| Research Background and Objective: The reactive oxygen species (ROS) is one of biological free radicals in the body, its oxygen-containing functional groups are chemically active. The free radicals can almost react with any cellular components, causing lipid peroxidation chain reaction, resulting in cell apoptosis and dysfunction. Under normal circumstances, ROS generation and removal keeps balance of the body. When the body suffers from oxidative stress, the amount of ROS was largely enhanced, and the antioxidant capacity decreased, leading to initiation of lipid peroxidation, which may cause DNA oxidative damage and abnormal protein expression. In many neurological diseases such as Alzheimer's disease, Parkinson's disease, ROS plays an important role.Acrylonitrile (AN) is an important raw material in chemical industries, widely used in synthesis of acrylic fibers, nitrile rubber, ABS engineering plastics, synthetic resins. The opportunities for occapational and daily life exposure increased significantly. Because of rapid development of AN industries, in order to protect the health of populational exposure to AN, its toxicity especially neurotoxicity have extensively explored. However, the characteristics of AN neurotoxicity and the exact underlying mechanism is still unclear so far. Several studies have shown that, AN caused oxidative stress, which palys key role in the toxic effects of AN. So from point of oxidative stress, how to explore deeply the mechanism of neurocoxicity and to seek for chemopreventive agents has been hot in the field of AN toxicology.Curcumin (CUR) is extracted from the rhizomes of the ginger family plant Curcuma longa (Cuma longa L.). Because of its color stability and low toxicity, it has been widely used as food additives and dyes. Studies suggest that CUR may produce anti-inflammatory, anti-atherosclerosis, anti-tumor, anti-oxidation, scavengering free radical and other pharmacological effects. Curcumin has a good protection for liver, kidney and immuno system. However, up to now, neuroprotection of CUR and its related mechanism has not been confirmed.Transcription factor Nuclear Factor-E2-related factor 2(Nrf2), as a member of zinc finger protein (bZIP) Cap'n'Collar, plays an important regulational role in the activation of genes coding antioxidant enzymes and phaseⅡdrug-metabolizing enzymes, which are regulated via antioxidant response element (ARE). Some studies showed that CUR may activate Nrf2, so the role of activation of Nrf2-ARE in neuroprotection of CUR is much more concerned.In this study, primary culture of neonatal rat astrocytes(AST) treated with AN was served as model of oxidative stress, and CUR as an inteventive agent. The cell viability, cell toxicity, oxidative stress and energy metabolism were evaluated for potential protection of CUR in rat AST; and the protective mechanism was aslo explored. Our study may offer biochemical evidence for the development of health food products for occupational protection. And also, as references for the prevention and treatment of neurodegenerative diseases as well as populations exposured to neurotoxic chemicals.Research methods: The AST were cultured primarily, and then were divided into several groups: blank control, solvent control (DMSO), 1.0mmol/L AN treated for 12 hour, CUR pretreated for 6 hour(2umol/L, 5umon/L, 10umol/L, 20umol/L). Cell viability and toxicity were evaluated by MIT assay and LDH assay. MDA content, GSH level, CAT and SOD enzymes activities were also determined. DCF-DA as fluorescence probe was used to detect ROS level by Flow Cytometry and Microplate fluorescence microplate reader; Cytochrome c oxidase activity was assessed by oxidation of reductive cytochrome C. Next, Nrf2 translocation was detected by Western Blot and immunohistochemical fluorescence; Western Blot was aslo used to detect expressions of Nrf2, keap1, HO-1 andγ-GCS proteins, and RT-PCR was used to detect expressions of Nrf2, HO-1 andγ-GCS mRNAs.Main results: 1) Compared with control, AST treated with AN alone significantly decreased the cell viability(P<0.05); However, CUR concentration>5umol/L could significantly inhibit the decline of cell viability induced by AN treatment(P<0.05).2) The LDH leakage in AST increased remarkably after treatment of AN compared with that of control group(P<0.05). CUR significantly stopped AN-induced increase of the level of LDH leakage(P<0.05), in a significant concentration dependent manner.3) Compared with control, AST in AN group, MDA content increased remarkably(P<0.05), ROS formation increased significantly (P<0.05), GSH level decreased significantly(P<0.05), CAT activity reduced significantly (P<0.05), SOD activity was some lower(P>0.05). However, Curcumin pretreatment signifantly inhibited the increase of MDA content and ROS generation induced by AN(P<0.05), CUR concentration >2umol/L signifcantly reversed the decrease of GSH level(P<0.05). When CUR concentration up to 20umol/L, SOD and CAT enzymes activities were aslo enhanced signifcantly compared with that of AN group(P<0.05).4) Cytochrome C oxidase activity was significantly lower in AN group compared with that of control(P<0.05). However, enzymes activity of the AST pretreated with CUR>2umol/L, were significantly higher compared with that of AN treated group(P<0.05), and with the concentration of Curcumin increasing, it gradually returned to normal levels.5) By application of immunofluorescence and Western Blot, it showed that there was only a little bit of Nrf2 in the nucleus of AST treated with AN compared with that of control(P>0.05); up to 10umol/L CUR pretreatment, the translocation of Nrf2 from cytoplasm to the nucleus signifacantly increased(P<0.05). It aslo showed that CUR upregulated the Nrf2 protein by Western Blot and Nrf2 mRNA by RT-PCR. However, the Keap1 protein expression wasn't effected by CUR.6) Compared with control, the expressions ofγ-GCS and HO-1 proteins in AN treatment group increased slightly(P>0.05) from Western Blot assay and the expressions of Nrf2,γ-GCS and HO-1 mRNAs increased slightly(P>0.05) from RT-PCR assay. However, after CUR pretreatment, they all became higher and higher with the concentration increasing. When the CUR concentration>5umol/L, they all showed a significant statistical difference(P<0.05), in a concentration dependent manner.Conclusions: Curcumin can significantly improve cell viability of AST, reduce cytotoxicity and improve antioxidant enzyme activity, reduce lipid peroxidation and inhibit ROS generation induced by AN. And Curcumin can enhance Nrf2 protein as well as increase its mRNA level. Curcumin can significantly stimulate Nrf2 from cytoplasmic translocate into nuclear, and activate its downstream genens such as HO-1 andγ-GCS, the protein and mRNA are upregulated by Curcumin. It can be concluded that Curcumin protects against AN neurotoxicity and its mechanism may be associated with the activation of Nrf2-ARE signal pathway.
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