Abstract:Objective:To explore the therapeutic targets and molecular mechanisms of Tiaozhi Oral Liquid for treating hyperlipidemia based on network pharmacology,molecular docking technology and molecular dynamics.Methods:The active ingredients of Tiaozhi Oral Liquid were retrieved based on the TCMSP database.The targets for hyperlipidemia were screened using the GeneCards,OMIM,TTD and DrugBank databases.The intersection targets for Tiaozhi Oral Liquid in treating hyperlipidemia were obtained.The protein-protein interaction(PPI)network was constructed using the STRING platform.Core targets were screened through network topological calculation using Cytoscape 3.10.1 software.Gene ontology(GO),Kyoto encyclopedia of genes and genomes(KEGG)signaling pathway enrichment analysis were performed using R 4.3.2.The "active ingredient-target" network was constructed.Molecular docking was carried out using AutoDock and the binding performance of the active ingredients and core targets was predicted.Molecular dynamics simulation was conducted on the molecular docking results using a supercomputer platform to verify further the accuracy of the docking results.Results:A total of 79 intersectional targets of Tiaozhi Oral Liquid for treating hyperlipidemia were screened out.The core targets were tumor necrosis factor(TNF),interleukin 6(IL6),and interleukin 1B(IL1B),and the main effective components involved Quercetin,Luteolin,and Kaempferol.The GO enrichment analysis showed that the biological process(BP)involved reactions to bacterial molecules,reactions to lipopolysaccharides,regulation of inflammatory responses,and responses to external stimuli.The cell composition(CC)involved membrane rafts,membrane sides,neuronal cell body membranes.The molecular function(MF)involved transcription factor activity and binding,receptor activity and binding,enzyme activity and binding.The KEGG enrichment results indicated that it was related to lipid and atherosclerosis,advanced glycation end products-advanced glycation end product receptor(AGE-RAGE)signaling pathway,tumor necrosis factor(TNF)signaling pathway,interleukin 17(IL-17)signaling pathway,nuclear factor κB(NF-κB)signaling pathway,and chemical carcinogenesis-receptor activation.The molecular docking results showed that the binding energies of quercetin,luteolin,and kaempferol to TNF were lower,the binding energies of quercetin and IL6 were lower,and the binding energy of quercetin to IL1B was lower,and the structure was more stable.The molecular dynamics simulation results confirmed that the study system showed better binding affinity and structural stability.Conclusion:Tiaozhi Oral Liquid for treating hyperlipidemia was related to lipid and atherosclerosis,the AGE-RAGE signaling pathway,TNF signaling pathway,IL-17 signaling pathway,NF-κB signaling pathway,and chemical carcinogenesis-receptor activation in diabetes complications.Quercetin,Luteolin,and Kaempferol might be the core active compounds of Tiaozhi Oral Liquid for treating hyperlipidemia,and the complex structure was relatively stable with better affinity.

Abstract:Objective:To observe the effects of effects of Catalpol-Tetramethylpyrazine Formula on the learning,memory abilities and brain energy metabolic disorders in Alzheimer's disease(AD)model mice.Methods:Fifty 3-month-old male APPswe/PS1△E9 mice were randomly divided into the model group,the low-dose Catalpol-Tetramethylpyrazine Formula group,the medium-dose Catalpol-Tetramethylpyrazine Formula group,the high-dose Catalpol-Tetramethylpyrazine Formula group,and the Aricept group,with 10 mice in each group.Another 10 3-month-old male C57BL/6J wild-type mice were selected as the normal group.The mice in each treatment group were gavaged with the corresponding dose of the drug,while the normal group and the model group were gavaged with the same volume of normal saline,once a day,for 8 weeks.After treatment,the behavioral characteristics of the mice were evaluated through novel object recognition test,step-down test,open field test,Y maze test and elevated plus maze test.The contents of adenosine triphosphate(ATP),adenosine diphosphate(ADP)and adenosine monophosphate(AMP)in the brain tissue of the mice were detected by high-performance liquid chromatography,and the energy charge(EC)level was calculated.Results:Compared with the model group,the resolution index and step down latency in the each treatment group were prolonged,the error times decreased,the total distance of movement and the movement speed increased,the spontaneous selection accuracy rate improved,the stay time on the open arm shortened,and the contents of ATP,ADP and EC in the brain tissue of the mice increased,while the content of AMP decreased(P<0.05 or P<0.01).Compared with the low-dose Catalpol-Tetramethylpyrazine Formula group,the resolution index in the medium-dose Catalpol-Tetramethylpyrazine Formula group,the high-dose Catalpol-Tetramethylpyrazine Formula group,and the Aricept group increased,step down latency prolonged,the error times decreased,the total movement distance and movement speed increased,the spontaneous selection accuracy rate improved,the time spent on the open arm shortened,the ATP,ADP contents and EC in the brain tissue of mice increased,and the AMP content decreased(P<0.05 or P<0.01).Compared with the medium-dose Catalpol-Tetramethylpyrazine Formula group,and the Aricept group,the resolution index in the high-dose Catalpol-Tetramethylpyrazine Formula group increased,step down latency prolonged,the error times decreased,the total movement distance and movement speed increased,the spontaneous selection accuracy rate improved,the time spent on the open arm shortened,the ATP,ADP contents and EC in the brain tissue of mice increased,and the AMP content decreased(P<0.05).Conclusion:Catalpol-Tetramethylpyrazine Formula could significantly enhance the learning and memory abilities of AD model mice,and its mechanism of action might be related to improvement of energy metabolism in the mice's brain.

Abstract:Objective:To explore the effects of Qihong San on myocardial fibrosis and the adenosine 5'-monophospe-activated protein kinase(AMPK)/peroxisome proliferator-activated receptor alpha(PPARα)signaling pathway in rats with heart failure.Methods:Forty-eight specific pathogen-free(SPF)male SD rats were randomly divided into the sham group,model group,Qihong San group,and Qihong San+AMPK inhibitor group,with 12 rats in each group.Except for the sham group,in all the other groups,the method of thoracic aorta constriction was used to establish the chronic heart failure model in rats.The Qihong San group was given 2 mL of 1.62 g/kg Qihong San working solution by gavage every day after the operation.The Qihong San group+AMPK inhibitor group was given 2 mL of 1.62 g/kg Qihong San working solution+500 μL of 0.15 mg/kg AMPK inhibitor working solution by gavage every day.The sham operation group and the model group were given the same volume of normal saline by gavage.Echocardiography was used to measure the left ventricular ejection fraction(LVEF),left ventricular shortening fraction(LVFS),left ventricular end-diastolic diameter(LVEDD),and left ventricular end-systolic diameter(LVESD)in rats.Hematoxylin-eosin(HE)staining was used to detect the pathological changes of rat myocardial tissue.Masson staining was used to detect the degree of fibrosis in rat myocardial tissue.The levels of amino-terminal brain natriuretic peptide precursor(NT-proBNP),myoglobin(Mb),and cardiac troponin I(cTnI)in rat serum were detected using the kit.Western Blot was used to detect the protein expressions of α-smooth muscle actin(α-SMA),type Ⅰ collagen(CollagenⅠ),typeⅢ collagen(CollagenⅢ),AMPK,phosphorylated AMPK(p-AMPK),and PPARα in rat myocardial tissues.Results:Compared with the sham group,the LVEF and LVFS in the model group decreased,while LVEDD and LVESD increased,the arrangement of myocardial fibers was disordered and the infiltration of inflammatory cells was more abundant,the levels of serum NT-proBNP,Mb,and cTnI elevated,and the protein expression levels of α-SMA,CollagenⅠ,and CollagenⅢ in the myocardial tissue increased,the protein expressions of p-AMPK and PPARα decreased(P＜0.05).Compared with the model group,the Qihong San group showed significantly increase of LVEF and LVFS,decrease of LVEDD and LVESD,reduced disordered myocardial fiber arrangement and inflammatory cell infiltration,decrease of serum NT-proBNP,Mb,and cTnI levels,decreased protein expression levels of α-SMA,CollagenⅠ,and CollagenⅢ in myocardial tissue,and increase of p-AMPK and PPARα protein expression(P＜0.05).Compared with the Qihong San group,the Qihong San+AMPK inhibitor group reversed the regulatory effects of Qihong San on the above indicators of myocardial fibrosis in rats with heart failure.Conclusion:Qihong San can improve the cardiac function of rats with heart failure,inhibit the synthesis of collagen,and alleviate myocardial fibrosis.Its mechanism of action may be related to the activation of the AMPK/PPAR α signaling pathway.

Abstract:Objective:To explore the effects of astragalus polysaccharides on the acid-sensitive ion channel 1(ASIC1)/calcium ion-calmodulin-dependent kinase Ⅱ(CaMKⅡ)pathway and cardiomyocyte pyroptosis in type 2 diabetic(T2DM)rats'myocardial tissues.Methods:Thirty SPF-grade male SD rats were randomly divided into the control group,the T2DM group,and the astragalus polysaccharide group,with 10 rats in each group.After the rats in the T2DM group and the astragalus polysaccharide group were fed high-fat and high-sugar diet for 4 weeks,streptozotocin(STZ)(35 mg/kg)was injected intraperitoneally.If the random fasting blood glucose(FPG)was>16.7 mmol/L 72 hours later,it indicated that the model was successfully established.The rats in the astragalus polysaccharide group continued to receive intragastric administration of astragalus polysaccharides(400 mg/kg)for 8 weeks.The control group was fed with regular feed.Blood glucose meter was used to detect FPG in rats.The kits was used to detect the levels of total cholesterol(TC),triglycerides(TG),interleukin(IL)-1β,and IL-18 in serum.Echocardiography was used to measure the left ventricular ejection fraction(LVEF),left ventricular short-axis shortening rate(LVFS),left ventricular diastolic end diameter(LVEDD),and the ratio of peak diastolic early velocity(E)to peak diastolic late velocity(A)of the mitral valve.The pathological morphology of rat myocardial tissue was examined using hematoxylin-eosin(HE)staining.Masson staining was used to detect the collagen deposition in the rat myocardial tissue.The protein immunoblotting method(Western Blot)was used to detect the protein levels of ASIC1,CaMKⅡ,NOD-like receptor protein 3(NLRP3)antibody,apoptosis-associated speck-like protein(ASC),and Cysteine protease(Caspase)-1 protein.The mRNA levels of ASIC1,CaMKⅡ,NLRP3,ASC and Caspase-1 were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).Results:Compared with the control group,the levels of FPG,TC,TG,IL-1β,and IL-18 in the T2DM group of rats increased(P<0.05).Compared with the T2DM group,the levels of FPG,TC,TG,IL-1β,and IL-18 in the astragalus polysaccharide group of rats decreased(P<0.05).Compared with the control group,the LVEF,E/A,and LVFS of the T2DM group rats decreased,while LVEDD increased(P<0.05).Compared with the T2DM group,the LVEF,E/A,LVFS in the astragalus polysaccharide group increased,and LVEDD decreased(P<0.05).Compared with the control group,the myocardial fibers disordered and broken in the T2DM group rats,and a large amount of collagen was deposited in the myocardial interstitium.Compared with the T2DM group,the rats in the astragalus polysaccharide group showed improvement of myocardial fiber damage and decrease of collagen deposition in the myocardial interstitium.Compared with the control group,the protein and mRNA expressions of ASIC1,CaMKⅡ,NLRP3,ASC,and Caspase-1 in the myocardial tissues of the T2DM group rats increased(P<0.05).Compared with the T2DM group,the expression levels of ASIC1,CaMKⅡ,NLRP3,ASC,and Caspase-1 proteins and mRNAs in the myocardial tissues of rats decreased in the astragalus polysaccharide group(P<0.05).Conclusion:Astragalus polysaccharides can improve the cardiac function and myocardial tissue pathological damage in T2DM rats,down-regulate the ASIC1/CaMKⅡ pathway,and inhibit cardiomyocyte pyroptosis.

Abstract:Objective:To investigate the regulatory effects of overexpression of sodium channel protein 1.5(Nav1.5)on the apoptosis and fibrosis of cardiomyocytes in mice with long QT syndrome induced by sea anemone toxin.Methods:Twenty-four SPF-grade 8-week-old male C57BL/6J mice were randomly divided into the control group,the sea anemone toxin group,the pcDNA-Nav1.5+sea anemone toxin group,and the pcDNA-null+sea anemone toxin group according to the random number table method,with 6 mice in each group.The sea anemone toxin group was injected with 10 nmo/L sea anemone toxin solution at a dose of 0.1 mL via the tail vein every day.The mice in the control group were injected with an equal volume of normal saline intravenously every day.The mice in the pcDNA-Nav1.5+sea anemone toxin group were intravenously injected with a 10 nmol/L sea anemone toxin solution and 0.1 mL of a mixed solution composed of 50 μg/L pcDNA3.1(+)and overexpressed Nav1.5.The pcDNA-null+sea anemone toxinin group was injected with 10 nmol/L sea anemone toxinin solution via the tail vein,and mixed with 50 μg/L pcDNA3.1(+)empty vector(pcDNA-null)solution in a 0.1 mL volume.Each group was injected once daily and the injections were carried out continuously for 7 days.The expression levels of Nav1.5,cleaved-Caspase-3,B-cell lymphoma-2 protein(Bcl-2),Bcl-2-associated X protein(Bax),type Ⅰ collagen(COL1A1),type Ⅲ collagen(COL3A1),alpha-subtype of smooth muscle actin(Alpha-Smooth Muscle Actin,α-SMA),TGF-β1,phosphorylated Smad3(p-Smad3),and Smad3 in cardiomyocytes were detected by Western Blot method.The apoptosis rate of cells in each group of myocardial tissues was detected by flow cytometry.Results:Compared with the control group,the mice in the hyaluronin group showed prolonged QT intervals,increased apoptosis rate of myocardial cells,upregulated expression of cleaved-Caspase-3 and Bax proteins,downregulated expression of Bcl-2 protein,upregulated expression of myocardial fibrosis-related proteins COL1A1,COL3A1,and α-SMA,and upregulated expression of TGF-β1 and p-Smad3 proteins(P＜0.05).After overexpression of Nav1.5,all these changes were significantly reversed.Compared with the pcDNA-null+sea anemone toxin group,the apoptosis rate of myocardial cells in the pcDNA-Nav1.5+sea anemone toxin group decreased,the expression of cleaved-Caspase-3 and Bax proteins was downregulated,the expression of Bcl-2 protein upregulated,the expressions of myocardial fibrosis-related proteins COL1A1,COL3A1,and α-SMA upregulated,and the expressions of TGF-β1 and p-Smad3 proteins downregulated(P＜0.05).Conclusion:Overexpression of Nav1.5 can effectively inhibit apoptosis and fibrosis of cardiomyocytes in mice with long QT syndrome induced by oculin.The mechanism may be related to the regulation of the TGF-β1/Smad3 signaling pathway.

Abstract:Objective:To investigate the expression and mechanism of microRNA(miRNA)-26 in atrial fibrillation.Methods:A total of 98 patients with atrial fibrillation were selected,and 98 healthy individuals who underwent physical examinations at the same time were chosen as the healthy control group.Forty male C57BL/6 mice were randomly divided into the control group,the atrial fibrillation group,the overexpression miR-26a(Adv-miR-26a)group,and the overexpression miR-26b(Adv-miR-26b)group.The control group and the atrial fibrillation group of mice were intraperitoneally injected with the same volume of normal saline.The mice in the Adv-miR-26a group and the Adv-miR-26b group were respectively injected intraperitoneally with the Adv-miR-26a lentiviral overexpression vector and the Adv-miR-26b lentiviral overexpression vector.Fourteen days later,except for the control group,the other three groups the atrial fibrillation model was established and given a tail vein injection of 1 mL/kg of the mixture of acetylcholine and calcium chloride,once a day for 7 consecutive days.The levels of miR-26a and miR-26b were detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR).The duration and induction time of atrial fibrillation in each group of mice,as well as the action potential duration,and cardiac function,included left ventricular end-diastolic diameter(LVEDD),left atrial effective refractory period(ERP),left ventricular fractional shortening(LVFS),and left ventricular ejection fraction(LVEF)were detected.Hematoxylin-eosin(HE)staining was used to observe the myocardial pathological conditions of the mice in each group.The protein levels of type Ⅰ collagen(CollagenⅠ),type Ⅲ collagen(CollagenⅢ),connective tissue growth factor(CTGF),and matrix metalloproteinase 2(MMP-2)in each group of mice were detected by Western Blot method.Results:The levels of miR-26a and miR-26b in the serum of patients with atrial fibrillation were lower than those of the healthy control group(P＜0.05).The area under the receiver operating characteristic(ROC)curve(AUC)for serum miR-26a and miR-26b levels in patients with atrial fibrillation were 0.812 and 0.784,with sensitivities of 77.49%and 76.53%,and specificities of 77.55%and 70.41%.Compared with the control group,the levels of miR-26a and miR-26b,ERP,LVFS,LVEF in the myocardial tissue and serum of the atrial fibrillation group,decreased,while APD50,APD90,LVEDD increased,and the protein expressions of CollagenⅠ,CollagenⅢ,CTGF,and MMP-2 were upregulated(P＜0.05).Compared with the atrial fibrillation group,the levels of miR-26a and miR-26b in the myocardial tissues and serum of mice in the Adv-miR-26a group and Adv-miR-26b group increased,while ERP,LVFS,and LVEF elevated.The duration of atrial fibrillation shortened,the induction time of atrial fibrillation prolonged,APD50,APD90,and LVEDD decreased,and the protein expressions of CollagenⅠ,CollagenⅢ,CTGF,and MMP-2 were downregulated(P＜0.05).Conclusion:The miR-26a and miR-26b are expressed at low levels in the serum of patients with atrial fibrillation,as well as in the myocardial tissues and serum of mice.Overexpression of miR-26a and miR-26b can improve the cardiac function and electrophysiological conditions of mice with atrial fibrillation,alleviate myocardial fibrosis,and inhibit the occurrence of atrial fibrillation.

Abstract:Objective:To identify the potential genes involved in the progression of atherosclerosis and explore the association between genes and immune cell infiltration.Methods:Two transcriptome profiles(GSE28829 and GSE41571)were analyzed using R packages for differential gene expression,gene ontology,pathway and disease ontology enrichment,protein-protein interaction,and candidate biomarker selection.The expression levels of candidate biomarkers were verified in a validation dataset,and the compositional patterns of immune cell fractions were calculated using CIBERSORT.The correlation analysis between diagnostic gene biomarkers and infiltrating immune cells was performed using Spearman's rank correlation analysis.Results:A total of 15 genes were determined as diagnostic gene biomarkers of advanced arteriosclerotic plaques.According to correlation analysis with immune infiltration,C1QA,C1QB,C1QC,LY86,CD14 and HCK were identified as hub genes,which were positively correlated with macrophage M2 and negatively correlated with CD8+T cell and activated dendritic cell.Conclusion:C1QA,C1QB,C1QC,LY86,CD14 and HCK were determined as hub genes which were identified to regulate immune-competent cells.These findings reveal light on the genes associated with immune cells in the progression of atherosclerosis and may aid the development of new treatments and medications.