Acid environment effected on autophagy and function of nucleus pulposus cells in rat

English Keywords:Nucleus pulposus cells  Acidic environment  Ovarian cancer G-protein-coupled receptor 1  Auophagy  Cellular function  Rat

English Abstract:

  【Abstract】 Objectives: To explore the autophagy activation and cell function of nucleus pulposus cells(NPCs) effected by acid environment, through observing the expression levels of ovarian cancer G-protein-coupled receptor 1(OGR1) and autophagy-related protein LC3 in rat. Methods: NPCs in normal nucleus pulposus tissues of SD rats were isolated and cultured, which were then identified by toluidine blue, alixin staining and Ⅱ collagen immunofluorescence. The cells were expanded, and the second generation NPCs were cultured in medium with pH 6.2, 6.4, 6.8, 7.0, 7.2 and 7.4 for 24h. Immunofluorescence was used to observe the expression of OGR1 in NPCs. Western blotting was used to examine OGR1. After incubation at pH 6.4 for 0h, 3h, 6h, 12h, 24h, 48h, the expression levels of LC3-Ⅰ and LC3-Ⅱ in NPCs were detected by Western blotting. The OGR1 interference sequences of three different targets were constructed, and the most suitable titer lentivirus was detected. NPCs cells were transfected with the optimal titer lentivirus, and the silencing effects of different interference sequences were detected by Western blotting and Real-time PCR. The interference series were used to construct the OGR1-shRNA lentiviral vector. The second generation NPCs were divided into 4 groups: normal group(DMEM medium), pH 6.4 medium group(blank group), pH 6.4 medium empty vector group(empty vector group), pH 6.4 medium OGR1 -shRNA lentiviral transfection group(transfection group), and the expressions of LC3-Ⅱ and p62 protein were detected by Western blotting after 24 hours of culture. Alcian staining was used to observe the expression of proteoglycan in NPCs. Results: (1)The isolated cultured cells highly expressed proteoglycan and type Ⅱ collagen, which were consistent with the NPCs phenotype. (2)The expression level of OGR1 protein was correlated with the pH value of the medium. When the pH value lower than 7.0, the expression levels of OGR1 and LC3-Ⅱ protein significantly increased(P<0.05), and the expression level was the highest at pH 6.4. (3)When cultured in a medium with a pH of 6.4, the expression levels of LC3-Ⅰ and LC3-Ⅱ in the cells were related to the culture time, which peaked at 24h and remained high at 48h. (4)Three interfering sequences had silent effect on OGR1, which was significantly different from the control group(P<0.05), and OGR1-shRNA1 had the best silencing effect. (5)After the cells were cultured for 24 hours in the medium with pH 6.4 in the transfection group, the expression level of LC3-II was significantly lower than that in the empty group(P<0.05); the p62 expression in the transfection group was significantly higher than that in the empty vector group(P<0.05). The expression of proteoglycan in the transfection group was lower than that in the empty group(P<0.05). Conclusions: The acid environment can promote OGR1 protein expression and autophagy of NPCs in rats. OGR1 mediates the activation of autophagy and affects the function of NP cells.