Study On Regulatory Mechanism Of MAPK/ERK Signal Pathway In Hippocampus Of Rats Intoxicated By Rotenone

Rotenone, as a widely used insecticide can damage the function of cell respiratory chain by inhibiting mitochondrial complexâ… . Epidemiological studies show that morbidity risk rate of Parkinson's disease in population group exposed to chronic low-dose rotenone is higher than that of non-exposed population group. Furthermore, it has been confirmed that rotenone could result in dopaminergic neuron apoptosis in vivo and in vitro. There is a tight connection between cholinergic neurons in hippocampus and dopaminergic neurons in substantia nigra and striatum. MAPK/ERK signal pathway plays a role in the formation and consolidation of memory and the process of neuron apoptosis in hippocampus. Thus, it is useful to learn how rotenone regulates MAPK/ERK cascade and how activated ERK1/2 contributes to neuron apoptosis. It is helpful for us to know more of mechanisms of environmental agents in neurodegenerative diseases and the specific way of how rotenone causes cell apoptosis.Objective: To investigate the effect of rotenone on upstream regulation of MAPK/ERK signal pathway combined with in vivo and in vitro studies, and it can be inferred that how the MAPK/ERK cascade contributes to the progress of hippocampal neuron apoptosis. Theoretical guideline of prevention and treatment of degenerative diseases could be provided by understanding more about specific molecular targets of environmental toxicants.Methods: (1) The animal model of rotenone intoxicated rats was established. The general state of animals health was observed. Morphological changes in heart, liver, spleen and kidney were detected by HE staining, so information of general injury of animals could be acquired. (2) Experiments such as HE staining, FJB combined with Nissl fluorescent staining, [Ca²⁺]_i detection, immunohistochemical staining, western blot and so on were used, and influence of rotenone on apoptosis of hippocampal neurons could be observed. Detection of activated ERK1/2 and its upstream regulatory factors would be obtained. (3) The model of cultured hippocampal neurons intoxicated by rotenone was established. Morphological changes of the cells treated with rotenone were observed by inverted microscope. The change of cell activity was detected by MTT method. Double labeled staining of Annexin-â…¤-FITC/PI was used to detect cell apoptosis rates, and changes of [Ca²⁺]_i were observed by laser confocal microscopy. Cultured cells pretreated with PD98059(MEK inhibitor), Nimodipine(L-type Ca²⁺ channel blocker), KT5720(PKA inhibitor), Chelerythrine(PKC inhibitor) and Sulindac Sulfide(raf-1 activation inhibitor) were compared with which was intoxicated by rotenone merely in order to determine the changes of phosphorylated ERK1/2.Results: (1)The animal model of rotenone exposure was successfully established in SD rats implanted with osmotic pumps. Symptoms similar to Parkinson's disease were found in the animals. HE staining indicated that pathological changes to different extents of heart, liver, spleen and kidney emerged in intoxicated rats. Injuries in liver and kidney were more evident. (2) Degenerative neurons were observed in hippocampus of intoxicated rats by FJB combined with Nissl fluorescent staining. Immunohistochemical and Western blot methods indicated that expressions of phosphrylated ERK1/2 and ras protein in hippocampus were up-regulated in the animals treated with rotenone. There was an obvious enhancement in hippocampal neurons of rats intoxicated by rotenone, and so did PKA and PKC. (3) The model of cultured hippocampal neurons intoxicated by rotenone was established successfully. The main morphological changes of the cells induced by rotenone were shrinkage, intensification of refraction and even flotation. Furthermore, MTT experiments verified that rotenone lead a damage to the cell activity in a time-dependent and dose-dependent manner. Rotenone could induce apoptosis in hippocampal neurons determined by flow cytometry, and there was an enhancement of apoptosis rates in the cells pretreated with PD98059 compared to which was treated with rotenone merely. Compared to that of control group, [Ca²⁺]_i in dose group increased depending on different doses and time. Pretreated with Nimodipine could decrease [Ca²⁺]_i significantly in specific time. It is found that the expression of phosphorylated ERK1/2 could be down-regulated by pretreatment with PD98059 and Sulindac Sulfide, whereas, not by Nimodipine, KT5720 and Chelerythrine. Conclusions: (1) Rotenone had an obvious general toxicity to rats, and damaged liver and kindey evidently. (2) Rotenone could cause apoptosis to hippocampal neurons in vivo and in vitro. MAPK/ERK signal pathway in hippocampus could be activated and the expression of phosphorylated ERK1/2 would be enhanced. Activated ERK1/2 may play a role in the process of cell apoptosis and can be considered as a protective factor against apoptosis. (3) The enhancement of [Ca²⁺]_i in hippocampus induced by rotenone in early stage didn't depend on L-type Ca²⁺ channel, and this channel was the main path for the enhancement of [Ca²⁺]_i in intermediate stage. Excitement of low-dose rotenone could result in an obvious [Ca²⁺]_i enhancement. However, [Ca²⁺]_i was increased to a limited extent and there was no time-dependent and dose-dependent relationships, especially no significant difference was found between intermediate stage and later stage. Regulation of ras protein by Ca²⁺ might be the indirect way for regulating ERK1/2. (4) Rotenone could activate many upstream protein factors of ERK1/2, and these activated protein factors participated in a series of correlated regulation, and among them ras and MEK played a key role in the activation of ERK1/2, therefore, activation of MAPK/ERK cascade by rotenone was mainly dependent on Ras→Raf-1→MEK→ERK1/2 classical pathway and then took its subsequent effect.