Based On The LncRNA H19/miR-148b-3p/ELF5 Pathway To Study The Effect Of Icariin In Inhibiting Endothelial-mesenchymal Transition And Improving Atherosclerosis

Atherosclerosis(AS)is a common cause of cardiac death worldwide.It is generally believed that AS is the deposition of lipids in the arteries and the loss of blood vessel elasticity,resulting in a decrease in blood flow.Unstable AS plaques can rupture,leading to thrombosis and interruption of blood flow,endangering the life of the patient.At present,statins commonly used in clinical practice but its side effects cannot be ignored.Therefore,seeking new therapeutic drugs and screening new drug intervention targets is particularly important for preventing and treating AS.Icariin(ICA)is one of the main active ingredients of traditional Chinese medicine Epimedium.It increases the blood flow of cardiovascular and cerebrovascular vessels,promote hematopoietic function,immune function and bone metabolism,invigorate the kidney and strengthen yang,anti-aging.Our previous research has been conducting on the protective effects of ICA on the cardiovascular system for many years.In vivo experiments have shown that ICA inhibit the progression of AS in ApoE^-/-mice fed with high-fat diet,and use Chip technology constructs gene expression profile.However,the specific mechanism of ICA has not been explored in depth,and little is known about the role of long non-coding RNA(lncRNA)and micro RNA(miRNA)in ICA in the occurrence and development of diseases.Based on the previous research of our research group,we found that lncRNA H19plays an important role in the inhibition of smooth muscle cell proliferation and migration by ICA,while the effect of lncRNA H19 on endothelial cells is not clear.To explore the role of ICA in improving AS through the lncRNA H19/miR-148b-3p/ELF5pathway,and to verify it in vivo and in vitro,in order to provide a scientific basis for the development and utilization of traditional Chinese medicine resources with ICA as the active ingredient.The research is divided into 3 parts:(1)Bioinformatics analysis of AS gene chip in ApoE^-/-mice treated with ICA and construction and validation of lncRNA H19/miR-148b-3p/ELF5 signaling pathwayThe previous study of our group confirmed that ICA can inhibit the progression of AS in ApoE^-/-mice,and the intervention effect of ICA may be closely related to the significant up-regulation of lncRNA H19 in the aorta of mice.In vitro experiments also confirmed that lncRNA H19 mediates the effect of ICA to inhibit the proliferation and migration of smooth muscle cells,but the effect of lncRNA H19 on endothelial cell function is still unclear.Therefore,this study determined lncRNA H19 as the research object.Then,bioinformatics analysis was carried out on the miRNA chip of ICA for the treatment of AS in ApoE^-/-mice constructed by the research group previously.Eligible miRNAs were screened,and miR-148b-3p,which has a binding site with lncRNA H19and is related to the progression of cardiovascular disease,was finally determined as the next research object.The KEGG signaling pathway enrichment analysis was performed on the target genes of miR-148b-3p,and it was found that the target genes of the ETS family were more in line with requirements.The target genes enriched by the ETS family were further predicted to have binding sites,and it was found that the3'-UTR of ELF5 m RNA had a binding site with miR-148b-3p.In addition,referring to the literature,it is known that ELF5 is related to cell proliferation,differentiation,cell-to-cell interaction and angiogenesis.Thus,the lncRNA H19/miR-148b-3p/ELF5pathway was constructed,and it was speculated that ICA may play its role in improving AS through the lncRNA H19/miR-148b-3p/ELF5 pathway.Subsequently,lncRNA H19,miR-148b-3p and ELF5 were preliminarily verified in the aorta of ApoE^-/-mice provided by the previous experiment of the research group,and it was found that the lncRNA H19 of ApoE^-/-mice aorta tissue and ELF5 m RNA and protein expression decreased,miR-148b-3p expression increased,and ICA could reverse the above changes.This indicated that the protective effect of ICA on AS might be achieved through the lncRNA H19/miR-148b-3p/ELF5 pathway.The dual-luciferase reporter gene experiments confirmed that lncRNA H19 can target and regulate miR-148b-3p;miR-148b-3p can target and regulate ELF5.(2)ICA inhibits endothelial-mesenchymal transition of human umbilical vein endothelial cells through lncRNA H19/miR-148b-3p/ELF5 pathwayTreating human umbilical vein endothelial cells(HUVECs)with oxidized low density lipoprotein(ox-LDL),results showed that ox-LDL could reduce the expression of lncRNA H19 and ELF5 in HUVECs,and increase the expression of miR-148b-3p,and ICA could reverse the above changes.Through scratch,transwell and western blot experiments,it was found that ICA can inhibit the ox-LDL-induced HUVECs migration ability,increase the expression of endothelial markers,reduce the expression of mesenchymal markers,and inhibit the endothelial-mesenchymal transition(End MT)induced by ox-LDL.HUVECs were transfected with lncRNA H19 overexpressing or silencing lentivirus to construct stable transfected cell lines.It was found that overexpression of lncRNA H19 could inhibit ox-LDL-induced End MT in HUVECs,while silencing lncRNA H19 promoted End MT.In order to further investigate whether ICA exerts the effect of inhibiting End MT through lncRNA H19,HUVECs were transfected with lncRNA H19 silencing lentivirus while ICA was given,and it was found that silencing lncRNA H19 could partially block the effect of ICA in inhibiting End MT,indicating that lncRNA H19 mediated the effect of lncRNA H19 on End MT.ICA inhibits ox-LDL-induced End MT in HUVECs.Subsequently,the effect of lncRNA H19 on its downstream miRNAs and target genes was detected,and it was found that overexpression of lncRNA H19 could down-regulate the expression of miR-148b-3p and up-regulate the m RNA and protein expression of ELF5;silencing lncRNA H19 could up-regulate the expression of miR-148b-3p and down-regulate ELF5 m RNA and protein expression,and miR-148b-3p mimics partially blocked the up-regulation of ELF5 expression by lncRNA H19.It was proved that lncRNA H19 has a targeted regulatory relationship with miR-148b-3p,and miR-148b-3p has a targeted regulatory relationship with ELF5 in HUVECs,which is consistent with the results of dual luciferase gene reporter experiments.In order to further confirm that lncRNA H19 inhibits End MT through miR-148b-3p/ELF5,this study found that overexpression of lncRNA H19 could inhibit End MT in HUVECs through rescue experiments;while miR-148b-3p mimics or si-ELF5 could promote End MT;when lncRNA H19 overexpressed lentivirus and miR-148b-3p mimics or si-ELF5 were simultaneously transfected in HUVECs,miR-148b-3p mimics or si-ELF5 partially blocked the effect of lncRNA H19 on End MT.This indicates that lncRNA H19 inhibits End MT through miR-148b-3p/ELF5.(3)The role of lncRNA H19 in the improvement of AS in ApoE^-/-mice by ICAApoE^-/-mice were selected as research subjects.After intragastric administration of ICA,serum lipid levels in ApoE^-/-mice were significantly improved,the area of aortic oil red O staining was reduced,and the degree of vascular intima thickening was reduced.This indicated that ICA could improve blood lipid levels,arterial lipid deposition and vascular pathological changes in ApoE^-/-mice.When ApoE^-/-mice were given ICA intervention by gavage,after lncRNA H19silencing lentivirus was injected into the tail vein,serum dyslipidemia,aortic oil red O staining area and vascular intimal thickening degree of mice were not significantly improved compared with the HFD group.This indicated that lncRNA H19 partially blocked the effect of ICA in improving blood lipid levels,arterial lipid deposition and vascular pathological changes.To further explore the role of lncRNA H19 in ICA in improving ApoE^-/-mouse aortic End MT,RT-q PCR,western blot and immunofluorescence techniques were used to detect the expression of lncRNA H19,miR-148b-3p,ELF5 and End MT related proteins(CD31,VE-Cadherin,?-SMA and S100A4).ICA increase the expression of lncRNA H19 and ELF5,decrease the expression of miR-148b-3p,increase the protein expression of endothelial markers CD31 and VE-Cadherin,and decrease the expression of interstitial marker?-SMA and S100A4 protein expression.This indicated that ICA could regulate the expression of lncRNA H19,miR-148b-3p and ELF5 in the aorta of ApoE^-/-mice and inhibited aortic End MT.While ICA intervention was administered to ApoE^-/-mice by gavage,after lncRNA H19 silencing lentivirus was injected into the tail vein,the expressions of lncRNA H19,miR-148b-3p and ELF5 in aortic tissue,and the expressions of endothelial markers and mesenchymal markers were not significantly improved compared with those in the HFD group.This indicated that lncRNA H19 partially blocked the role of ICA in regulating the expression of lncRNA H19,miR-148b-3p,and ELF5 and inhibited aortic End MT.In summary,this study has obtained the following conclusions through in vivo and in vitro experiments:1.Through the bioinformatics analysis of the gene chip of ICA for the treatment of AS in ApoE^-/-mice obtained from the previous research of our group,lncRNA H19was selected as the research object,and the target gene was predicted to construct the lncRNA H19/miR-148b-3p/ELF5 signaling pathway.2.In vitro studies confirmed that ICA inhibits ox-LDL-induced End MT in HUVECs through the lncRNA H19/miR-148b-3p/ELF5 signaling pathway.3.In vivo studies confirmed that the protective effect of ICA on AS in ApoE^-/-mice was achieved by lncRNA H19,indicating that lncRNA H19 is an important molecular target of ICA to inhibit AS.