Effects Of Bromoxynil On The Cytotoxicity And Signaling Pathways Of NF-κB And MAPKs In SH-SY5Y Cells

Part I The effects of bromoxynil on the cellular proliferation and apoptosis ofSH-SY5Y cells in vitroObjective: To explore the effect of bromoxynil on the cellular proliferation and apoptosis ofSH-SY5Y cells in vitro.Methods: SH-SY5Y cells were cultured and then the effect of bromoxynil on cell damagewas detected by MTT assay. We screen appropriate dose base on cell viability. At last, weselected four dose groups (0,10,50,100μmol/L) and stimulated SH-SY5Y cells for24h.Themorphological changes were observed under inverted microscope. In addition, Cell apoptosiswas measured by double labeled staining with Hoechst33342/Propidium iodide (PI) and flowcytometry. Finally, the effect of bromoxynil on cell growth curve and doubling time weremeasured by cell counting. We used MTT assay to detect cell inhibition rate and IC50whenSH-SY5Y cells was treated by bromoxynil for24h and48h.Results: After exposure to bromoxynil (0,10,50,100μmol/L) for24h, SH-SY5Y cells lostcontact with their neighbor and displayed a spindle-shape. The impacts of bromoxynil onSH-SY5Y cell apoptosis: Hoechst33342/PI staining showed that a small amount of nucleardebris or lobule, and the nuclear appeared light blue in100μM of bromoxynil group. Theapoptosis rates of SH-SY5Y cells in the bromoxynil treated groups were no significant higherthan those in the control group（P＞0.05）. The impacts of bromoxynil on SH-SY5Y cellproliferation: cell proliferation inhibition rate (CPIR) of SH-SY5Y cells with50or100μM ofbromoxynil was higher than that of the vehicle control group(P＜0.05)， in aconcentration-dependent manner. IC50of cell proliferation in cells exposed to bromoxynil for24h was267.00±29.54μmol/L，95%CI（193.06～340.40μmol/L).And for48h, it was54.33±8.08μmol/L，95%CI（34.25～74.41μmol/L). IC50of cell proliferation of24h washigher than48h (P＜0.05). The cell growth was inhibited with different degree when the cellexposure to10,50or100μM of bromoxynil (P＜0.05).Conclusion: It was concluded that bromoxynil could restrain the proliferation of SH-SY5Ycells but it has little effect on cell apoptosis. Part II The effects of bromoxynil on NF-κB and MAPKs Signaling Pathways bybromoxynil in SH-SY5Y cellsObjective: To establish bromoxynil neurotoxicity model in SH-SY5Y cells and observeproteins expressions of NF-κB and MAPKs signaling pathways.Methods: SH-SY5Y cells were cultured and then stimulated with indicated dose ofbromoxynil (0,10,50, or100μmol/L). Western-blot was performed to detect the expressionof NF-κB, IκBα, Bcl-2, XIAP, p-p38, and p-JNK. Besides, the expressions of NF-κB andCytc-c were analyzed by immunocytochemistry.Results: After exposure to bromoxynil for24h, The expression of NF-κB,p-p38and Cytc-cwere up-regulated, while that of IκBα and Bcl-2were down-regulated (P＜0.05). The levelsof p-JNK and XIAP in the bromoxynil treated groups were no significant higher than those inthe control group（P＞0.05）.Conclusion: In our laboratory conditions, bromoxynil can activate the NF-κB andp38MAPKs signaling pathways and decrease the anti-apoptosis proteins Bcl-2. It wasconcluded that NF-κB, p38MAPKs and Bcl-2were involved in bromoxynil neurotoxicity thatbromoxynil restrains the proliferation of SH-SY5Y cells.