The Mechanism Of Sesamin Effect On Intervertebral Disc Degeneration Based On RNA-seq Technique

Objective: In In this study,RNA sequencing(RNA-Seq)technology was used to explore the differentially expressed genes(DEGs)of sesamin in the degenerative cartilage endplate cell model.To explore the effect of DEGs on the cell model of degenerative cartilage endplate,and further clarify the molecular mechanism of sesamin affecting intervertebral disc degeneration.Methods: 1.The control group(group C)and sesamin group(group S)were set up.In group C,10μg/mL lipopolysaccharide(LPS)was used to induce mouse chondrogenic cell ATDC5 to establish a degenerative cartilage endplate cell model;In group S,the cells were cultured in medium containing 1μM sesamin for 24 hours after the degenerative cartilage endplate cell model was established as the control group.RNA-seq technology was used for mRNA sequencing of the two groups of cells.Deseq2 software was used to analyze the two groups of DEGs,and GO and KEGG enrichment analysis of DEGs was performed.2.After screening DEGs,the base Mean expression of DEGs in the cell samples of group S and group C was analyzed again to further screen DEGs.At the same time,combined with relevant literature reports,5 DEGs were selected.The blank control group(Normal group,N group),C group and S group were established.The blank control group was untreated ATDC5 cells.Five DEGs were verified by RT-qPCR.3.The Becn2 overexpression vector was constructed and transfected into ATDC5 cells to over-express Becn2,and RT-qPCR and WB were used to verify whether BECN2 overexpression was successful.4.Group N,group C and control +Becn2 overexpression group(group C+Becn2)were established.In C+Becn2 group,ATDC5 cells were transfected with Becn2 overexpression vector for 48 hours,and cells were cultured in medium containing 10μg/mL LPS for 24 hours.The cell proliferation rate and apoptosis rate of the three groups were detected by flow cytometry.Western blotting was used to detect the expression of apoptosis-related proteins Caspase-3,Bax and Bcl-2,and the expression of extracellular matrix(ECM)degradation related proteins MMP-13,ADAMTS-5 and ECM synthesis related protein Collagen Ⅱ.Results: 1.Compared with group C,there were 117 DEGs in group S,including 54up-regulated genes and 63 down-regulated genes.GO and KEGG enrichment analyses were also performed for DEGs.In GO enrichment analysis,the molecular functions of DEGs were mainly related to actin binding,iron ion binding,viral receptor activity,etc.The cellular components of DEGs were mainly located in the cell process membrane,basolateral plasma membrane,cytoplasmic microtubule,etc.DEGs were mainly involved in biological processes such as cellular calcium ion homeostasis,cellular divalent inorganic cation homeostasis,and divalent metal ion transport.KEGG enrichment analysis showed that DEGs were mainly related to signaling pathways such as neuroactive ligand-receptor interaction,complement and coagulation cascades,vascular smooth muscle contraction and tight junction.2.The up-regulated genes Becn2,Cckbr,Ace2,Ric3 and down-regulated gene Pappa2 were verified by RT-qPCR.Compared with the C group,the expression of Pappa2 and Ric3 in the S group was opposite to the sequencing results.Ace2 expression was not statistically significant.The expression of Cckbr was consistent with the sequencing results,but compared with the N group,the expression of Cckbr in the C group was not statistically significant,suggesting that there was no difference in the expression of Cckbr before and after LPS-induced degeneration of ATDC5 cells.Compared with group N,the expression of Becn2 in group C was down regulated,and that in group S was up regulated compared with group C,suggesting that the expression of Becn2 in LPS-induced ATDC5 cells was down regulated,and sesamin could promote the expression of Becn2 in LPS-induced ATDC5 cells,which was consistent with the sequencing results,indicating that sesamin might alleviate cartilage endplate degeneration by targeting Becn2.3.The Becn2 overexpression vector was successfully constructed,and the overexpression of Becn2 in ATDC5 cells was verified by RT-qPCR and WB.4.Flow cytometry was used to detect the proliferation rate of ATDC5 cells.Compared with group N,the proliferation rate of group C decreased,suggesting that LPS could inhibit the proliferation of ATDC5 cells.Compared with group C,the proliferation rate of cells in group C+Becn2 was increased,suggesting that overexpression of Becn2 could promote the proliferation in LPS-induced ATDC5 cells.Flow cytometry showed that compared with the N group,the apoptosis rate of the C group increased,suggesting that LPS could promote the apoptosis of ATDC5 cells.Compared with the C group,the apoptosis rate of the C+Becn2group decreased,suggesting that overexpression of Becn2 can inhibit the apoptosis in LPS-induced ATDC5 cells.5.WB detection of apoptosis-related proteins showed that compared with the N group,the expression of Bcl-2 in the C group was decreased,and the expression of Caspase-3 and Bax was increased,suggesting that LPS could promote ATDC5 cell apoptosis.Compared with group C,the expression of Bcl-2 was increased and the expression of Caspase-3 and Bax was decreased in group C+Becn2,suggesting that overexpression of Becn2 can inhibit the apoptosis in LPS-induced ATDC5 cells.6.Western blotting was used to detect ECM degradation and synthesis related proteins.The results showed that compared with the N group,the expression of MMP-13 and ADAMTS-5in the C group was increased,and the expression of Collagen Ⅱ was decreased,suggesting that LPS can promote ECM degradation and inhibit ECM synthesis in ATDC5 cells.Compared with group C,the expression of MMP-13 and ADAMTS-5 was decreased,and the expression of Collagen Ⅱ was increased in group C+Becn2,suggesting that overexpression of Becn2 can inhibit the degradation of ECM and promote ECM synthesis in LPS-induced ATDC5 cells.Conclusion: Sesamin may promote cell proliferation,inhibit apoptosis,inhibit ECM degradation and increase ECM synthesis by promoting the expression of Becn2 in LPSinduced ATDC5 cells,thus alleviating cartilage endplate degeneration and playing a role in delaying intervertebral disc degeneration.