In Vitro Study on the Mechanism of Autophagy of Nucleus Pulposus Cells Induced by Glycosylation of O-GlcNAc

Objective: The aim of this study was to determine the mechanism of autophagy of nucleus pulposus (NP) cells induced by glycosylation of O-GlcNAc in vitro. Methods: NP cells were extracted and cultured from 12-week-old, healthy male SD rats. The nucleus pulposus cells of second generations SD rats were divided into two groups: control group and experimental group. The control group after the second generation cultured NP cells of SD rats; the experimental group was divided into OGT group and OGA group, respectively using O-GlcNAc transferase and O-GlcNAcase intervention. The proportion of autophagic vacuolar cells, positive cells and NP cells were analyzed by flow cytometry, and the viability of cells was analyzed by OGT/OGA. The expression of autophagy and apoptosisrelated genes in NP cells was detected by real-time fluorescence quantitative PCR (RFQ-PCR) at each time point. The expression of autophagy-related genes (LC3B and Beclin-1) and apoptosisrelated genes (Caspase-3, Bcl-2 and Bax) were detected by extracting NP mRNA from each group. Result: Flow cytometry showed that within the duration of incubation, the number of positive cells of autophagic vacuoles increased gradually, reached the peak at 36 hours, and decreased after 48 hours. Compared with the control group, the positive rate of, 36 hours and 48 hours in OGA group was higher than that in control group (P < 0.05). The number of apoptotic cells increased gradually and reached the peak at 48 hours. The ratio of apoptotic cells in OGA group, 24 hours, 36 hours and 48 hours was lower than that of control group (P < 0.05). The number of viable cells gradually decreased and reached trough values at 48 h. Compared with the control group, the percentage of viable cells in the 24, 36 and 48 hours was significantly higher in the OGA group than in the latter group (P < 0.05). The expression of Bcl-2 gene in rat NP cells was decreased with the prolongation of culture time in NP cells of the second generation SD rats. The expression of Bcl-2 gene in NP cells was decreased by RFQ-PCR (P < 0.05). The expression of Caspase-3 and Bax gene increased gradually (P < 0.05). The expression of Caspase-3 and Bax gene was significantly increased (P < 0.05). The expression of Bcl-2 gene was significantly decreased in OGA group compared with control group (P < 0.05). The expression of LC3B and Beclin-1 gene in NP cells of NP rats increased gradually (P < 0.05), and the expression of autophagy-related genes in NP cells of OGT group and control group showed that the expression of LC3B and Beclin-1 (P < 0.05). The expression of OGT in the OGT group was significantly higher than that in the control group (P < 0.05) at the time of 12 hours and thereafter. Conclusion: As an O-GlcNAc inhibitor, OGA can promote autophagy in NP cells by inhibiting the glycosylation process of O-GlcNAc, thereby inhibiting the process of NP cell apoptosis and increasing the proportion of NP cell survival.