Guan Xin Ⅱ And Its Major Absorbed Components Anchored By High-fat-fed ApoE^-/- Mice With Vascular-brain-Intestinal Multimorbidity And Its Regulatory Networks

Research Background:In the field of medical research of the 21st century,diseases such as cardiovascular diseases(CVDs),depression,and gastrointestinal dysfunction are not only showing a high incidence trend worldwide,but their comorbidity phenomenon has also attracted widespread attention.As one of the main causes of death globally,the continuous rise in the incidence and mortality rates of CVDs has had a profound impact on individual health,family well-being,and the economic burden on society.At the same time,there is a close interaction between CVDs,depression,and gastrointestinal dysfunction,significantly increasing the risk of CVDs.Various stressors in modern life are also important factors exacerbating symptoms of various chronic diseases.Therefore,comprehensively understanding and effectively intervening in the comorbid mechanisms of these diseases at molecular,cellular,tissue,and whole physiology levels has become one of the key issues urgently to be addressed in current medical research.The Apolipoprotein E-deficient(ApoE-/-)mouse model,due to its genetic absence of Apolipoprotein E leading to abnormal lipid metabolism,has not only become a valuable model in the field of CVDs research but,with further studies,is increasingly considered an ideal choice for studying the comorbid state of multiple diseases.This model,in simulating the development of human atherosclerosis(AS),displays lipid metabolism disorders,providing a unique perspective and deep understanding of the complex interactions between CVDs and other diseases(such as depression,gastrointestinal dysfunction,etc.).Studies have shown that plaque increase after restraint stress(RES)is similar to the aggravation of chronic diseases after various stresses in modern life,facilitating a deeper understanding of these complex disease interaction mechanisms and the development of new therapeutic strategies.Guan Xin Ⅱ(GXⅡ)is a classic traditional Chinese medicine formula,with studies already proving its significant effects in the prevention and treatment of CVDs,depression,and gastrointestinal dysfunction.However,its key active ingredients and their specific mechanisms of action have not been fully revealed.This study explores the potential therapeutic value of GXⅡand its main absorbed components in the ApoE-/-mice comorbid model of AS,depression,and gastrointestinal dysfunction,and their regulatory effects and mechanisms on organ phenotypes under stress conditions.This study aims to provide a scientific basis for the application of GXⅡ in the management of multiple comorbidities,and also explores a new perspective for understanding and addressing the impact of psychological stress on chronic diseases in modern society,providing theoretical foundations and experimental support for the development of new therapeutic strategies.Methods:1.Establishment of comorbidity model:High-fat diet(HFD)fed ApoE-/-mice for 12 weeks to establish an AS model,with daily gavage of GXⅡ at 9g/kg.RES was applied to some mice to simulate a psychological stress environment,exploring the effect of psychological stress on the interaction between CVDs,depression,and intestinal flora imbalance.2.Pharmacokinetics(PK):On the basis of successful model establishment,blood samples were collected through orbital bleeding,and five main absorbed components of GXⅡ,Ferulic acid,Tanshinol,Hydroxysafflor yellow A,Paeoniflorin,and Senkyunolide H(FTAPH),were selected based on preliminary screening.The pharmacokinetics of FTAPH in ApoE-/-mice were studied using Ultra-Performance Liquid Chromatography-Mass Spectrometry(UPLC-MS/MS),calculating pharmacokinetic parameters such as half-life time(T1/2),time of maximum concentration(Tmax),peak concentration(Cmax),area under curve(AUC),and mean residence time(MRT)based on DAS software.3.Anti-AS pharmacodynamics study:Mouse weight changes were recorded,and plasma total cholesterol(TC),triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),and high-density lipoprotein cholesterol(HDL-C)were analyzed using biochemical tests.Mouse aortic AS plaque areas were analyzed using Oil Red O staining.Plasma interleukin-6(IL-6)and endothelial nitric oxide synthase(eNOS)levels were measured using ELISA.4.Anti-depression pharmacodynamics study:The sucrose preference test(SPT),open field test(OFT),light-dark box test(LDBT),tail suspension test(TST),and forced swim test(FST)were conducted to detect changes in depressive-like behaviors in mice.Plasma levels of 5hydroxytryptamine(5-HT)and brain-derived neurotrophic factor(BDNF)were measured using ELISA.5.RNA sequencing(RNA-seq):RNA-seq was used to analyze differentially expressed genes(DEGs)in the aorta(AO)and prefrontal cortex(PFC)of ApoE-/-mice before and after model establishment,medication,and stress,with Gene Ontology(GO)annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of DEGs functions,and protein-protein interaction networks(PPI)for functional analysis of common DEGs regulatory proteins.6.Blood oxygenation level dependent-functional magnetic resonance imaging(BOLD-fMRI)study:BOLD-fMRI was used to quantitatively evaluate changes in brain function of ApoE-/-mice before and after model establishment,medication,and stress,through regional homogeneity(ReHo)analysis.7.16S ribosomal RNA(16S rRNA):Changes in ghrelin levels and intestinal flora composition before and after model establishment,medication,and stress were assessed.Alpha and Beta diversity analysis and 16S functional prediction evaluated the mechanisms of GXⅡ and its main absorbed components on the balance of intestinal microbiota in ApoE-/-mice.8.Macrophage experiment:RAW264.7 cell line was cultured in 1640 medium with 10%fetal bovine serum and 1%double antibody,maintained at 37℃ and 5%CO2.The experiment included a blank group(untreated),a model group(treated with 100μg/ml ox-LDL),and groups pre-treated with different concentrations of Ferulic acid(FA)followed by ox-LDL stimulation.Oil Red O staining was used to assess intracellular lipid accumulation,and ELISA was used to quantitatively analyze IL-6,tumor necrosis factor α(TNFα),intercellular cell adhesion molecule-1(ICAM-1),monocyte chemotactic protein-1(MCP-1),and ghrelin.Additionally,the intracellular reactive oxygen species(ROS)levels were measured using the DCFH-DA probe.Results:1.UPLC-MS/MS detection and pharmacokinetic analysis:The distribution of FTAPH,the five main absorbed components of GXⅡ,in ApoE-/-mice was successfully detected by UPLC-MS/MS method.Pharmacokinetic analyses showed that FA had a high Cmax and fast Tmax,indicating rapid absorption;danshensu had a long T1/2,implying slow metabolism and long-lasting efficacy;and hydroxy saffron yellow pigment A had a large AUC and MRT,indicating a high overall exposure,wide distribution and long-lasting efficacy.2.Anti-AS and antidepressant efficacy:The body weights of mice were stable after GXⅡ,FTAPH and FA treatments.Behavioural tests(SPT,OFT,LDBT,TST,FST)showed that GXⅡ,FTAPH and FA significantly improved depression-like behaviour.Biochemical tests showed significant improvement in plasma levels of TC,TG,LDL-C and HDL-C.Oil red O staining showed a reduction in AS plaque area in AO.ELISA assay showed changes in IL-6 and eNOS levels,reflecting anti-inflammatory and vasculoprotective functions;5-HT and BDNF levels were increased,verifying the antidepressant effect.GXⅡ was also effective in reversing behavioural and biochemical abnormalities caused by RES.3.RNA-seq analysis:HFD led to many gene expression changes in PFC and AO samples,which were significantly regulated by different interventions.Cdhrl and Ryr3 were regulated in PFC,and 14 genes such as Cacng1,Myh8,and others were regulated in AO samples as the key regulated genes of GXⅡ.GO analysis revealed that these genes were mainly involved in transmembrane transporter,calcium import,and lipid metabolism,etc.KEGG analysis revealed that these genes act in circadian rhythm,aldosterone synthesis and PPAR signalling pathway.PPI analysis identified Cacnalc,Acly and Kcna5 as potential regulatory nodes,providing molecular targets under the influence of high-fat diet.4.BOLD-fMRI study:ReHo analysis reveals that GXⅡ regulates activity changes in brain regions of ApoE-/-mice after HFD and RES.Increased activity in brain regions such as the preoptic nucleus and decreased activity in the motor cortex after HFD;GXⅡ reverses these changes.Increased activity in the insula cortex and decreased activity in the thalamus after RES;GXⅡ similarly reverses these changes.5.16S rRNA sequencing:Serum Ghrelin levels were reduced in the HFD group of mice compared to the normal diet group;GXⅡ,FTAPH,and FA treatments elevated Ghrelin levels,and GXⅡ reversed the stressinduced decline.Gut flora analysis showed that GXⅡ,FTAPH,and FA treatments increased the alpha diversity of gut microbes and adjusted the flora composition to approach normal dietary status.Functional potential analyses showed the interventions activated key metabolic pathways and improved host metabolic health.6.oil red O staining and ROS measurements:The FA treatment significantly increased intracellular lipid accumulation.ox-LDL significantly increased intracellular lipid accumulation,and FA treatment decreased lipid deposition(200 ng/ml was the most effective).FA significantly decreased ox-LDL-induced increases in TC and HDL-C levels(40 ng/ml was effective).FA significantly decreased ox-LDLinduced enhancement of ROS,restored the levels of inflammatory factors,and increased Ghrelin,demonstrating that it attenuates potential benefits of cellular damage and inflammatory response,improving lipid metabolism and antioxidant status.Conclusion:This study successfully applied UPLC-MS/MS technology to detect the distribution of five main absorbed components of GXⅡ,FTAPH,in ApoE-/-mice,and revealed their absorption,distribution,and metabolism characteristics in the body through pharmacokinetic analysis,especially FA showing rapid absorption rates and higher peak plasma concentrations.The evaluation of anti-AS and anti-depression effects showed that GXⅡ and its components could significantly improve behavioral indicators,lipid levels,and inflammatory status in animal models,while reducing arterial plaque area.RNA-seq analysis and BOLD-fMRI studies revealed the effects of GXⅡ on gene expression and brain region activity,while 16S rRNA sequencing results emphasized its potential positive effects on intestinal flora balance.Additionally,Oil Red O staining and related biochemical analyses in macrophages further confirmed the efficacy of FA in reducing ox-LDL-induced lipid accumulation,inflammatory response,and ROS production.Overall,these comprehensive results highlight the potential therapeutic efficacy of GXⅡ and its components in improving AS-related pathological features and regulating gastrointestinal and depression,providing a scientific basis for its use as a potential therapeutic agent.