A study of the effect of the endoplasmic reticulum stress in protection the human nucleus pulposus cells from the acid-induced injury

English Keywords:Intervertebral disc degeneration  Nucleus pulposus  Endoplasmic reticulum stress  Acid

English Abstract:

  【Abstract】 Objectives: To investigate the role of endoplasmic reticulum(ER) stress played in acid-induced injury of nucleus pulposus cells. Methods: Normal human nucleus pulposus cells(NPCs) were cultured in vitro, pH 7.4 was used as the control, pH 7.0 and 6.5 were used to simulate the normal and degenerative intervertebral disc acidic environment. All cells were cultured for 12-72h, acid-induced damage model of NPCs was established. CCK8 was used to detect the proliferation of NPCs and transmission electron microscopy(TEM) was used to detect the stress activation of endoplasmic reticulum in NPCs. Immunofluorescence assay was performed to detect the expressions of glycoregulin 78(78 kDa glucose-regulated protein, GRP78) and C/EBP homologous protein(CHOP), the stress specific markers of endoplasmic reticulum. Apoptosis and cell cycle were detected by flow cytometry after 4-PBA(endoplasmic reticulum stress blocker) was used to block endoplasmic reticulum stress. SA-β-gal staining kit was used to observe aging cells. Acidity-induced changes in autophagy-, aging- and apoptosis-related markers were studied by using Western blotting analysis, including LC3, GATA4, p53, p21, p16, Bax, Bcl-2 and Caspase3. Results: Compared with the control group, the overall proliferation capacity of myeloid nucleus cells under acid stimulation (pH6.5 group) was significantly lower than that of the control group. Under TEM, the endoplasmic reticulum expanded significantly, the membrane surface area increased, and the mitochondrial membrane structure of the endoplasmic reticulum was formed, accompanied by mitochondrial swelling. Immunofluorescence detection of cells indicated that GRP78 and CHOP expression increased significantly(P<0.05). After 4-PBA was applied, the apoptosis rate of cells increased, and the acid induced G1 stagnate and transglutaminase positive staining rate significantly increased(P<0.05). Western blot assay showed that the expressions of LC3, GATA4, p53, p21, p16, Bax and Caspase3 increased under acid stimulation(P<0.05), while Bcl-2 expression decreased(P<0.05). After applying 4-PBA, LC3 ratio and bcl-2 expression level decreased, and GATA4, p53, p21, p16, Bax and Caspase3 significantly increased(P<0.05). Conclusions: Acidic microenvironment can activate endoplasmic reticulum stress and play a protective role in acute injury of human medullary nucleus cells induced by acid.