Network Pharmacology Combined With Metabolomics To Explore The Effects Of Dihuang Yinzi On Brain Energy Metabolism In APP/PS1 Double Transgenic Mic

Objective:Alzheimer’s disease(AD)is one of the most common neurodegenerative diseases and the main form of elderly dementia.The pathogenesis of AD is complex,involving multiple pathways and links,and there is still a lack of effective treatment methods.Energy metabolism disorders are also one of the important pathological events in the early stages of AD and potential therapeutic targets for AD,making them a hot topic of international research.Traditional Chinese medicine formulas have the characteristics of multiple components,pathways,and targets,and have shown good clinical efficacy and application potential in long-term clinical practice.Dihuang-Yinzi is a traditional Chinese medicine formula,derived from the "Huangdi Suwen Xuanming Lunfang" and composed of 15 traditional Chinese medicines.It is a classic kidney tonifying and marrow filling formula and has been used for long-term treatment of neurological degenerative diseases including AD.However,the integrated mechanism of multiple pathways and targets in the treatment of AD with Dihuang-Yinzi is still unclear.This study aims to explore the integrated regulatory mechanism of Dihuang-Yinzi in the treatment of AD through technical methods such as network pharmacology,metabolomics,and molecular biology.Method:1.Ultrahigh pressure liquid chromatography(UPLC)was used to identify some active ingredients in Dihuang-Yinzi.Evaluate the effect of Dihuang-Yinzi on the learning and memory abilities of mice through Morris water maze.2.After screening through TCMSP,BATMAN,SwissADME,and SwissTargetPrediction databases,the effective active ingredients and corresponding targets of 15 traditional Chinese medicines in Dihuang-Yinzi formula were obtained.Use the GeneCards database to obtain key targets for AD,and then intersect the targets involved in the drug and disease to obtain key targets for the treatment of AD with Dihuang-Yinzi.The protein interaction network analysis of the intersection target between Dihuang-Yinzi and AD was conducted through the STRING 11.0 database,and a visualized protein protein interaction network(PPI)was constructed.Afterwards,KEGG analysis enrichment analysis and GO functional annotation of intersection targets were conducted using the Metascape database.Finally,using Cytoscape 3.8.2 software to construct a visualized "Traditional Chinese Medicine-Active Ingredients-Targets"relationship network of Dihuang-Yinzi.3.After collecting the urine of control group,model group,and Dihuang-Yinzi group mice,UPLC-HRMS was used for metabolomics detection and analysis to screen for differential metabolites in the urine of the three groups of mice.MetaboAnalyst 5.0 software was used for KEGG pathway analysis.4.Using MetScape from the Cytoscape 3.8.2 software package for network pharmacology and metabolomics joint analysis.Construct a network of "metabolites-target genes-pathwaysenzymes".5.The molecular biology,biochemistry,histopathology and other experimental techniques were used to verify the mechanism of Dihuang-Yinzi in regulating nicotinic acid/nicotinamide metabolism,glycerol phospholipid metabolism,glycolysis,tricarboxylic acid cycle and energy metabolism in APP/PS1 mice.Result:1.The Morris water maze experiment results showed that Dihuan-Yinzi can shorten the escape latency of APP/PS1 mice and improve the spatial learning and memory ability of AD model mice.2.Through database screening,123 effective ingredients and 826 related targets of Dihuang-Yinzi and 1119 AD related targets were identified.Obtained 192 potential targets for intervention in AD with Dihuang-Yinzi.The GO and KEGG enrichment analysis results show that the 192 therapeutic targets mentioned above are mainly related to energy metabolism,oxidative damage,protection of mitochondrial function,oxidative phosphorylation,oxidoreductase system,etc.,mainly involving neurodegenerative diseases such as AD,Parkinson’s disease(PD)and Huntington’s disease(HD).Construct an action network of"Traditional Chinese medicine-Active ingredients-Targets" for Dihuang-Yinzi,and screen out 15 core genes for anti AD of Dihuang-Yinzi.3.The results of non targeted urine metabolomics analysis showed that there were 64 different metabolites between the model group and the control group mice;There are 44 different metabolites between the model group and the Dihuang-Yinzi group mice.Through comprehensive analysis,22 differential metabolites of Dihuang-Yinzi in treating APP/PS1 mice were screened.The main metabolic pathways involved in these differential metabolites include tricarboxylic acid cycle,pyruvate metabolism,glycolysis/gluconeogenesis,arginine biosynthesis,nicotinic acid,nicotinamide metabolism and glycerol phospholipid metabolism.4.The joint analysis of network pharmacology and metabolomics found that the main targets of Dihuang-Yinzi intervention in APP/PS1 mice are D-amino acid oxidase(DAO)and hypoxia inducible factor 1-α(HIF1A),Acetaldehyde dehydrogenase 3(ALDH3B2),Poly(ADP ribose)polymerase 1(PARP-1),and Acetylcholinesterase(ACHE).It mainly involves 14 differential metabolites such as pyruvate and L-lactic acid,as well as 4 metabolic pathways closely related to energy metabolism,including TCA cycle,glycolysis,niacin/nicotinamide metabolism,and glycerol phospholipid metabolism.5.Dihuang-Yinzi can regulate the metabolism of glycerol phospholipids,increase the levels of acetylcholine and cardiolipin in the brain of APP/PS1 mice,improve the structure and function of mitochondria,and reduce the release of ROS;By increasing the levels of niacin,niacinamide,and NAD+,downregulating the expression of PARP-1 and improving the metabolism of niacin/niacinamide in APP/PS1 mice;By downregulating the expression of HIF1A and pyruvic acid dehydrogenase kinase 1(PDHK1)in APP/PS1 mice,inhibiting the phosphorylation of pyruvic acid dehydrogenase(PDH),promoting TCA circulation,improving energy metabolism,and increasing the energy charge level of APP/PS1 mice brain tissue;By improving the glycolysis metabolism of APP/PS1 mice,further improving the energy metabolism disorders in the brain of model mice.Conclusion:The traditional Chinese medicine formula Dihuang-Yinzi can exert anti AD therapeutic effects in multiple pathways and targets,improving the learning and memory abilities of APP/PS1 mice.This mechanism of action involves pathways closely related to energy metabolism,including glycerol phospholipid metabolism,nicotinic acid/nicotinamide metabolism,glycolysis,and tricarboxylic acid cycle.