Therapeutic Effect Of Qingda Granule On Cardiovascular Endothelium In Hypertensive Mice Induced By High Fructose And High Salt Diet

Hypertension is a major manifestation of cardiovascular disease.With the accelerated pace of life,refined diet,and an increasing number of unhealthy dietary behaviors,a high fructose and high salt diet is one of the main unhealthy dietary habits that induce hypertension.At present,the clinical treatment of hypertension is mainly based on conventional Western medicine,which has a certain therapeutic effect,but there are also side effects.Among the current traditional Chinese medicine formulas for treating hypertension,Qingda granules are one of the representatives for treating hypertension.Modern research has found that Qingda granules have effects such as lowering blood pressure,protecting myocardial cells,antiinflammatory,and inhibiting the proliferation,migration,and apoptosis of vascular smooth muscle.This study aims to explore the endothelial therapeutic effect of Qingda granules on high fructose and high salt induced hypertension in mice through in vitro and in vivo experiments and network pharmacology methods.ObjectiveQingda Granule is one of the representative formulas for treating hypertension.This study aims to construct a vascular endothelial injury model by using high fructose and high salt intervention to investigate its injury mechanism,and explore the effective components and mechanism of Qingda granules in treating high fructose and high salt induced hypertension through network pharmacology.Furthermore,a model of high fructose and high salt diet induced hypertension in mice will be established to preliminarily explore the therapeutic effect of high fructose and high salt on mouse cardiac vascular endothelium,Provide reference for the clinical medication and basic research of Qingda granules in the treatment of high fructose and high salt induced hypertension.Methods(1)Exploring the Mechanism of Vascular Endothelial Injury in High Fructose and High Salt Induced Hypertension Models: Referring to Drugbank,OMIM,and Genecard disease databases,obtain disease targets for "High Fructose","High Salt",and "Hypertension",and then draw online Venn maps to obtain the relevant target genes for Hypertension(HFSD).Draw Venn maps to obtain the intersection targets for hypertension(HFSD).Set different concentrations of sodium chloride+fructose: 25 m M+12.5m M,50 m M+25m M,100 m M+50m M,200 m M+100m M,400 m M+200m M.Use MTT method to detect the effect of different concentrations of sodium chloride+fructose on human umbilical vein endothelial cells,and use LDH reagent kit to detect lactate dehydrogenase activity of HUVEC;Stimulating HUVEC with high fructose(50m M)and high salt(100m M)for 24 hours to establish a vascular endothelial injury model.Western Blot(WB)was used to detect MMP-2,MMP-9,and HIF-1α、 Expression of Bcl-2.(2)Preliminary exploration of the effects and mechanisms of Qingda granules on hypertension: Based on in vitro experimental results,preliminary exploration of the effects and mechanisms of Qingda granules on hypertension(HFSD).Obtain the chemical composition of Qingda granules through the TCMIP platform,BATMAN data,and TCMSP database,and use Pubchem to obtain the Canonical SMILES formula for the effective chemical composition;And predict its corresponding target through the Swiss Target Prediction platform,and screen effective chemical components using Swiss Target ADME;Refer to Gene Cards,OMIM,and Drugbank databases to obtain disease targets related to high fructose and high salt diet models,draw Venn diagrams,obtain intersection targets between disease targets and effective component targets of Qingda granules,and use STRING online mapping to construct an intersection target protein interaction network(PPI),And use Cytoscape version 3.8.2 software to modify PPI images and organize them,and use the DAVID database for intersection target enrichment analysis of GO and KEGG pathways.Select the effective components of Nelumbinis Plumula,including methylneferine,liensinine,and isoliensinine,and set the concentrations to 0,0.1% DMSO,and 0.1μM.0.25μM.0.5μM.1μ M.2.5μM.10μM.20μM(0.01%DMSO as solvent control group),MTT was used to detect the effects of different concentrations of methylliensinine,liensinine,and isoliensinine on human umbilical vein endothelial cells.(3)Confirming the effect of Qingda granules on high fructose and high salt induced hypertension in mice: Based on the results of previous in vitro and network pharmacology experiments,a mouse model of high fructose and high salt induced hypertension was constructed.48 mice were divided into 6 groups,with 8 mice in each group.The group names were blank control group,high fructose and high salt model group,low dose group of Qingda granules(57.25mg/g),medium dose group of Qingda granules(114.5mg/g)The high-dose group(229mg/kg)and positive control group(10mg/kg)of Qingda granules were treated with losartan potassium as the positive control,and Qingda granules were administered by gavage at the same time as modeling.After 28 consecutive days of administration,blood pressure was measured in mice using a non-invasive blood pressure meter.the mice were sacrificed and their hearts and blood were collected.After separating serum from blood,use a reagent kit to measure the serum NO concentration;After timely freezing of the mouse heart,use frozen sections and cut them into a thickness of 8μm’s frozen sections were attached to adhesive glass slides and stored in a-80℃ medical refrigerator.Immunofluorescence dual color co localization method was used to detect the expression of inflammatory factors NLRP3,Caspase-1,and adhesion factors ICAM-1 and VCAM-1 in mouse cardiac vascular endothelium,in order to explore the therapeutic effect of Qingda granules on the cardiac vascular endothelium of hypertensive mice.Protein immunoblotting(WB)was used to detect the expression of NLRP3 and HMGB1 in the mouse kidney homogenate.Results(1)Exploring the Mechanism of Vascular Endothelial Injury Induced by High Fructose and High Salt in a Hypertensive Model: After the intersection of "High Salt","High Fructose",and "Hypertension",a total of 107 target genes related to Hypertension(HFSD)were obtained.This includes HIF-1α、 Disease targets such as MMP-2,MMP-9,and Bcl-2.In vitro experiments,this study found that the culture medium containing lower concentrations of sodium chloride and fructose(sodium chloride concentration+fructose concentration:25m M+12.5m M,50 m M+25m M)can promote the proliferation of human umbilical vein endothelial cells;If the concentration is high(sodium chloride concentration+fructose concentration: 100 m M+50m M,200 m M+100m M,400 m M+200m M),proliferation is inhibited,morphology shrinks,and even death occurs.LDH detection found that lower concentrations of sodium chloride and fructose(sodium chloride+fructose concentration:25m M+12.5m M,50 m M+25m M)increased the activity of LDH in human umbilical vein endothelial cells,but not significantly;When the concentration is high(sodium chloride concentration+fructose concentration: 100 m M+50m M,200 m M+100m M,400 m M+200m M),the LDH activity significantly decreases;Western Blot assay showed that under high fructose and high salt conditions,The expression of HIF-1α,MMP-9 and MMP-2 significantly increased,while Bcl-2 significantly decreased in HUVEC,confirming the disease targets of Hypertension(HFSD)obtained earlier.(2)Preliminary exploration of the therapeutic effects and mechanisms of Qingda granules on hypertension: After Swiss Target screening,a total of 82 effective components and 960 drug related targets of Qingda granules were obtained.Draw a Venn diagram and intersect 960 drug targets with 107 Hypertension(HFSD)disease targets obtained in result(1),resulting in a total of 42 core intersection targets.Import 42 core intersection targets into the String database and Cytoscape 3.8.2 drawing software to obtain a protein interaction network with 42 nodes and 404 edges.Key core intersection targets include NLRP3 and HIF-1α、 Bcl-2,IL-6,TNF,NOS3,MMP-2,MMP-9,etc.GO analysis of core intersection targets using the DAVID database.The results showed that 297 biological processes(BP),38 cell components(CC),and 68 molecular functions(MF)were involved.The effect of Qingda Granules on hypertension(HFSD)is mainly related to protein binding,RNA polymerase II transcription factor activity,sequence specific DNA binding and sequence specific DNA binding activated by ligands.KEGG enrichment found that the treatment of hypertension induced by high fructose and high salt with Qingda Granule was related to HIF-1 signaling pathway,cancer related pathway,AGE-RAGE signaling pathway in diabetes complications.MTT detection found that methylneferine,neferine,and isolienne can all alleviate the cytotoxic side effects of DMSO on HUVEC.And the concentration of methylneferine is less than 10μM has no effect on the proliferation rate of HUVEC,and the concentration of liensinine is less than 5μM has no effect on the proliferation rate of HUVEC,and the concentration of isoleucine is less than 2.5μM has no effect on the proliferation rate of HUVEC.(3)Confirming the effect of Qingda granules on high fructose and high salt induced hypertension in mice: To construct a model of high fructose and high salt induced hypertension in mice for 28 days,non-invasive tail cuff method was used to measure blood pressure in mice.The blood pressure results showed that the blood pressure of mice modeled with a high fructose and high salt diet was significantly increased.Qingda granules and positive control losartan potassium can effectively reduce hypertension caused by a high fructose and high salt diet in mice,and the efficacy of Qingda granules is not dose-dependent.The serum NO assay kit was used to determine the concentration of NO in mouse serum.The concentration of NO in the serum of hypertensive mice was significantly reduced,while the concentration of NO in the serum of mice in the middle dose of Qingda granules and the positive control group of losartan potassium significantly increased.This indicates that Qingda granules can increase the concentration of NO in the serum,and Qingda granules can relax blood vessels by increasing the content of NO in the blood,thereby playing a role in reducing blood pressure.The immunofluorescence co-localization experiment results showed that the fluorescence intensity of NLRP3,Caspase-1,ICAM-1,and VCAM-1 in hypertensive mice was significantly enhanced,indicating that high fructose and high salt induced activation of inflammatory bodies and enhanced adhesion of endothelial cells in hypertensive mice;The expression of NLRP3,Caspase-1,ICAM-1 and VCAM-1 in cardiac vascular endothelial cells of mice given Qingda Granule significantly decreased,while ICAM-1 and VCAM-1 in losartan group did not change significantly,indicating that Qingda Granule can reduce blood pressure in hypertensive mice,inhibit the activation of inflammatory bodies in vascular endothelium,and reduce vascular endothelial cell adhesion.Immunoblotting of mouse kidney protein showed that under a high fructose and high salt diet,the expression of NLRP3 and HMGB1 in the kidney of mice increased.After administration,the expression of NLRP3 decreased,but there was no significant difference.The expression of HMGB1 in the high-dose group of Qingda granules and the positive control group also decreased,with only the high-dose group of Qingda granules showing a significant decrease Conclusion(1)Low concentrations of fructose and sodium chloride can promote the proliferation of human umbilical vein endothelial cells,while high concentrations can damage cells,inhibit growth,and even cause death;High fructose and high salt environment,vascular endothelial cells HIF-1 α、 MMP-2 and MMP-9 proteins were upregulated,while Bcl-2 was downregulated.(2)In the results of network pharmacology,it was found that the treatment characteristics of Qingda granules for high fructose and high salt induced hypertension are multi target and multi pathway,with core targets including NLRP3,IL-6,TNF,NOS3,MMP-2,MMP-9,Bcl-2,etc;The main signaling pathways include HIF-1 signaling pathway,AGERAGE signaling pathway,lipid and atherosclerosis related pathways,etc.The effective components of Nelumbinis Plumula in Qingda granules,including methylneferine,liensinine,and isoliensinine,have no significant toxic or side effects on HUVEC.(3)Qingda granules can improve blood pressure in mice with hypertension(HFSD)model and inhibit the activation of NLRP3 inflammasomes induced by this model.At the same time,the expression of ICAM-1 and VCAM-1 is reduced,reducing the adhesion of vascular endothelial cells.However,the effect of Qingda granules on the expression of NLRP3 in the kidney is not significant.