| With the changes of the energy structure in the large and middle cities and the changes in atmospheric pollution in China, the harmful effects of air pollution from coal fired sources on the environment and human health will become more subtle and more easily overlooked. In China, the sulfur dioxide (SO2) emissions which still are ahead of dust, nitrogen oxides and carbon dioxide and other atmospheric pollutants, contribute 85% of the air pollution caused by burning coal. As Shanxi province is a leading producer of coal in China, atmospheric SO2 pollution is widespread over the industrial and mining areas and areas of concentrated population. Therefore, long-term effect of exposure to low-concentrations of SO2-induced health hazards is particularly prominent. In addition, an increasing body of literature provides evidence that SO2 pollution in atmospheric environment is involved in neurotoxicity and increased risk for hospitalization and mortality of many brain disorders; however, our understanding of the mechanisms by which SO2 caused harmful insults on neurons remains elusive.Firstly, in order to investigate the effects of SO2 exposure on CNS, Wistar rats were treated with SO2 at various concentrations (7,14,28 and 56 mg/m3, 6h/day, for 7days). The PCO contents in rat hippocampus were measured by spectrophotometry; the levels of interleukin-1β(I1-1β) and tumor necrosis factor-α(TNF-α) in rat hippocampus were measured by ELISA; and the mRNA and protein expression of apoptosis-related genes (p53, bax, bcl-2, c-fos and c-jun) in rat hippocampus were measured by real-time RT-PCR analysis and Western blotting technique. The results demonstrate that SO2 inhalation significantly increased the PCO content in rat hippocampus in a concentration-dependent manner; and elevated the levels of I1-1βand TNF-αin rat hippocampus, and the effect was obvious after lower concentrations exposure (7 and 14 mg/mj). Also, the mRNA and protein levels of c-fos and c-jun significantly elevated in proportion to exposure concentration. In addition, primary hippocampal neurons from rat P1 pups were cultured and treated with 30,100 and 300μM SO2 derivatives (bisulfite and sulfite,3:1 M/M) for 6h,12h and 24h separately. The transcription and translation of several apoptosis-related genes (p53, bax, bcl-2, c-fos and c-jun) in rat hippocampal neurons were used real-time RT-PCR analysis and western blotting techniques, respectively. The results showed that p53, c-fos, c-jun mRNA expression and the ratio of bax to bcl-2 augmented as functions of SO2 derivative concentration and exposure time, and confirm that SO2 affected the transcription and translation process of apoptosis-related genes in central nervous system via its derivatives in vivo. The present data provided further evidences for SO2-caused neurological insults, and imply that two major pathways associated with p53 and AP-1 might play important roles in the pathogenesis. The results implied that SO2 inhalation could cause injury to the central neuronal system, and suggestted that the molecular mechanisms might be involved in oxidative stress, inflammation, and neuronal apoptosis.Secondly, the possible neurotoxicity of SO2 has been implicated by determining morphological change, oxidative stress, inflammation, and neuronal apoptosis in previous studies, however, the detailed mechanisms of the signaling pathway of SO2-induced neuronal injury in rat hippocampus remain unclear. In the present study, we investigated SO2 induced neuronal insults in primary hippocampal neurons, and tested the possibility for using COX-2 as its biomarker. The primary hippocampal neurons from rat P1 pups were cultured and treated with 100μM SO2 derivatives (bisulfite and sulfite, 3:1 M/M) for 24h in the absence and presence of antioxidant agents, Vc (500μM) and Ve (100μM); COX-2 inhibitor, NS398 (20μM), and silencing COX-2 expression by shRNA; COX-1 inhibitor, SC-560 (10μM); EP2 and EP4 antagonists, AH6809 (20μM) and AH28348 (30μM); PKA inhibitors, H89 (1μM) and KT5720 (1μM); and NMDA receptor inhibitors, MK-801 (10μM) and DL-2-amino-5-phosphonovaleric acid (D-APV) (25μM). The levels of PGs and cAMP in culture were measured by EIA; the mRNA expressions of COX-2 were measured by real-time RT-PCR analysis; and the protein expression of COX-2, p-NF-KB, EP2 & EP4, NR2B and cleaved caspase-3 in neurons were measured by Western blotting technique. The results indicated that (1) SO2 derivatives up-regulated COX-2 expression and activated caspase-3 to induce apoptosis in primary cultured hippocampal neurons; and COX-2 inhibitor NS398 significantly reduced SO2 derivatives-caused neuronal apoptosis. It is suggestted that SO2 exposure produced neuronal insults, and the neurotoxic effect was likely via stimulating COX-2 elevation; (2) the action of SO2 on elevating COX-2 ultimately appeared to be dependent on the increased production of arachidonic acid-derived prostaglandins, mainly PGE2, and functioning of its EP2 & EP4 receptors. Also, the molecular modulating process might be triggered by free radical attack from SO2 metabolism in vivo, and followed by activating cAMP/PKA pathway and enhancing probability of the release of glutamate, up-regulating NMDA receptor expression, stimulating over-influx of Ca2+ and causing neuronal apoptosis.Thirdly, in order to demonstrate the key role of COX-2 in the possibly signaling pathway of SO2 derivatives-induced neuronal injury in rat hippocampus, the Wistar rats were treated with SO2 at various concentrations (7,14 and 28 mg/m3) for 6 h/day for 7 days, and intraperitoneally injected with NS398 (3 mg/kg bw/day), then exposed to SO2 (14 mg/m3) 1.5 h after injection for 6 h/day for 7 days. The levels of PGE2 and cAMP in culture were measured by EIA; and the protein expressions of COX-2, COX-1, p-NF-κB, EP2&4, NR2B and cleaved caspase-3 in neurons were measured by Western blotting technique. The results showed that SO2 inhalation statistically enhanced COX-2 expression, PGE2 content, EP2 & EP4 receptor expression, cAMP level and NMDA receptor expression, and induced neuronal apoptosis.Finally, the above evidences suggest that SO2 induced neuronal injury, and COX-2-mediated AA metabolism in synaptic signal transduction is the key of regulation at the cellular and molecular levels. In addition, in synaptic signal transduction, COX-2 plays an important role. In order to demonstrate the synaptic transmission participates in SO2-induced neuronal toxicity in rat hippocampus, Wistar rats were treated with SO2 at various concentrations (7, 14,28 and 56 mg/m3,6h/day, for 7days). The synaptic structural parameters of hippocampal CA1 area were observed, measured and analyzed by transelectron microscopy and image analysis instruments in rat hippocampus were measured by spectrophotometry; and the protein expression of synaptic plasticity-related gene (Syp, PSD-95, p-ERK1/2 and p-creb) in rat hippocampus were measured by Western blotting technique. The results showed that SO2 inhalation statistically decreased the width of synaptic cleft, and enhanced the thickness of synaptic active zone (PSD), the length of synaptic active zone, and synaptic vesicles at presynaptic; SO2 inhalation statistically elevated presynaptic and postsynaptic markers (Syp and PSD-95) expression, and inhibited synaptic plasticity-related factors (p-ERK1/2 and p-creb) expression, and induced neuronal injury. The results implied that SO2 inhalation could cause neuronal injury, and suggestted that the molecular mechanisms might be involved in the changes of synaptic transmission.In conclusion, this work reveals a mechanistic basis for exploring an association between SO2 inhalation and increased risk for neurologic disorders, and opening up therapeutic approaches of treating, ameliorating, or preventing brain injuries resulting from SO2 exposure in atmospheric polluting environment. |
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