The Preventive Effect Of Compound Danshen Dripping Pills On Cognitive Impairment In KK-Ay Mice Based On Network Pharmacology And Transcriptomics

ObjectiveUsing network pharmacology and transcriptomics sequencing technology to explore the effect of Compound Danshen Dripping Pills on cognitive impairment in spontaneous type 2 diabetes model KK-Ay mice.MethodThirty-two 6-week-old male KK-Ay mice were randomly divided into model group(DM),low-dose Compound Danshen Dripping Pills group(CL,13.5 mg/kg/d),medium-dose Compound Danshen Dripping Pills group(CM,27 mg/kg/d)and high-dose Compound Danshen Dripping Pills group(CH,40.5 mg/kg/d).Eight male C57BL/6J mice of the same age were employed as the normal control group(NC).After one week of adaptive feeding,the normal control group was fed with normal food,and the other groups were fed with high-sugar and high-fat food.Four weeks later,blood samples were collected from the caudal vein of the mice to measure the random blood glucose.Blood glucose>13.9 mmol/L for two consecutive times was regarded as the mouse model of type 2 diabetes established successfully.Every day,each treatment group was given Compound Danshen Dripping Pills(CDDP)with different concentrations,and the model group and the normal control group were given the same volume of normal saline.After 12 weeks of gavage,the Morris water maze space exploration experiment was used to test the spatial learning and memory ability of the mice,and the changes in the hippocampus volume of the mice were detected by MRI imaging.The hippocampal specimens of mice were isolated immediately after the mice were killed,part of them were used for mice hippocampus morphology observation,the left part was used for the application of the transcriptome study in mice hippocampal tissue specificity by high-throughput sequencing,screened the differential genes of CDDP improving diabetic cognitive dysfunction,and bioinformatics analysis.Meanwhile,network pharmacology was applied to explore the effect of CDDP on diabetic cognitive impairment(DCI).Genes related to cognitive dysfunction of diabetes were retrieved from six databases,including TTD,DrugBank,OMIM,GAD,PharmGKB,and DisGeNET.Using TSMSP database to retrieve the three taste traditional Chinese medicine(salvia miltiorrhiza,notoginseng,and borneol)of CDDP,the effective components of target genes were investigated by TCMSP database,using Cytoscape3.7.1 software and plug-ins to analyze target genes and build targets network graph of CDDP composition.Combined the above results with the GO analysis and KEGG pathway annotations to build component-targets-channel visualization network.Finally,combined the results of transcriptomics and network pharmacology,and verified the finding in hippocampal tissues of mice.Results1.The body weight and blood glucose of KK-Ay mice were higher than that of C57BL/6J mice(P<0.001).Compared with DM group,the weight decreased in the group treated with CDDP(P<0.05),and the blood glucose also declined,but there was no significant difference(P>0.05).2.The results of Morris water maze experiment showed that compared with NC group,the escape latency period of mice in the DM group was significantly prolonged(P<0.01),and the times of crossing the platform were reduced(P<0.001).Compared with DM group,the escape incubation period of mice in CL?CM and CH groups was shortened(P<0.05),and the number of times of crossing the platform were increased(P<0.05).3.MRI results showed that,compared with NC group,low signal areas were observed in the hippocampus of KK-Ay mice,and the hippocampal volume of the DM group was significantly decreased(P<0.001).Compared with DM group,the hippocampal volume of mice in CM and CH group was increased(P<0.05).4.Compared with NC group,the expression levels of TNF-?,IL-1?,and IL-6 in DM group were increased(P<0.01).Compared with the DM group,the expression levels of the above inflammatory factors in mice treated with CDDP were decreased to different degrees(P<0.05).5.For the results of HE staining,Nissl staining,and TEM images of the hippocampal tissues,the neurons in the CA1 region of the hippocampus from the DM group were loosely arranged and disorganized,showing a large number of abnormal cells with reduced nuclei,cytoplasm,and Nissl bodies.The nuclear membrane of neurons was wrinkled,the nucleoli were hyperchromatism,the mitochondria were hypertrophy and swelling,the vacuolar degeneration could be seen.However,compared with the DM group,the pathological state of hippocampal neurons in mice treated with CDDP were alleviated to different degrees.6.One hundred and twenty-one effective components of CDDP were found in the TCMSP database.Two hundred and nineteen diabetic cognitive dysfunction disease gene targets were got in the six diseases gene-related database(TDD,DrugBank,OMIM,GAD,PharmGKB,and DisGeNET).Based on the aboved results to build the Protein-Protein Interaction(PPI)network of potential gene targets for improved DCI by CDDP.GO and KEGG analyses showed that the neurotrophin signaling pathway,MAPK signaling pathway,Toll-like receptor signaling pathway,and PI3K-Akt signaling pathway were the main pathways that contribute to the pathophysiologic process.Using the disease analysis function in the NIMNT database,we found that possible indications for Compound Danshen Dripping Pills mainly included coronary artery disease,arteriosclerotic cardiovascular disease,Alzheimer's disease,and Type 2 diabetes,etc.7.The mRNA expression profiles of the hippocampus of the NC,DM,and CM group were analyzed by using high-throughput sequencing technology.GO enrichment analysis and KEGG pathway annotation were performed.There were 7 pathways at the intersection of 131 signaling pathways enriched by network pharmacology and 136 signaling pathways enriched by transcriptome.The expression of the Toll-like receptor pathway,which is associated with DCI,was verified in the hippocampal tissues in mice.The mRNA and protein expression levels of TLR-4,MyD88,and NF-?B p65(main components of Toll-like receptor pathway)were all increased in DM group compared with the NC group(P<0.05),while the expression levels of these genes were decreased in the treatment groups compared with DM group(P<0.05).ConclusionCompound Danshen Dripping Pills can improve the diabetic cognitive impairment in KK-Ay mice,and the mechanism may be related to the inhibition of TLR-4/MyD88/NF-?B signaling pathway.