Mechanism Of Ox-LDL Induced NF-?B Signaling Pathway Promoting Osteogenic Differentiation Of Rat Bone Mesenchymai Stem Cells

Oxidized Low Density Lipoprotein(ox-LDL)and its physiologically active phospholipids are the main initiating factors of atherosclerosis(AS).It has been suggested that mesenchymal stem cells(MSCs)may participate in the development of atherosclerosis.However,to date,study about the effects of ox-LDL on the physiological functions and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSC)during atherosclerosis is scant.Therefore,this study sought to reveal the molecular mechanism of the role of ox-LDL in the proliferation and osteogenic differentiation of BMSCs.In order to demonstrate the direct effect of ox-LDL on BMSCs,we cultured and differentiated rat bone marrow mesenchymal stemcells(rBMSCs)in vitro.Firstly,the rBMSCs were isolated and purified by whole bone marrow adherence method;the expression of surface marker proteins CD73,CD90 and CD106 were detected by flow cytometry,and the rBMSCs were induced to differentiate into osteoblasts,adipogenic and chondrogenic cells to verify their differentiation potential.rBMSCs were cultured in osteogenic induction fluid with an addition of ox-LDL,and osteogenic detection was performed by determining alkaline phosphatase activity and calcium deposition.The results showed that oxLDL dose-and time-dependently promoted osteogenic differentiation of BMSCs.To elucidate the mechanism of ox-LDL promoting osteogenic differentiation of rBMSCs,we performed transcriptome sequencing,which showed significant changes in more than 20 signaling pathways including NF-? B signaling pathway.Previous research from our group and others showed that NF-?B signaling pathway plays an important role in the development of atherosclerosis.However,the molecular mechanism of NF-?B signaling pathway in the process of ox-LDL induced osteogenic differentiation of rBMSCs has not been fully understood.Therefore,to illustrate the mechanism,we focused on the expression and function of three complements associated with the NF-?B signaling pathway,NF-?B-p65,uPAR,and C5 aR.The results of real-time PCR and Western blot showed that the expression of NF-?B-p65,uPAR and C5 aR was significantly up-regulated during osteogenicdifferentiation of rBMSCs,which was consistent with the results of RNA-seq sequencing.Adding C5 aR complement-specific antagonists significantly inhibited the ability of rBMSCs to differentiate into osteoblasts and the expression levels of C5 aR,uPAR and NF-? B-p65 were down-regulated.However,the similar results were obtained with the NF-? B-p65 antagonist for osteogenic induction experiments.These results indicate that the NF-?B signaling pathway plays an important role in the differentiation of rBMSCs into osteoblasts.A number of recent studies have reported that histone modifications play an important role in the osteogenic differentiation of mesenchymal stem cells.Therefore,we focused on the dynamic changes and effects of histone H3K27 trimethylation in the genome of osteogenic differentiated rBMSCs.The results of Western blot showed that rBMSCs significantly up-regulated the expression of histone demethylase JMJD3 during osteogenic differentiation and specifically decreased the methylation level of H3K27me3.Following the intervention of NF-?B and C5 aR specific antagonists,the expression of JMJD3 was significantly down-regulated and the level of H3K27me3 modification was significantly increased.These results indicate that the NF-?B signaling pathway promotes osteogenic differentiation of rBMSCs by regulating the expression of histone demethylase JMJD3.To conclude,the results of this study indicate that NF-?B-Jmjd3 signaling pathway promotes osteogenic differentiation of rBMSCs in oxLDL-mediated high-lipid culture.