Study On The Role Of ERK1/2,p38MAPK On Neural Cell Apoptosis Apoptosis And INOS Expression Induced By Long-term Rotenone Intoxication

Rotenone is a naturally occurring chemical with insecticidal and acaricidal, obtained from the roots of several tropical and subtropical plant species belonging to the genus Lonchocarpus or Derris. It is a non-specific insecticide, used in home gardens for insect control, for lice and tick control on pets, and for fish eradications as part of water body management. It has been demonstrated that rotenone exerts its toxic action by inhibiting the function of mitochondrial complex I and blocking the oxygen utility of organix cells. The recent results from etiological studies and from administering rotenone by continuous infusion into the jugular vein of rats indicate a relationship between rotenone exposure and dopamine neuron degeneration. Furthermore, the effects of chronic exposure to sodium cyanide and sodium azide, two cytochrom oxidase inhibitors, produce hippocampus dysfunction and learning or memory deficits in rats. These results suggest a close relation of mitochondria dysfunction to neurodegenerative disease. Although MAPK and NO were both reported participating in the process of neuroapoptosis, the relationship of them remains unclear. In the present study ERK1/2, p38MAPK and iNOS expressions were investigated in animals and cells treated with rotenone. The main aim of this study was to elicit the molecular mechanism of rotenone-induced nerodegeneration through MAPK-NO signaling.Materials and Methods:1. SD rats were administrated for 42 days by sc injections with rotenone solutions at the doses of 1/3,1/6 and 1/12 LD50 daily. Working memory and reference memory tasks of the rats treated with rotenone or placebo were tested by Morris water maze. The brain tissues were prepared and the pathological changes in the hippocampus were observed with light or scanning electronic microscopy. Acetylcholine esterase activity and the neuroapoptosis in the hippocampus tissue of the rats were measured with biochemistry and TUNEL, respectively.2. The concentrations of NO, MDA and the activities of NOS,SOD and GSH-Px inhippocampus of the rotenone-treated rats were determined using bilogical kits and chlormetric analysis method. RT-PCR and Western blot were employed in these study to measure the mRNA and protein expressions of ERK1/2 and p38MAPK.3. In this study PC 12 cell line was served as an established model system for studying mechanisms of neuroapoptosis induced by rotenone treatment. The cells were cultured, treated with different concentrations of rotenone and collected for apoptotic analysis with TUNEL method. ERK1/2 and p38MAPK protein expressions were detected with Western Blot. The catalytic activity of Capase-3 was tested by fluorimetric analysis method and the transcriptional activity of AP-1 and CREB were determined by the method of EMSA.4. PD98059 and SB203850, the ERKl/2 and p38MAPK inhibitors, were also used to study the role of MAPK regulations in rotenone-induced neuroapoptosis. NO concentration, NOS activity, iNOS expression and AP-1 activity in the PC 12 cells treated with rotenone were extensively determined.Results:1. 42 days rotenone exposure of the rats at various doses produced a obviours decline of animal body weight growing. Some histological injuries of the hippocampus were observed including less staining and positive cells in CA1 and CA3 areas than in dendate gyrus area. Neuroapoptosis was also detected in hippocampus. Long-term treatment with rotenone did not affect acetylchone esterase activity obviously. In the animals administrated with rotenone, furthermore, no significant neurobehavior change was detected in the Morris water maze tests by calculating the parameters of escape latency, navigating distance, plate seaching time, plate crossing frequency and the retention time in the plate quadrant. These results suggest that long-term exposure to rotenone can not affect the ability of learning or memory of rats obviously.2. Long-term rotenone exposure produced up-regulations in expressions of phosphorylated ERKl/2 and p38MAPK protein in rats hippocampus. In the animals treated with lower dose of rotenone, the change of the expressions was about 20%. Higher dose rotenone treatment induced more than 50% up-regulations in the protein and mRNA expressions of ERK1/2 and p38MAPK. Compared with normal group, the phosphorylated p38MAPK protein and mRNA level are up-regulation to 202.91% and 189.59%.TUNEL method and immunohistochemistry method all suggested that long term exposure to rotenone can lead to a enhancement of ERKl/2 and p38MAPK phosphorylation in rats hippocampus. The most number of stained cell is dentate gyrus positive cells. At the sametime, long-term exposure to rotenone the NO, MDA and NOS content increased significantly in rats hippocampus tissue. But the activity of SOD and GSH-PX decreased remarkably. The activity of SOD and GSH-PX of rats hippocampus in 1.2mg/kg rotenone administration group decreased 40.47% and 37.43% than normal group, respectively.3. Rotenone treatment presented morphological and functional changes in PC 12 cells, including the decrease of survival rate, the increase of apoptotic rate, the increase of caspase3 activity and the decrease of mitochondria membrane potential as well. Results from immunohistology and immunobloting experiments indicated that the ERK1/2 phospho relation and the p38MAPK expression can be induced by rotenone administrations. Rotenone also produced increases of the levels of NO, MDA and NOS in PC 12 cells. However, decreased activities of SOD and GSH-PX were determined. After rotenone intoxication the binding activity of AP-1 to DNA was increased. No significant change was observed in CREB.4. Pretreatment with PD98059 did not only affect the apoptotic rate of the PC 12 cell line. SB203850 pretreatment significantly decreased PC 12 cell apoptosis (43%). The pretreatments of the PC 12 cells with PD98059 plus SB203850 presented the same kind of changes as in the cells pretreated with SB203850 only. Decreased NO concentration, NOS activity, iNOS expression were observed either in PD98059 treated cells or in SB203850 treated cells. The combination of PD98059 and SB203850 significantly enhanced the changes induced by PD98059 or SB203850 only. The binding activity of AP-1 to DNA were decreased by PD98059 or by SB203850, especially in the cells pretreated with SB203850. Pretreatment with PD98059 and SB203850, The binding activity of AP-1 to DNA significantly decreased more significantly than rotenone intoxication group, and had no difference with SB203850 pretreatment group.Conclusion1. Long-term rotenone intoxication could inhibit the increase and injure the hippocampus of SD rats , and could induce hippocampus apoptosis. Furthermore, these results suggested that long-term rotenone intoxication could not affect the ability of learning and memory of Morris water maze of rats on the experiment condition.2. Long-term rotenone intoxication could enhance the expressions of phosphorylated proteins and the mRNA of ERK1/2 and p38MAPK in rats hippocampus, and could affect the metabolism of NO and free radical of rats hippocampus.3. Long-term rotenone intoxication could increase the apoptosis rate and caspase 3activity, and could decrease the activites of mitochondria membrane potential of PC 12 cells; The enhancement of ERK1/2 and p38MAPK phosphorylated protein expression,unbalanced the metabolism of NO and free radical and the AP-1 activities of PC 12 cells also could be induced by long-term rotenone intoxication.4. The binding activity of AP-1 to DNA activated by the p38MAPK signal cascade 's activation could enhance the apoptosis rate and iNOS expression of PC 12 cells. Except the ERK1/2 and p38MAPK signal cascade, there was another signal cascade in PC 12 cells that participated in the regulation of the AP-1 activation induced by long-term rotenone intoxication.Significance: The study suggested that long-term rotenone intoxication could activate the ERKl/2 and p38MAPK signal cascade, the binding activity of AP-1 to DNA activated by the p38MAPK signal cascade 's activation could enhance the apoptosis rate and iNOS expression of PC 12 cells. That may play an important role in the hippocampus injury mechanisms induced by long-term rotenone intoxication.