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Introduction Earlier work has suggested that the p38 MAPK, JNK1/2, and ERK1/2 signal pathway existed in nucleus pulposus cells and the cell growth, differentiation, and apoptosis were regulated by them. Because osmotic fluctuations are inevitable in the physicochemical environment of intervertebral disc cells, high osmolality could activate p38 MAPK, JNK1/2, and ERK1/2 signal pathway. The effects of high osmolality on the catabolic program and proliferation of nucleus pulposus cells are still not clear. Materials and Methods Rabbit nucleus pulposus cells were cultured and divided into different group at random. The cells were pretreated with inhibitor for p38 MAPK, JNK1/2, and ERK1/2 signal pathway, respectively. In next step, the cells were cultured in different osmolality environment for different time at 37°C in 5% carbon dioxide incubator. After treatments, ratio of apoptosis was measured by flow cytometry, and Western blotting was performed to quantify the expression of the activated forms of p38 MAPK, JNK1/2, and ERK1/2. Furthermore, immunofluorescence analysis with confocal microscopy was performed to confirm the hyperosmolality effects on activation of p38 MAPK, JNK1/2, and ERK1/2 signal pathways in nucleus pulposus cells. Results Our results show that in 500 and 600 mOsm/kg medium, rabbit nucleus pulposus cell apoptosis increased, and a persistent phosphorylation of p38 MAPK, JNK1/2, and ERK1/2 proteins were observed. In the same condition, the apoptotic cells death remarkably decreased when the p38 MAPK and JNK1/2 signal pathways were blocked by their inhibitors SB203580 and SP600125, respectively. On the other side, the apoptotic cells death rate reraised greatly when the ERK1/2 signal pathways were blocked by its inhibitor PD98059. 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