Estrogen-like Effects And Mechanisms Of Icariin On SHR And Endothelial Progenitor Cells In Castrated Females

Hypertension is a common clinical chronic cardiovascular disease,with relatively insidious onset.There are usually no obvious symptoms or mild symptoms in the initial stage of hypertention.However,long-term hypertension can cause abnormalities in the structure and function of blood vessels,multiple organ damage such as heart,kidneys,brain and etc.and has become the first risk factor for death in the world.Hypertension has threatened the function of vascular endothelial cells and blood vessels as a circulatory system disease.And vascular endothelial dysfunction and the occurrence of hypertension promote each other,thus forming a vicious circle.As the endothelial precursor cells,Endothelial progenitor cells can proliferate,differentiate into endothelial cells,repair and improve endothelial function,thereby delaying the development of hypertension.Studies have pointed out that decreased estrogen levels,which may independently induce the occurrence of hypertension by affecting vascular function,renin-angiotensin system and sympathetic nervous system,has become an important reason for high incidence of female menopausal hypertension.Estrogen has protective effects on cardiovascular system such as vasodilation,regulation of blood lipids,anticoagulation,anti-inflammatory effect,and improvement of endothelial progenitor cells'function.However,estrogen replacement therapy still remains controversial in terms of contraindications,adverse reactions,application dosage and time.Icariin is the main component of the traditional Chinese medicine Epimedium.It belongs to phytoestrogens and has protective effects on endothelial progenitor cells and vascular endothelial cells.We tried to provide a theoretical basis for the application of the kidney-tonifying method in menopausal hypertension by exploring whether icariin can protect endothelial progenitor cells and reduce blood pressure of ovariectomized female hypertensive rats through estrogen-like effect.And we conducted the following research:First,we conducted the network pharmacology study of Erxian decoction on menopausal hypertension.Secondly,we continued to explore the effect and mechanism of Epimedium on menopausal hypertension based on network pharmacology and molecular docking.Further,in vivo experiment was carrried out to explore the effect of icariin on ovariectomized female hypertensive rats and endothelial progenitor cells.Finally,in vitro experiment was conducted to explore the effect and mechanism of icariin on angiotensin ?-injured endothelial progenitor cells.Study 1 Network pharmacology study on mechanism of Erxian decoction in the treatment of menopausal women with hypertensionObjevtive:To explore the mechanism of Erxian decoction in the treatment of menopausal hypertension based on network pharmacology.Methods:Traditional Chinese medicine systems pharmacology database and Analysis platform(TCMSP)was used to screen the main active ingredients and targets of Erxian decoction.And Uniprot database was used to convert target names to standard gene names.By means of the Human Gene Database(GeneCards)and Online Mendelian Inheritance in Man(OMIM),the targets of menopausal hypertension were collected.The intersecting targets between menopausal hypertension and Erxian decoction were obtained by using the venn map.The Cytoscape software was used to construct the "herbs-active ingredients-intersection targets" network,and STRING online database was used to build protein-protein interactions(PPI)network.DAVID database was used to perform Gene ontology(GO)function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The "drugs-components-targets-disease network" was constructed by Cytoscape software.Results:A total of 92 active ingredients,218 predicted targets of Erxian decoction,1189 menopausal hypertension-related disease targets and 127 intersection targets were obtained after screening.Through "herbs-active ingredients-intersection targets" network,treatment of menopausal hypertension with Erxian decoction may be related to active ingredients,such as quercetin,luteolin,kaempferol,etc.GO analysis and KEGG enrichment analysis result showed that biological process mainly included cell apoptosis,cell proliferation,response to bacterium,etc,KEGG pathways mainly including Toll-like receptor signaling pathway,TNF signaling pathway,PI3K/Akt signaling pathway,etc.Conclusion:Erxian decoction may mainly play a role in regulating endothelial function,proliferation of smooth muscle cells,anti-inflammatory reactions and anti-oxidation,etc.,thereby reducing blood pressure levels through quercetin,luteolin,kaempferol and Toll-like receptor signaling pathway,TNF signaling pathway,PI3K/Akt signaling pathway,etc.Study 2 Network pharmacology study on mechanism of Epimedium in the treatment of menopausal hypertensionObjevtive:To explore the mechanism of Epimedium in the treatment of menopausal hypertension based on network pharmacology and molecular docking.Methods:TCMSP was used to screen the main active ingredients and targets of Epimedium.And Uniprot database was used to convert target names to standard gene names.By means of GeneCards and OMIM,the targets of menopausal hypertension were collected.The intersecting targets between menopausal hypertension and Epimedium were obtained by using the venn map.The Cytoscape software was used to construct the "Epimedium-active ingredients-intersection targets" network,and STRING online database was used to build PPI Network.DAVID database was used to perform GO function enrichment analysis and KEGG pathway enrichment analysis.The "Epimedium-components-targets-disease network" was constructed by Cytoscape software.Autodock vina software was used to conduct molecular docking between targets and active ingredients with higher degrees in the"Epimedium-components-targets-disease network".Results:Totally 23 active components,1 99 targets of Epimedium and 118 related disease intersecting targets.Through "Epimedium-active ingredients-intersection targets"network,treatment of menopausal hypertension with Epimedium may be related to quercetin,luteolin,kaempferol,etc.GO analysis and KEGG enrichment analysis result showed that biological process mainly included cell cell apoptosis,cell proliferation,response to bacterium,etc,KEGG pathways mainly including Toll-like receptor signaling pathway,TNF signaling pathway,PI3K/Akt signaling pathway,etc.The results of molecular docking showed that main active ingredients in Epimedium including quercetin,luteolin,kaempferol had high binding activities with PTGS2?PTGS1?MAPK1?AKT1?AR?RELA.Conclusion:Epimedium may mainly exert effects on the treatment of menopausal hypertension by anti-inflammatory effect,,anti-oxidation,and anti-proliferation of smooth muscle cell,regulation of endothelial function,etc,thereby reducing blood pressure levels through quercetin,luteolin,kaempferol and Toll-like receptor signaling pathway,TNF signaling pathway,PI3K/Akt signaling pathway,etc.Study 3 Estrogen-like effect of Icariin on endothelial progenitor cells and ovariectomized female hypertensive ratsObjective:To explore the effect of icariin on body weight,blood pressure,heart rate and endothelial progenitor cells(EPCs)function in ovariectomized female hypertensive rats.Methods:Ten 9-week-old Wister Kyoto female rats(WKY)and fifty 9-week-old spontaneously hypertensive female rats(SHR)were given ovariectomy.WKY rats were used as the control group(WKY).SHR group rats were randomly divided into model group(SHR),estrogen group(SHR-E2,0.1 mg/kg),icariin low-dose group(SHR-1,20 mg/kg),icariin high-dose group(SHR-Hl,40 mg/kg)and the icariin low-dose+estrogen receptor antagonist group(SHR-1+ICI,52 mg/kg/month).Rats were observed for one week after ovariectomy,and then given drug for 8 weeks respectively.The body weight(BW)of rats in each group was measured every week.The mean arterial pressure(MAP)and heart rate(HR)of rats in each group were measured before and 8 weeks after drugs treatment.After 8 weeks,blood was taken from the abdominal aorta to isolate peripheral blood EPCs.And then their hind legs were isolated bone marrow EPCs.EPCs were cultured and identified in vitro.Meanwhile,cell viability and nitric oxide(NO)content were tested.Results:(1)there was no difference in BW of rats in each group before drug treatment.Compared with SHR group,the BW of SHR-E2 group was significantly reduced after drug treatment.(2)Before drug treatment,compared with WKY group,MAP of other groups were remarkebly increased,but there was no difference in MAP between SHR groups.After 8 weeks of drug treatment,MAP of WKY and SHR groups were increased significantly than before.After 8 weeks of drug treatment,compared with WKY group,MAP of SHR group was significantly increased.Compared with SHR group,MAP of SHR-E2,SHR-1 and SHR-H1 groups were markedly reduced.Compared with the SHR-1 group,MAP of the SHR-1+ICI group was significantly increased.(3)There was no significant difference in the HR of each group before treatment.After 8 weeks of drug treatment,HR of each group was markedly increased than before,and compared with SHR group,HR of SHR-1 and SHR-H1 groups were significantly reduced.Compared with SHR-1 group,HR of SHR-1+ICI group was increased.(4)After 8 weeks of drug treatment,compared with WKY group,cell viability and NO secretion of EPCs derived from both bone marrow and peripheral blood of the SHR group were significantly decreased.Compared with SHR group,cell viability and NO secretion of EPCs of SHR-E2,SHR-1 and SHR-H1 groups were remarkebly increased.Compared with SHR-1 group,cell viability and NO secretion of EPCs of SHR-1+ICI group were markedly reduced.Conclusion:Icariin can decrease the level of blood pressure,heart rate,and improve viability of EPCs,nitric oxide of ovariectomized hypertensive rats.This mechanism may be related to estrogen-like effect.Study 4 Estrogen-like effect of Icariin on Angiotensin ?-injured rat bone marrow-derived endothelial progenitor cellsObjevtive:To explore the estrogen-like effect of icariin on Angiotensin?(Ang-?)-injured bone marrow-derived EPCs in rat.Methods:EPCs were isolated,cultured and identified.These cells were divided into seven groups:control group,model group(Ang-?,10 ?mol/L),estradiol group(E2,0.01?mol/L),and low,medium and high concentration groups of icariin(ICA-L,ICA-M and ICA-H correspond to 0.01 ?mol/L,0.1 ?mol/L and 1 ?mol/L),estrogen receptor antagonist group(ICA-H+ICI,1 ?mol/L).The corresponding drug intervention was given 24 hours.The viability of EPCs was detected by MTT.The level of NO and endothelin-1(ET-1)were detected by nitrate reductase method and ELISA respectively.The protein expression of eNOS,P-eNOS and iNOS were detected Western-blot method.Results:(1)Compared with control group,the EPCs' viability of Ang-? group was significantly decreased.Compared with Ang-? group,ICA-L,ICA-M and ICA-H groups can improve the viability of EPCs.(2)Compared with control group,the level of NO in Ang-? group was decreased.Compared with Ang-? group,the level of NO in E2,ICA-L,ICA-M and ICA-H groups were remarkebly increased.The level of NO in ICA-H+ICI group was significantly increased when compared with ICA-H group.Compared with control group,the level of ET-1 of Ang-? group was markedly increased.The secretory capacity of ET-1 in E2 and ICA-H groups were significantly decreased when compared with Ang-? group.The secretion level of ET-1 in ICA-H+ICI group was markedly increased compared with ICA-H group.(3)Compared with control group,the protein expression of iNOS of Ang-? group was significantly increased.The protein expression of iNOS in E2 and ICA-M groups were decreased compared with Ang-? group.Compared with ICA-H group,the expression of iNOS in ICA-H+ICI group was markedly decreased.There was no significant difference inthe protein expression of eNOS among groups.Compared with control group,the protein expression of P-eNOS of Ang-? group was remarkebly increased.The protein expression of P-eNOS in E2,ICA-L,ICA-M and ICA-H groups were significantly decreased compared with Ang-? group.Compared with ICA-H group,the expression of P-eNOS in ICA-H+ICI group was markedly decreased.Conclusion:Icariin has an estrogen-like effect on EPCs induced by Ang-?,which can increase cell viability,NO levels,the expression of eNOS protein and reduce ET-1 levels,and down-regulate the expression of iNOS and P-eNOS protein.