Modulation Of PC12Cells Injury And BV2Microglial Activation By5-lipoxyenase In Rotenone-induced Cell Model

Parkinson disease (PD) is a chronic neurodegenerative disorder characterized by the selective degeneration of the nigrostriatal dopaminergic (DA) neurons in the central nervous system. Neuroinflammation plays a prominent role in the pathogenesis of PD, and microglial activation contributes to initiating and maintaining brain inflammation and neuronal death.5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to produce cysteinyl leukotrienes (CysLTs; LTC4, LTD4, LTE4). CysLTs are potent proinflammatory mediators, and their actions are mediated by activating CysLT receptors. The role of5-LOX in brain inflammation has been recently reported. However, whether5-LOX regulates neuronal damage and inflammatory responses associated with PD, and whether5-LOX represents a potential therapeutic target for PD remain unknown.In this project, we plan to study following problems.1) To reveal whether the change of5-LOX expression is related to rotenone-induced PD-like injury in PC12cells.2) To clarify whether5-LOX/CysLT] receptor play roles in rotenone-induced microglial activation and release of inflammatory factors in mouse microglial cell line BV2cells.3) To observe the effects of5-LOX inhibitor and CysLT1receptor antagonist on PD-like neuronal injury and microglial inflammation. Part15-lipoxygenase is involved in rotenone-induced injury in PC12cellsOBJECTIVE:To determine whether5-LOX is involved in rotenone-induced neurotoxicity in PC12cells, which is a cell model of Parkinson disease.METHODS:After rotenone treatment for various durations, cell viability was determined by the colorimetric MTT reduction assay, and5-LOX translocation was detected by immunocytochemistry. The effect of the5-LOX inhibitor zileuton was also investigated.RESULTS:●Rotenone (0.3-30μmol/L) induced PC12cell injury, and zileuton (3-100μmol/L) attenuated cell injury.●Rotenone time-and concentration-dependently induced5-LOX translocation into the nuclear envelope (a key event for5-LOX activation), and zileuton (1-30μmol/L) significantly inhibited rotenone-induced5-LOX activation.CONCLUSION:5-LOX is involved in rotenone-induced injury in PC12cells, and the5-LOX selective inhibitor zileuton can reduce rotenone-induced5-LOX activation and cell injury.Part2Role of5-lipoxygenase/cysteinyl leukotriene receptor1in rotenone-induced BV2microglial activationOBJECTIVE:To determine the role of5-LOX/CysLT1receptor in rotenone induced BV2microglial activation.METHODS:BV2cells, a murine BV2microglia cell line, were cultured in media with or without rotenone for24h. The morphplogical and proliferation changes in BV2 cells were observed. Phagocytotic activity of BV2cells was evaluated using immunofluorescence technique and flow cytometry. The expression and distribution of5-LOX and CysLT1receptor were determined by western blotting and immunocytochemical analysis. The levels of5-LOX products CysLTs (LTC4, LTD4and LTE4) and cytokine interleukin (IL)-1β, tumor necrosis factor-a (TNF-a) were detected by ELISA method. The effects of the5-LOX inhibitor zileuton and CysLT1receptor antagonist montelukast were investigated.RESULTS:●The low doses of rotenone (0.001-0.03μmol/L) induced cell proliferation and microglial phagocytosis, and increased release of cytokine production IL-1β and TNF-a in BV2cells. Selective5-LOX inhibitor zileuton (0.01-1μmol/L) significantly inhibited rotenone-induced cell proliferation and microglial phagocytosis, and decreased IL-1β and TNF-a release.●Furthermore, we found that5-LOX expression up-regulated in a concentration-dependent manner, and5-LOX translocated into the nuclear envelope/cytoplasm in rotenone-activated BV2cells, The levels of CysLTs increased in the culture medium.5-LOX inhibitor zileuton (0.001-1μmol/L) significantly inhibited rotenone-induced5-LOX activation and reduced the production of5-LOX products, CysLTs.●In addition, CysLT1receptor antagonist montelukast (0.001-1μmol/L) inhibited rotenone-induced microglial phagocytosis and reduced cytokine IL-1β release; The expression of CysLT1receptor increased after rotenone treatment at0.001and0.003μmol/L; Rotenone caused time-dependent translocation of CysLT1receptor from the cytosol to the nucleus.CONCLUSION:These results suggest an involvement of the5-LOX/CysLT1receptor in rotenone-induced BV2microglial activation. The5-LOX/CysLT1receptor signaling pathway might therefore be a potential therapeutic target for modulating microglial-mediated inflammation of PD.