ICA Regulates MiR-672-5p/AKT3/NF-κB Signaling Pathway To Inhibit The Inflammation Of RAW264.7 Induced By Ox-LDL

With the improvement of living standards and changes in lifestyle,the incidence of cardiovascular diseases such as atherosclerosis(AS)has shown an upward trend year by year.AS is a chronic inflammatory disease involving smooth muscle cells,endothelial cells,macrophages and other cells,and is accompanied by a series of complications,which brings many troubles to people’s lives.Epimedium is a traditional medicinal plant in my country.Its main chemical components are flavonoids,lignans,phenol glycosides,alkaloids,polysaccharides,volatile oils,anthraquinones,tannins,sterols,sesquiterpenes,etc.Icariin(ICA)is the main chemical component of Epimedium.It is a flavonoid compound.It has anti-inflammatory,anti-tumor,anti-aging,anti-osteoporosis,anti-myocardial ischemia and hypoxia,and promotes immune regulation.Promote multiple pharmacological effects such as reproductive endocrine function.MicroRNA(microRNA,miRNA)is a short-chain non-coding RNA.miRNA plays an increasingly important role in cell proliferation and differentiation,organism growth and development,and disease treatment.Studies have shown that miRNAs can become therapeutic targets and diagnostic markers for cardiovascular diseases.Therefore,it provides us with new ideas and methods for finding the treatment and diagnosis of AS.The previous experimental studies of this group have confirmed that ICA has the effect of anti-AS,and constructed ApoE-/-mouse thoracic aortic microRNA(miRNA)expression profile,but the specific molecular mechanism of ICA’s anti-AS needs further exploration and the study.In this study,starting with miRNA chips,the miR-672-5p/AKT3/NF-KB signaling pathway was constructed using bioinformatics analysis,and this signaling pathway was used as an entry point to study ICA’s response to ox-LDL-induced RAW264.7 inflammation and Molecular mechanism.This research is divided into 3 parts:(1)ICA anti-AS miRNAs chip bioinformatics analysis and miR-672-5p/AKT3/NF-κB signaling pathway constructionFirst,compare the miRNA chip results of ApoE-/-mouse thoracic aorta MOD group/CON group,set the condition of differential genes to p<0.05 and |log2 fold change|>1.32,and screen a total of 16 differentially expressed miRNAs.In order to find the key miRNAs that ICA plays against AS in RAW264.7,6 miRNAs were randomly selected,the expression of miRNAs was verified by qPCR technology,and miR-672-5p was identified as the research object.Through TargetScan and DAVID database to predict its target genes,GO analysis and KEGG analysis,we found that miR-672-5p is closely related to cell proliferation,apoptosis,protein transcription and translation and other biological processes.In miR-672-5p enriched signaling pathway,PI3K-AKT pathway has a smaller p value and more target genes enriched in it.At the same time,the 3’UTR of AKT3 mRNA has a binding site with miR-672-5p and has better targeting.Therefore,we built the miR-672-5p/AKT3/NF-KB signaling pathway.(2)Study on the Effect and Mechanism of ICA on the Inflammatory Response of RAW264.7 Induced by ox-LDLRAW264.7 was induced by ox-LDL to construct macrophage-derived foam cells.At the same time,ICA was added to intervene.The effects of ICA on ox-LDL-induced inflammation of RAW264.7 were investigated by CCK-8,Oil Red O staining,ELISA,qPCR and Western Blot As a result,it was found that ox-LDL can reduce the viability of RAW264.7 cells,induce foaming of RAW264.7,and increase the content of IL-1β,IL-6,and TNF-α in the culture supernatant.ICA can reverse the changes in the above reaction.It shows that ICA can inhibit the inflammatory response induced by ox-LDL in RAW264.7.The expression of miR-672-5p/AKT3/NF-κB signaling pathway-related molecular genes and proteins were further detected by qPCR and Western Blot.The results showed that ox-LDL can reduce the expression of RAW264.7 miR-672-5p,and increase AKT3 mRNA and Protein expression,and up-regulate the phosphorylation levels of IKK,IκBα and p65,and ICA can reverse the changes in the expression of the above genes and proteins.This indicates that ICA may regulate the miR-672-5p/AKT3/NF-κB signaling pathway,thereby inhibiting the ox-LDL-induced inflammatory response of RAW264.7.(3)The effect of miR-672-5p on the inflammatory response of RAW264.7 induced by ox-LDLConstruction of miR-672-5p overexpression/silencing transient RAW264.7,oil red O staining,ELISA,qPCR and Western Blot technology to explore the role of miR-672-5p in ox-LDL-induced RAW264.7 inflammation.ox-LDL can induce foaming of RAW264.7,increase the content of IL-1β,IL-6 and TNF-α in RAW264.7 culture supernatant,reduce the expression of RAW264.7 miR-672-5p,and increase AKT3 mRNA and protein And up-regulate the phosphorylation levels of IKK,IκBαand p65.Overexpression of miR-672-5p can reverse the effect of ox-LDL,while silencing miR-672-5p can enhance the effect of ox-LDL.This indicates that miR-672-5p mediates the NF-κB signaling pathway by regulating AKT3,thereby inhibiting ox-LDL-induced RAW264.7 inflammation.This experiment proves that ICA may inhibit ox-LDL-induced RAW264.7 inflammation by regulating the miR-672-5p/AKT3/NF-KB signaling pathway,thereby treating AS.miR-672-5p is a molecular target for ICA to treat AS.