Research On The Regulation Of Water Extract Of Eucommia Ulmoides Leaves On Osteoblast MC3T3-E1

Objective:To study the regulatory effect and molecular mechanism of Eucommia ulmoides leaf extract on osteoblast MC3T3-E1.Methods:Single factor and orthogonal experiments were used to investigate the effects of different material-to-liquid ratios,temperature,time,and extraction times on the yield and content of Eucommia ulmoides leaf extract.The preparation process of Eucommia ulmoides leaf extract was established;it was determined by high performance liquid chromatography.The content of the core components of Eucommia ulmoides leaves;the CCK-8 detection kit is used to determine the effects of different concentrations of Eucommia ulmoides leaves extract on the proliferation of osteoblast MC3T3-E1;the eucommia leaves are investigated by alkaline phosphatase staining,alizarin red staining and quantitative analysis The effect of the extract on the proliferation and differentiation of MC3T3-E1 cells;the use of transcriptome sequencing,bioinformatics analysis and qRT-PCR and other methods and methods to study the effect of Eucommia leaf extract on MC3T3-E1 cell gene expression,and establish its regulation of osteogenic The molecular mechanism of the cell.Result:(1)Through the screening of different temperature,time,extraction times and material-to-liquid ratio and other influencing factors,the results show that the extract of Eucommia ulmoides leaves at 100℃,15min,three extractions,and the material-to-liquid ratio of 1:800 The yield is the highest,up to 47.60%.(2)The content of active ingredients in Eucommia ulmoides leaf extract was determined by HPLC,and the results showed that the core components of Eucommia ulmoides leaf extract were mainly aucubin,geniposide,trifoside,rutin,and isoquercitrin.,Chlorogenic acid and Astragalus glycosides,the contents were 1.18%,4.29%,0.47%,0.13%,0.06%,0.43%,0.13%,respectively.(3)The CCK-8 method was used to determine the effect of Eucommia ulmoides leaf extract on the proliferation of osteoblasts.The results showed that Eucommia ulmoides leaf extract had a significant proliferation-promoting effect on MC3T3-E1 cells at a concentration of 0.05 mg/ml.Promoting proliferation is the best at 0.15mg/ml,so the concentration of 0.15mg/ml is chosen for subsequent experiments.(4)Compared with the blank group by transcriptome sequencing and bioinformatics analysis,the Eucommia ulmoides leaf extract treatment group significantly up-regulated 30 genes,mainly involved in regulating the proliferation of osteoblasts;significantly down-regulated 604 genes,mainly involved in negative Genes that regulate osteoblasts.Through GO function enrichment,these differentially expressed genes are mainly classified as regulating primary metabolism and regulating cell metabolism.Further KEGG enrichment analysis,the main metabolic pathways include functional classification;through KEGG metabolic pathway analysis,these differentially expressed genes are mainly classified into Ampk signaling pathway,cell cycle,TCA cycle,mRNA monitoring pathway,NOD-like receptor signaling pathway,P53 signaling pathway,RNA transport and TNF signaling pathway.(5)Through the qRT-PCR verification,the results show that the measured gene expression levels are basically consistent with the transcriptome sequencing results,which proves that the experimental results of the transcriptome sequencing are true and credible.Conclusion:The water extract of Eucommia ulmoides leaves prepared in this study basically covers its core components that regulate bone growth,such as aucubin,geniposide acid,trichoside,rutin,chlorogenic acid,etc.,and the yield is Higher.Subsequent activity experiments show that the water extract of Eucommia ulmoides leaves can promote the proliferation,differentiation and mineralization of osteoblasts MC3T3-E1,and inhibit apoptosis.At the same time,the regulation of the water extract of Eucommia ulmoides leaves on the osteoblast MC3T3-E1 is mainly by up-regulating genes involved in the proliferation and differentiation of osteoblasts,and down-regulating genes involved in inhibiting the proliferation and differentiation of osteoblasts,thereby affecting the cell area and intracellular And biological processes in organelles,as well as through the Ampk signaling pathway,cell cycle,TCA cycle,mRNA monitoring pathway,NOD-like receptor signaling pathway,p53 signaling pathway,RNA transport and TNF signaling pathways.These research results will be The development of bone disease treatment drugs represented by Eucommia ulmoides provides a solid data basis and potential therapeutic targets.