Based On The Transcriptome Sequencing,the Effects Of The Therapy Of “Peiyuan Jiannao Anshen Yizhi” On Cognitive Function And MAPKs、cGAS-STING Pathway In APP/PS1 Mice Were Investigated

Purpose:This dissertation aims to search the theoretical connotation of traditional Chinese medicine prevention and treatment of dementia by consulting and sorting out a large amount of classical Chinese medical literature represented by the Chinese Medical Code.Meanwhile,it also attempts to explore Shenghui granule,the representative of the therapeutic method of"Peiyuan Jiannao Anshen Yizhi",on its improvement of the biological cognitive function of the Alzheimer’s disease model constructed by APP/PS1 male double transgenic mice,and investigate its impact on the immune response and neuroinflammation related mechanisms based on transcriptome sequencing technology.As a result,the study is respected to provide certain research value for finding new therapeutic targets for AD,developing the diagnosis and treatment ideas for AD,and inheriting and expanding the mission of traditional Chinese medicine.Methods:1.Theoretical research:We will review and sort out the classic literature of traditional Chinese medicine through the combination of online library and offline reading,in order to excavate the theoretical basis of the prevention and treatment of dementia in traditional Chinese medicine.Besides,we attempt to explore the theoretical connotation of the prevention and treatment of AD by the method of“invigorating energy,strengthening brain,calming nerves and improving intelligence”.Based on the characteristics of its representative method Shenghui granules,together with the relevant research theories of modern medicine on AD,it is also our desire to highlight the advantages of this prescription for AD in traditional Chinese medicine.2.Experimental studies:Experiment 1:The 4-month-old APP/PS1 male double transgenic mice were used in the prevention experiment.Eighteen APP/PS1 mice were randomly divided into model group,Shenghui granule group and positive control group with 6 in each group.The 6 4-month-old male C57/BL6mice were in normal control group.The 6-month-old APP/PS1 mice were used in the treatment experiment.Thirty-six mice were randomly divided into model group,Shenghui granule group and positive control group with 12 in each group,and 12 male C57/BL6 mice were in normal control group.Shenghui granule group was orally administrated with Shenghui granule water by 13.715 g/kg/d,with a volume of 0.3 ml;the positive control group was administrated by Donepezil Hydrochloride water solution in 0.65 mg/kg/d,with a volume of 0.3 ml;normal control group and model group were gavaged with the same volume of normal saline.All administrations had lasted for 8 weeks.（1）From first day of the 8^thweek of intervention,Morris water maze was used to observe the cognitive function of mice in both prevention and treatment experiments.（2）The mice being sacrificed at the 8^thweek to collect experimental samples,Immunofluorescence was used to observe the quantitative expression of Aβand p-Tau protein in hippocampus.（3）Detection on the expression of sAPPα,sAPPβand p-Tau protein in hippocampus was applied by western blot.（4）The morphology of hippocampal CA1 region was observed by H&E staining.（5）Nissl staining was used to observe the Nissl morphology of neurons in hippocampal CA1 area.（6）Golgi staining was used to observe the density and type of dendritic spines in the primary branches of hippocampal CA1neurons.（7）The mitochondrial ultrastructures in hippocampus were observed by electron microscope.Experiment 2:After taking samples at the end of experiment 1,three fresh hippocampal tissues of mice in each group were randomly selected from the treatment experiment for transcriptome sequencing（RNA-seq）:（1）Gene expression statistics:fastp software cleaned the data of fastq files,HISAT2 software was used for reference genome comparison,HTSeq-count calculated the number of reads of each gene,and R language obtained gene expression（FPKM）.（2）The influence of thermal map and volcanic map analysis on gene expression and signal pathway enrichment:the differential expression genes（DEGs）were calculated by DESeq2,with q value<0.05 and fold change>2,and the thermal map and volcanic map were drawn by R language.（3）GO and KEGG enrichment analysis:import the DEGs of Shenghui granule treatment group and model group into metascape website for GO and KEGG enrichment analysis.（4）TRRUST analysis:The differential signal pathway between treatment group of Shenghui granule and model group was screened to determine the subsequent research pathway.（5）Screening of research mechanism:the relevant mechanism of the follow-up experimental study was screened by combining the analysis results of GO,KEGG enrichment,TRRUST and the incidence of AD.Experiment 3:After taking materials from experiment 1,three fresh hippocampal tissues of mice were randomly selected from each group in the treatment experiment for the following tests:（1）Western blot was applied to detect cGAS,STING,p-IRF3,p-ERK,p-p38MAPK and IL-1βin hippocampus for protein expression and statistic analysis.（2）The fluorescence localization and expression of dsDNA,cGAS and STING protein in hippocampus of mice in each group after fixation were observed by immunofluorescence.（3）The content of IL-1βin serum of mice in each group was analyzed by ELISA.Experiment 4:Preparation of drug-containing serum:15 2-month-old SPF grade SD rats were adaptively fed for 1 week,10 rats were given 9.495g/kg/d Shenghui granule water,1 ml per 100g body weight by gavage,and 5 rats were given equal volume of normal saline by gavage for10 days.On the 10th day,blood was taken to obtain drug-containing serum.Cell intervention:After pretreatment of BV-2 cells,Shenghui granule contained medicated serum.BV-2 cells were then divided into four groups,namely,normal control group,model group,2.5%Shenghui granule medicated serum group,and 10%Shenghui granule medicated serum group.When the cell density reached 60%,model group,2.5%Shenghui granule medicated serum group and 10%Shenghui granule medicated serum group were given 1μg/ml LPS to induce cell inflammation respectively.The subsequent intervention were implemented in 2.5%and 10%Shenghui granule medicated serum groups,which were given separately the medium containing 2.5%and 10%Shenghui granule medicated serum（without FBS）,while normal control group and model group were treated with medium containing10%control serum（without FBS）for intervention.Both interventions had lasted for 24 hours.（1）CCK-8 kit was used to determine the cell activity of different concentrations of Shenghui granule medicated serum.（2）Western blot was used for protein expression and statistical detection on extracted p-p38MAPK,p-JNK,p-ERK,IL-1βin four groups of cells.（3）Immunofluorescence was used to observe and analyze the quantitative fluorescence of p-p38MAPK protein in the four groups of cells after fixation.Results:1.Results of theoretical research:AD belongs to the category of encephalopathy in Chinese medicine and is similar to dementia and other abnormalities of the mind.In the classical literature of Chinese medicine,there are numerous records of mental disorders caused by deficiency and aging,of which deficiency is the root cause of AD.The therapeutic method of"Peiyuan Jiannao Anshen Yizhi"has a deeper connotation in TCM theory.The treatment method is based on the principle of seeking the root cause of the disease in TCM,and is aimed at the etiology and pathogenesis of AD due to the deficiency of vital energy and the deficiency of internal organs,resulting in the loss of vital energy and brain,and the decline of the mind.Based on this,we used this formula to intervene in APP/PS1 mice to elucidate the prevention and treatment mechanism of AD,which is of high value for medical research on AD.2.Results of experiment:Results of experiment 1:（1）Morris water maze resultsCompared with the normal control group,the latent escape time of the model group was prolonged（P<0.01）,while the latent escape time of the Shenghui granule group was shortened（P<0.01）.Considering the results of the number of times the prevention experiment crossed the platform,the number of times the model group crossed the platform was decreased（P<0.01）,compared with the normal control group,while the Shenghui granule group was increased（P<0.01）in comparison with the model group.The latency results of the treatment experiment showed that the latent escape time of model group was longer than that of the normal control group（P<0.01）;the latency escape time of the Shenghui granule group was shorter than that of the model group（P<0.01）.Results of the times of platform crossing in the treatment experiment showed that compared with the normal control group,the times of platform crossing in model group were decreased（P<0.01）;while in comparison with model group,the times of platform crossing in Shenghui granule group were increased（P<0.01）.（2）Results of ImmunofluorescencePrevention experiment:Compared with normal control group,the expression of Aβand p-tau in hippocampus of model group was significantly promoted（P<0.01）,while in hippocampus of Shenghui granule prevention group,the expression of Aβand p-tau was evidently declined（P<0.01）,in comparison with the model group,Treatment experiment:The expression of Aβand p-tau in hippocampus of model group,compared with normal control group,was significantly increased（P<0.01）;however,compared with the model group,the expression of Aβand p-tau in hippocampus of Shenghui granule treatment group was obviously decreased（P<0.01）.（3）Results of Western blotIn the prevention experiment,compared with normal control group,the expression of sAPPβand p-tau protein in the hippocampus of model group was significantly ascended（both P<0.01）while sAPPαdescended（P<0.05）.On the contrary,in comparison with model group,the expression of sAPPβand p-tau protein in hippocampus of Shenghui granule group and positive control group was descended（both P<0.05）,while the expression of sAPPαsignificantly was ascended（both P<0.01）.In the treatment experiment,compared with normal control group,the expression of sAPPβand p-tau protein in the hippocampus of model group significantly went upward（both P<0.01）,while the expression of sAPPαwent downward evidently（P<0.01）.In comparison with model group,the expression of sAPPβand p-tau protein in hippocampus of Shenghui granule group and positive control group was both clearly decreased（both P<0.01）,while the expression level of sAPPαboth was obviously increased（both P<0.01）.（4）Results of H&E stainingIn normal control group,the number of neurons in hippocampal CA1 area showed its abundant quantities,normal morphology and regular arrangement.In contrast,in model group,the number of neurons in CA1 area demonstrated its significantly reduced quantities,abnormal morphology（plenty of broken cytoplasm,shrunk cell membrane and deepened and deformed color of nucleus）,and very scattered arrangement.Compared with model group,the number of neurons in CA1 area of Shenghui granule group and positive control group was increased,with relatively normal morphology（with a small amount of ruptured cytoplasm and part of the membrane shrinkage,and deepened and deformed color of the nucleus）and slightly regular arrangement.（5）Results of Nissl stainingIn terms of the morphology and distribution of Nissl bodies in hippocampal CA1 area,it was basically regular in normal control group.However,in model group,this measure was decreased significantly and the morphology was different.Compared with model group,the number of Nissl bodies in CA1 area of Shenghui granule group and positive control group was increased,and the structure and arrangement were more integrated and regular.（6）Results of Golgi stainingIn model group,compared with normal control group,the density and types of the first branches of the dendritic spines in the CA1 area of the hippocampal neurons were significantly decreased（P<0.01）.On the contrary,in the hippocampus CA1 region of the Shenghui granule group and the positive group,in comparison with model group,the density and species of the first branches of the dendritic spines were significantly increased（both P<0.01）.（7）Results of electron microscopeIn terms of the morphology and structure of mitochondria in the cytoplasm,it was observed in normal control group as normal,regular,oval,with lamellar cristae in the inner plate,without fracture and defect.In contrast,it was presented in model group as abnormal,with irregular distribution,a lot of swelling and deformation,and other bubble-like changes as well,and the lamellar ridge in the inner plate was basically disappeared.While in Shenghui granule group and positive control group,it was acceptable to observe that most of them were in normal elliptical shape and regular arrangement.However,a small amount of mitochondria with abnormal swelling could be seen,and the lamellar cristae of the inner plate were scattered and broken.Results of experiment 2:（1）Statistical results of gene expressionAccording to the statistics of the basic information of transcriptome,the number of sequences in each group was more than80 million,and the proportion of total mapped reads located on the genome was more than 70%,indicating that the sample is pollution-free and the reference genome selection is appropriate,and the proportion of the number of sequences in reads mapped in proper pairs was more than 85%（see Table 1）.The log10（FPKM+1）values of 50%genes in each group were concentrated between 0.1 and1.1,and there was no significant difference between groups.The trend of curves in each group was similar,with a peak value of about1,and the value of log10（FPKM）was mainly concentrated in the range of 0-2.（2）Results from the influence of thermal map and volcanic map analysis on gene expression and signal pathway enrichmentCompared with normal control group,84 genes in model group were revealed significant differences,including Abcb11,Epb42,Gm40447and so forth.Compared with model group,there were also considerable dissimilarities in 151 genes in Shenghui granule group,including Adgre4,Il1b,Il17f,etc.（3）Results of GO and KEGG enrichment analysisThe differential gene analysis of GO and KEGG enrichment between Shenghui granule group and model group found that the differences were demonstrated rather obviously in immune system through the biological process,cell membrane in cell composition and the receptor binding in molecular function.Meanwhile,the relevant signal pathways such as G protein-coupled receptor binding,inflammatory response,defense response to fungus,and antimicrobial humoral response might play an important role and participate in the therapeutic mechanism of Shenghui granule on AD.（4）Results of TRRUST analysisThrough the differential gene analysis and comparison of TRRUST between treatment group of Shenghui granule and model group,it was found that Sp1,Jun,Sox9,Nfkb1 and other transcription factor-related signaling pathways played a major regulatory role after intervention.（5）Screening results of research mechanismTo combine the analysis of GO and KEGG enrichment and TRRUST,together with the incidence of AD,MAPKs and cGAS-STING were selected as the focus of this study.Results of experiment 3:（1）Results of Western blotCompared with normal control group,in model group,the expression level of cGAS,STING,p-IRF3,p-ERK,p-p38MAPK and IL-1βprotein were all significantly enhanced（all P<0.01）.In comparison with model group,however,the expression of cGAS,STING,p-IRF3,p-p38MAPK and IL-1βprotein in Shenghui granule group and positive control group,together with p-ERK protein in Shenghui granule group,were all evidently diminished（all P<0.01）,while the expression of p-ERK protein in positive control group showed no difference（P>0.05）.（2）Results of ImmunofluorescenceCompared with normal control group,the expression of dsDNA,cGAS and STING protein in the hippocampus in model group was significantly increased（all P<0.01）.In contrast,to compare with model group,the expression of dsDNA,cGAS and STING protein in the hippocampus of mice in Shenghui granule group and positive control group was decreased evidently（all P<0.01）.（3）Results of ELISACompared with normal control group,the content of IL-1βof mice serum in model group was significantly increased（P<0.01）.However,in comparison with model group,the content of serum IL-1βin Shenghui Granule group and model group was evidently decreased（both P<0.01）.Results of experiment 4:（1）Results of CCK-8Compared with 10%medicated serum,the cell activity of 20%medicated serum was significantly decreased（p<0.05）.In comparison with other groups,there was no obvious difference in the cell activity of 10%medicated serum and the difference was revealed with no statistical significance（p>0.05）.The results showed that the serum containing 0.1%,1%,5%and 10%of Shenghui granule produced no effect on cell activity.（2）Results of Western blotIn model group,to compare with normal control group,the expression of p-p38MAPK,p-ERK,p-JNK and IL-1βprotein of BV-2 cells was significantly up-regulated（all P<0.01）.In contrast to model group,both in 2.5%and 10%of Shenghui granule medicated serum groups,the expression of p-p38MAPK,p-ERK,p-JNK and IL-1βprotein was evidently down-regulated（all P<0.01）.（3）Results of ImmunofluorescenceCompared with normal control group,the expression of p-p38MAPK protein in BV-2 cells in model group was significantly increased（P<0.01）.While in 2.5%and 10%Shenghui granule-containing serum groups,in comparison with model group,the expression of p-p38MAPK protein was significantly decreased（both P<0.01）.Conclusion:1.As a neurodegenerative disease,AD is similar to the symptoms of many of the mental abnormalities represented by dementia in Chinese medicine.Based on syndrome differentiation and treatment in traditional Chinese medicine,the basic pathogenesis is the deficiency in origin.From the perspective of the concept of the whole,its pathogenesis is mainly due to the deficiency of the five internal organs and vital energy.To synthesize the correlation between deficiency and spirit in classic Chinese medicine,we discussed AD from deficiency and treated AD by supplementing deficiency,established the principle of treatment of deficiency and the method of invigorating energy,and used Shenghui granule,the representative of the therapeutic method of"Peiyuan Jiannao Anshen Yizhi"to treat AD according to the symptoms.2.Shenghui granule,the representative of the therapeutic method of"Peiyuan Jiannao Anshen Yizhi",can protect and improve the cognitive function of the AD model mice constructed by APP/PS1,reduce the accumulation of Aβand p-Tau in their hippocampus,and repair their neuronal damage.3.Shenghui granule has a significant regulative effect on immune response,fungal defense,anti-inflammatory and other aspects of AD model mice constructed by APP/PS1,including the remarkable influence on transcription factors such as JUN and NF-κB.4.The improvement of cognitive function of AD model mice constructed by APP/PS1,the clearance of Aβand p-tau,and the repair of neuronal damage,all constructed by Shenghui granules,may be related to the inhibition of neuroinflammation mediated by MAPKs and cGAS-STING pathway.5.The dislocation gene caused by excitatory neurotoxicity and the immune response to neurons may be the main cause of neuroinflammation.While Shenghui granule may inhibit the secretion of neuro IL-1βto block the continuous effect of the pathway of MAPKs on neurons,and alleviate the inflammatory polarization of microglia to improve the occurrence of AD neuroinflammation.