Bioinformatics Analysis Of Intervertebral Disc Gene Expression Profiling And The Mechanism Of TonEBP Regulated Autophagy Involves In Hyperosmotic Condition Of Intervertebral Disc

Objective: To investigate the microarray data on gene expression profiles of intervertebral disc related to hyperosmotic condition and inflammatory response.To reveal the differentially expressed genes(DEGs)and regulatory mechanisms using bioinformatics methods.To detect the expression of autophagy in intervertebral disc tissue under hyperosmotic condition,and to investigate the mechanism of Ton EBP regulated autophagy involves in the modulation of intervertebral disc degeneration.Methods: The gene expression profiles treat or without treat with hyperosmotic condition,IL-1β or TNF-α disc cells were downloaded from Gene Expression Omnibus(GEO)database.DEGs were screened by T test.Me V,DAVID,STRING and Enrichr were used to analysis the DEGs.Based on upon results,we detected the protein expression of autophagy related genes and Ton EBP using Acridine orange staining,Immunofluorescence staining of endogenous LC3 and Western Blot.Addition of Bafilomycin A1 to block the combination of autophagy and autophagy lysosomal,and to detect the effect of hypertonic on autophagy.Then tandem-tagged-m Cherry-EGFP-LC3 B transduction were used to detect the autophagosomes and autophagolysosomes.Stable knockdown of Ton EBP was performed to confirm the role of Ton EBP in autophagy through detect the activation of AMPK and m TOR signaling pathway.Results:(1)Total of 111 DEGs were screened in gene expression profiles treated with hypertonic environment compared with isotonic,including 28 up-and 83 down-regulated genes.The DEGs were enriched in Erb B signaling pathway,Regulation of autophagy,and NFAT,MYB in TFs.In GSE27494,total of 324 DEGs were screened,including 187 up-and 137 down-regulated genes.The DEGs were enriched in Apoptosis,Cytokine-cytokine receptor interaction and Chemokine signaling pathway.And NF-κB,JUN in TFs.753 DEGs(458 up-regulated and 295 down-regulated)were found in the intervertebral disc cells treated with TNF-α,including compared with those untreated.These DEGs were mainly associated with Cytokine-cytokine receptor interaction,Apoptosis,and Chemokine signaling pathway.And NF-κB,STAT1 in TFs.(2)Autophagy related proteins of NP tissue from organ culture showed that there is no change after hypertonicity treatment,similar results in cells experinments.Western blot showed the expression of Ton EBP in NP tissue in an organ culture system and with increased osmolarity treatment the accumulation of Ton EBP can be detected.Western blot showed accumulation of Ton EBP with increasing osmolarity and time course,but the level of autophagy related proteins did not change with increasing osmolarity.Baf A1 treatment leads to further accumulation of LC3 II and SQSTM1.AO staining of NP cells does not show appreciable increase in the number of acidic punta in cells under hyperosmotic condition compared to isotonic condition.Immunofluorescence staining of endogenous LC3 shows the number of LC3-positive autophagosomes remains same under hyperosmotic condition.NP cells transduced with retrovirus expressing tandem-tagged m Cherry-EGFP-LC3 B showed autophagosomes in green/yellow did not change under hyperosmotic condition.When treated together with Baf A1,the formation of autophagosomes in green/yellow increased,but autophagolysosomes in red decreased.Western blot of autophagy related proteins in NP cells with or without stable Ton EBP knockdown,treated with hyperosmotic medium resulted in increased level of SQSTM1 and the activation of m TOR/ULK-1ser757 signaling pathway.Conclusions:(1)It indicated that regulation of autophagy related NFAT pathway may contribute to hyperosmotic-related disc degeneration.Inflammatory factors may be involved in the intervertebral disc degeneration through the NF-κB signaling pathway,Cytokine-cytokine receptor interaction and Chemokine signaling pathway,which provide potential therapeutic targets for the treatment of intervertebral disc degeneration.(2)In the current study,we show that there is a high level of basal autophagy that occurs in healthy rat NP cells.Importantly however,in NP cells,hyperosmotic stress neither induces autophagy.Additionally,hyperosmolarity may affect autophagic flux,indicate that hyperosmotic stress could maintain the baseline of autophagy through Ton EBP/m TOR/ULK-1ser757 signaling pathway.