CPN-9 PROTECTS RABBIT NUCLEUS PULPOSUS CELLS FROM XANTHURENIC ACID-INDUCED CYTOTOXICITY BY ACTIVATING THE NRF2/ARE PATHWAY

Xanthurenic acid as the metabolite of tryptophan can induce the cytotoxicity and damage of nucleus pulposus cells. In addition, it was reported that N-(4-(2-pyridyl)(1,3-thiazol-2-yl))-2-(2,4,6-trimethylphenoxy) acetamide (CPN-9) played a pivotal role in oxidative stress-induced cell death by activating the Nrf2/ARE pathway. Therefore, it was of significance to investigate on mechanism of CPN-9 resisting xanthurenic acid-induced cytotoxicity. Cells used in this research were isolated from rabbit nucleus pulposus, analyzed by the growth curve and staining identifications. Hematoxylin eosin staining, toluidine blue, immunohistochemistry and immunofluorescent staining were performed to observe the cellular morphology. Furthermore, cell proliferative and apoptosis activities were detected by CCK-8 methods and AV-PI double-staining, respectively. Simultaneously, the expressions of Nrf2, ATF3, HO-1 and NQO1 were measured by western blot in this investigation. By analysis of growth curve plotting, cells isolated from nucleus pulposus growed in pattern of "S" in 1-5 generation. Staining observations revealed that nucleus pulposus cells were not only triangular, polygonal and short spindle shaped, but also cell nucleus was relatively large. The CCK-8 research demonstrated that xanthurenic acid had adverse effects on cell proliferative activity. Simultaneously, CPN-9(<= 40 mu M) resisted the impairments induced by xanthurenic acid and exerted protective effect on proliferative activity, consistent with the apoptosis results. Furtherly, the expressions of Nrf2, ATF3, HO-1 and NQO1 were elevated by injection of CPN-9. CPN-9 protects rabbit nucleus pulposus cells against xanthurenic acid-induced cytotoxicity by activating the Nrf2/ARE pathway.