Long Non-coding RNA 042398 Regulates MAPK3 To Promote Macrophage Foaming And Its Role In Icariin Against Atherosclerosis

Atherosclerosis(AS) is the main pathological basis of various vascular diseases such as coronary heart disease,stroke and peripheral atherosclerotic occlusive disease.Atherosclerotic lesions not only involve large blood vessels and microvessels and data shows that the consequences of atherosclerotic disease are the main cause of disability and death among young and middle-aged people in my country.It endangers huma n health seriously that needs to be solved urgently.The exploration of the pathogenesis of AS has always been a hot topic in many studies.AS involves a variety of cells,including endothelial cells and smooth muscle cells in the arterial system wall,as well as monocytes-macrophages derived from the blood circulation.The activation of the monocyte-macrophage system,the transformation of macrophages into foam cells and the inappropriate apoptosis of foam cells are the key links in the formation of AS plaques and the main factors of plaque instability.Therefore,proper blocking transformatio n of monocyte to macrophage and macrophage foaming will effectively delay the occurrence and progression of AS.Long non-coding RNAs(lncRNAs)have been gradually recognized in the last few years.The definition of lnc RNA is that its sequence length is more than 200nucleotides and it does not have protein-coding function.The number of lnc RNA far exceeds the number of m RNA in the body,and they play an irreplaceable regulatory role in many life activities,including cell proliferation and apoptosis,cell and tissue differentiation,cell and organ aging.Many scholars have confirmed that lncRNAs are abnormally expressed in cardiovascular diseases,including endothelial dysfunction,abnormal lipid metabolism,inappropriate proliferation of smooth muscle cells,foam cell formation,inflammatory response,oxidative stress.LncRNAs may become indicators for the prediction,diagnosis and monitoring of atherosclerotic disease.Icariin(ICA)is the active ingredient extracted from the traditional Chinese medicine Epimedium.Previous studies have shown that ICA play various effects such as anti-osteoporosis,maintaining immune system balance,inhibiting inflammatory response,and anti-cancer.Resent several years have found that it plays an important role in protecting cardiovascular diseases including anti-atherosclerosis.The previous experimental study of our group found that ICA has the effect of delaying the progression of AS in Apo E^-/-mice,but the specific mechanism still needs to be further improved.This experiment includes animal level research and in vitro cell level research,using gene chip technology,sh RNA technology,gene overexpression technology,FISH,Oil Red O staining,HE staining,ELISA,immunohistochemical staining,CCK,flow cytometry,Western blot and RT-q PCR to explore the role of lnc RNA042398 in regulating MAPK3 in the pathogenesis of AS.The results confirm that ICA has an anti-atherosclerotic effect and the mechanism of ICA regulating MAPK3 through lnc RNA042398 to inhibit the proliferation and foaming of macrophages.This study consists of three parts.1.Apo E^-/-mouse atherosclerotic tissue gene and protein expression profiling,bioinformatics analysis and verification of differentially expressed genesIn this part of the experiment,the atherosclerosis animal model was successfully established by feeding Apo E^-/-mice with high-fat diet.The expression profiling of aortic lncRNAs and m RNAs was analyzed by gene chip.GO and Pathway enrichment analysis was performed on the significantly different genes,and the LncRNA target pathway network map and the LncRNA-m RNA co-expression network map were constructed.GO enrichment analysis showed that MAPK3 was closely related to cholesterol metabolism and AS;Pathway enrichment analysis showed t hat NONMMUT042398 was involved in chemokine signaling pathway,MAPK signaling pathway,m TOR signaling pathway,PI3K-Akt signaling pathway,Ras signaling pathway,and TGF-?signaling pathway,TNF signaling pathway,toll-like receptor signaling pathway,these signaling pathways are closely related to monocyte-macrophage chemotaxis,activation of inflammation,lipid metabolism,cell proliferation,differentiation and apoptosis.The LncRNA target pathway network diagram shows that LncRNA 042398 is the gene with the highest degree in the network;LncRNA 042398 has a co-expression relationship with MAPK3 in the LncRNA-m RNA co-expression network.It indicated that LncRNA 042398 and MAPK3 play an important role in the occurrence and development of AS.RT-q PCR in the aorta of Apo E^-/-mice confirmed that the expression trends of LncRNA 042398and MAPK3 were consistent with the gene chip results.2.LncRNA 042398 regulates MAPK3 and affects macrophage foamingFoam cell model was constructed by oxidized low density lipoprotein(ox-LDL)induced of RAW264.7 cells.The expression of lnc RNA 042398 and MAPK3 was verified in the foam cell model.The results showed that the expression of lnc RNA042398 was up-regulated and the expression of MAPK3 was down-regulated in the foam cell model,which confirmed that the expression trend was consistent with the gene chip results.To further clarify whether the regulation of MAPK3 by lnc RNA 042398 affects the proliferation and foaming of macrophages,the subcellular localization of lnc RNA042398 was identified by FISH.The subcellular localization of lnc RNA 042398 is mainly located in the cytoplasm,which helps to predict its mechanism of action.RAW264.7 cells were transfected with lnc RNA 042398 overexpressing/silencing lentivirus to construct stable transfected cell lines.Overexpression of lnc RNA 042398could promote the proliferation of RAW264.7 cells induced by ox-LDL;The foaming of phagocytes was aggravated;the contents of intracellular total cholesterol and free cholesterol were increased;the expressions of MAPK3 and CD36 were up-regulated,and the expression of SR-BI was inhibited;the levels of cytokines IL-1?,IL-6 and TNF-?were increased in the cell culture supernatant;lnc RNA The above results were the opposite after 042398 was silenced.It is suggested that the regulation of MAPK3by lnc RNA 042398 promotes foaming of macrophages.3.Icariin inhibits macrophage foaming and anti-atherosclerosis by regulating MAPK3 through lnc RNA 042398In vivo experiments it was confirmed that ICA can significantly reduce the levels of TC,LDL-C and ox-LDL in the serum of Apo E^-/-mice fed with high fat,reduce the atherosclerotic AS plaque area ratio,and alleviate the pathology of aortic AS.Change;increase the expression of MAPK3 and scavenger receptor SR-BI in aortic tissue,down-regulate the expression of CD36,ICA has anti-atherosclerosis effect.In vitro experiments confirmed that ICA can increase the expression of MAPK3 and SR-BI by inhibiting lnc RNA 042398,and down-regulate the expression of CD36;reduce the content of intracellular total cholesterol and free cholesterol;inhibit the proliferation and foaming of macrophages,and reduce the IL-1?,IL-6 and TNF-?levels.ICA reversed the changes in macrophage proliferation and foa ming induced by lnc RNA 042398 overexpression.In summary,this study draws the conclusions as following.(1)LncRNA 042398 and MAPK3 are differentially expressed genes in the expression profile of AS lesions in mouse aorta,and bioinformatics analysis show ed that the two genes played an important role in the progression of AS.(2)In vitro experiments,it was shown that lnc RNA 042398 negatively regulates MAPK3.Overexpression of lnc RNA 042398 promoted proliferation and foaming of macrophages.ICA inhibited the proliferation and foaming of RAW264.7 cells induced by ox-LDL,and its inhibitory effect was achieved by regulating lnc RNA042398 resulting of MAPK3 and CD36 down-regulation but up-regulation of SR-BI expression.(3)In vivo experiments it was proved that ICA has an anti-atherosclerotic effect,and its mechanism of action is achieved through the regulation of MAPK3 by lnc RNA042398 to down-regulate the expression of CD36 and up-regulation of SR-BI.