containing serum group decreased（P<0.05）,while the expression of Bcl-2protein increased（P<0.05）.Compared with APPsw cell group,after using p38 activator U46619,the expression of p-p38,APP and Bax protein in APPsw+U46619 group increased significantly（P<0.05）,and the expression of Bcl-2 protein decreased significantly（P<0.05）.Compared with APPsw+U46619 group,the expression of p-p38,APP and Bax protein in APPsw+U46619+medicated serum group decreased to a certain extent（P<0.05）,and the expression of Bcl-2 protein increased to a certain extent（P<0.05）.Experiment 3:（1）By comparing the mass spectrum information data in the standard sample,drug-containing serum and blank serum,identify four components of Shenghui Granule,including spironoside,mononoside,loganine and magnolia alkaloid.（2）The four active components of Magnolia alkaloid,loganin,mononoside and spironoside were predicted in TCMSP,Pharm Mapper and Swiss Target Prediction databases.After screening,173 component action targets were obtained,and 9599 anti-AD targets were obtained from Gene Cards database.Input compound target and disease target into Venny 2.1 to make Venny map,and obtain 146 intersection targets.（3）The signal pathway was obtained by KEGG enrichment analysis,and 133 pathways were screened according to P<0.05.These include MAPK signal pathway.There are 11 targets enriched in this pathway,including MAPK14,CSF1R,EGFR,EPHA2,FGFR1,HRAS,HSPA8,KDR,PDGFRA,PDGFRB,PPP5C,PRKCG,MAP2K1,TNF,TP53,MAPKAPK2,IRAK4.（4）The results of molecular docking showed that loganin had the best binding with MAPK14,the binding energy was-12.7 kcl/mol,and the residues were ASP168 GLU71,LYS53,ARG173,VAL83,LEU75,ILE84,MET78,LEU74,ILE146,and ILE147;Spinozol and TNF-αThe binding energy is-12.7 kcl/mol,and the residues are B:LYS89,B:GLY112,B:TYR142,B:SER51,B:LEU111,A:GLY112.Experiment 4:（1）The conformation of p38 gradually stabilized after 20 ns;After 11 ns of binding between loganin and p38,loganin began to gradually disengage from the binding site;TNF-αThe conformation is gradually stable after 17 ns,and in the 30 ns simulation,Spinozol and TNF-αThe combination is relatively stable.（2）In the simulation results of50ns loganin and p38,no small molecule was found to bind to the binding site;Spinoxone and TNF at 50ns-αIn the simulation results,lig8 was found to bind to the binding site at45-50 ns.（3）Four time points were selected for sampling analysis,which were 40.00 ns,42.15 ns,44.31 ns and 46.46 ns respectively,and the distance between the molecule and the binding site was 2.47 nm,1.62 nm,0.97 nm and 0.49 nm respectively.（4）For Spinozol and TNF-αThe movement trajectory of the complex was calculated using MM-GBSA method to calculate the receptor-ligand binding energy.The results showed that the van der Waals free energy contributed the most to the binding free energy;The binding energy is-12.1544kcal/mol at 46.46 ns;The lowest binding energy in the whole motion trajectory is-24.0964kcal/mol,and the time point is 46.14 ns.Conclusion:1.The"Shen"and"Zhi"in traditional Chinese medicine are linked by the five internal organs,and the two are closely related.The important theoretical basis for the treatment of dementia with the method of calming the mind and benefiting the mind is to regulate the five internal organs to calm the five spirits,and to calm the five spirits to treat dementia.2.Shenghui granule has protective effect on EC and CA1 regions of AD model rats,and may play a therapeutic role through p38 MAPK signal pathway.3.Shenghui Granule medicated serum can reduce APP expression and p38 protein phosphorylation in APPsw-SHSY-5Y cells,and the mechanism may be related to the regulation of p38 MAPK pathway.4.Identify 4 blood components of Shenghui Granule,which are magnolian,loganine,mononucleoside and spironoside.Network pharmacology research shows that the mechanism of Shenghui Granule in treating AD may be related to MAPK pathway.Loganin may have MAPK14 inhibition,and spironoxone may have TNF-αinhibition.5.Spinosok and TNF-αThe dimer binding site binds,suggesting that spironolactone may inhibit TNF-αActive to treat AD.