A new method to detect nicotine's effect in alleviating rotenone-induced toxicity of ubiquitin-proteasome system

Objective: To establish a new method for dynamically detecting the activity of ubiquitin-proteasome system (UPS) in living cells and observe the influence of nicotine on rotenone-induced dysfunction of UPS. Methods: By adding a sequence coding CL1, a degradation signal for UPS, the plasmids pEGFP-C1 expressing GFP were reconstructed into CL1-pEGFP-C1 expressing GFP. PC12 cells were transfected with CL1-pEGFP-C1, and then the stable cell lines expressing CL1-pEGFP-C1 were obtained by G418 selection. After treatment with the proteasome inhibitor MG132, rotenone and nicotine + rotenone, the level of GFP in CL1-pEGFP-C1 stable cell line was analyzed by immunoblotting and the fluorescent intensity of GFP was examined under fluorescent microscope. Results: It was found that MG132 and rotenone treatment obviously elevated the level and fluorescent intensity of GFP in CL1-pEGFP-C1 stable cell line in a dose-dependent manner (P<0.05). Nicotine could block the toxic effect of rotenone on UPS partially by alleviating the level and fluorescent intensity of GFP. Conclusion: A new method for dynamically detecting the activity of UPS in living cells has been successfully established. Rotenone can reduce the activity of UPS and nicotine can alleviate rotenone-induced dysfunction of UPS.