The Effect Of Geniposide On The Gene Expression Of Foam Cells Derived From RAW264.7 Cells Induced By Ox-LDL Based On RNA Sequencing

With the development of social economy and the improvement of living standards,cardiovascular diseases have became human killers whichhaves high incidence,mortality and recurrence rate,and many complications,as one of the major public health problems which human beings have to face and solve in a long time of future period.Atherosclerosis(AS)is a chronic systemic disease with progressive lipid deposition,fibrous tissue hyperplasia and inflammatory cell infiltration of large and medium elastic and muscular arteries,which is the common pathological cause of coronary heart disease,cerebral infarction,fundamentals of abdominal aortic aneurysm,chronic kidney disease and intermittent claudication.The pathogenesis of AS is explained as the theory of lipid infiltration,endothelial injury,inflammatory reaction,oxidative stress and so on.But the continuous disorder of lipid metabolism is an important reason of AS formationin the condition of high blood lipids which may increase the expression of various inflammatory cytokines,adhesion between cells,and mononuclear cells migrating from vascular endothelial surface into subendothelium,inducing macrophages to become foam cells.So inhibiting the formation of foam cells for avoiding the occurrence and development of AS is a critical and hotspot research field about cardiovascular disease.In recent years,the new generation of high-throughput sequencing technology including high throughput RNA sequencing,namely RNA-seq,has developed rapidly.Compared with the technology of gene chip,RNA-seq is able to detect and quantify transcription fragments with single nucleotide resolution in a genome-wide,and not need to design probes which can be used in the detection of uncompleted species in genome.RNA-seq have many advantages which not only high signal-to-noise but also high resolution,being an important experiment means to study gene expression.Geniposide is the iridoid glycosides extracted from traditional Chinese medicine gardenia,and the present study showed that geniposide has anti-atherosclerosis function with antioxidant,anti-inflammatory,protecting endothelial cells,regulating blood lipid,inhibiting smooth muscle cell proliferation,while the mechanism is not clear.We speculate that geniposide can regulate gene expression of foam cells.Based on this,we designed this experiment to known its role in gene expression of foam cells with RNA sequencing,and explore the new mechanism of Geniposide in terms of anti-atherosclerosis.Objective:With RNA sequencing,we studied the effect of geniposide,which is the main active ingredient of Gardenia jasminoides Ellis,on the gene expression of the foam cells induced from RAW264.7 cells by ox-LDL.Methods:The foam cell model is induced from RAW264.7 cell by ox-LDL;cell toxicity test—detecting the inhibition effect of different concentrations of geniposide on foam cell with CCK-8 reagent to select the most appropriate concentration;dividing into groups—blank control group,model group,low dose group,moderate geniposide,high dose geniposide group;cells were collected after 24 hours of incubation which were intervened with different concentrations of Gardenia;thus RNAs were extracted from them,and the differential expression was detected with RNA sequencing,and we analyzed the relevant data in GO and Pathway gene database.Results:The results of cell oil red O staining:the most obvious foamy of the RAW264.7 source cells were treated by 80ug/ml ox-LDL,and a large number of red lipid particles gathered on the inside of the cells,which exceeded the cell circumference.1.CCK-8 results:geniposide had no cytotoxic effect on RAW264.7 derived foam cells;RNA sequencing results:compared with the blank control group,there were a large number of abnormal high expression and low expression genes in the model group.GO and Pathway enrichment analysis of these abnormal genes showed that these altered genes involved multiple AS related functions and pathways,such as lipid metabolism related pathways,MAPK signaling pathways,TOLL like receptor signaling pathways,vascular endothelial cell migration,smooth muscle cell proliferation and migration,PPAR signaling pathway,Inflammatory signaling pathway,etc.Compared with the model group,it was found that the large and small dose geniposide(80 g/ml,30 mu g/ml)could change the abnormal high expression or low expression genes in the foam cells,and then regulate the MAPK signaling pathway and lipid metabolism related pathways,and the large dose of Jasminoidin can regulate the abnormal expression of genes in the foam cells while regulating the MAPK signaling pathway Nr4algene and Fos gene are targeted up to achieve AS resistance.Conclusion:1.Geniposide may improve the foamy of ox-LDL induced RAW264.7 cells by regulating the MAPK target signal pathway.2.Geniposide may achieve the aim of anti AS by regulating the abnormal expression of Nr4al and Fos genes in foam cells.