RESUMO
The microenvironment of degenerative intervertebral disk (IVD) is characteristic of a high concentration of lactic acid and low pH levels, whereas the underlying mechanism has not been clearly defined. Endoplasmic reticulum (ER) is the hub of interactions between environmental signals and cellular biological functions, the malfunction of which is closely involved in the pathogenesis of multiple disorders, including IVD degeneration (IVDD). This research mainly aims at exploring what role ER stress plays in the natural process of IVDD and pH-induced apoptosis of rat nucleus pulposus (NP) cells (NPCs). The IVD of Sprague-Dawley rats at different ages was stained by Hematoxylin-Eosin staining to visualize the histocytological changes during the nature process of IVDD. Immunohistochemical staining was performed to evaluate the expression of ER stress markers within normal and degenerated NP. The ER stress markers were also quantified by quantitative real-time-polymerase chain reaction (PCR) and Western blotting analysis, respectively. NPCs were exposed to the culturing media with acidity of pH 7.4, 7.0, 6.5, or 6.0 for 24-72 h, with or without the supplement of 4-phenylbutyrayte (4-PBA, the blocker of ER stress pathways). Changes in cell viability were evaluated by CCK-8 assay and neutral red assay, whereas apoptosis was stained by Annexin-V/PI staining and quantified by flow cytometry analysis. The acidity-induced changes in the expression of ER stress markers were studied by immunofluorescent staining, qRT-PCR, and Western blotting analysis. In vivo, the expression of GRP78 and XBP1 was downregulated whereas CHOP and Caspase12 were upregulated in natural degeneration. In vitro, low pH induced apoptosis of rat NPCs; prolonged exposure of acid reduced cell viability and caused upregulation of ER stress markers. 4-PBA was used to alleviate ER stress, and it promoted acid-induced apoptosis of NPCs. ER stress is involved in NP natural degeneration and attenuates low-pH-induced apoptosis of NPCs.
