Leptin inhibits apoptosis of nucleus pulposus cells via promoting autophagy

W. Zhang, Z.-L. Li, Q. Zhang

Department of Orthopedics, General Hospital of PLA, Surgical Building, Haidian District, Beijing, China. weizhang_99@163.com

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• Pharmacology

OBJECTIVE: To investigate whether leptin can regulate the apoptosis of nucleus pulposus cells by adjusting the autophagy of human degenerative nucleus pulposus cells and its possible molecular mechanism.

PATIENTS AND METHODS: The human degenerative nucleus pulposus cells were extracted and cultured, then treated with leptin, leptin inhibitor and leptin neutralizing antibody; the expressions of light chain 3 (LC3) II/I and Beclin-1 were detected by Western blot, and the change of apoptosis rate was detected by flow cytometer. After the nucleus pulposus cells were treated with leptin, bafilomycin A, the autophagy inhibitor, was used to inhibit the autophagy. Western blot was used to detect the expressions of LC3II/I and cleaved caspase 3, and the apoptosis rate was detected by flow cytometer. Finally, Western blot was used to detect the expressions of Akt and extracellular regulated protein kinases 1/2 (Erk1/2) signal pathway-associated proteins after nucleus pulposus cells were treated with LY294002, the phosphatidylinositol 3-kinase (PI3K) inhibitor, and PD98059, the extracellular regulated protein kinases (MEK) inhibitor.

RESULTS: After the cells were treated with leptin, the expressions of LC3II/I and Beclin-1 could be increased, and the apoptosis rate of nucleus pulposus cells could be decreased (p<0.05). After the degenerative nucleus pulposus cells were treated with leptin inhibitor or neutralizing antibody, the expressions of LC3II/I and Beclin-1 were decreased, and the apoptosis rate of nucleus pulposus cells was increased (p<0.05). Bafilomycin A could increase the expression of LC3II/I (p<0.05), increase the apoptosis rate (p<0.05), and offset the effect of leptin of decreasing the apoptosis rate of nucleus pulposus cells. The results of Western blot showed that LY294002 could inhibit the expression of LC3II/I by inhibiting the Akt phosphorylation (p<0.05), but leptin could partially offset the inhibiting effect of LY294002 on the expression of LC3II/I. PD9805 could partially decrease the expression of LC3II/I via inhibiting the Erk1/2 phosphorylation, but leptin could not reverse the inhibiting effect of PD98059 on the expression of LC3II/I.

CONCLUSIONS: Leptin inhibits the apoptosis of degenerative nucleus pulposus cells via promoting autophagy, and leptin regulates the autophagy of human degenerative nucleus pulposus cells through the phosphorylated Erk1/2 signal.

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