Study On The Mechanism Of Deltamethrin-induced Neurocytes Apoptotic Signal Transduction

As one of the popular applied pesticide, the toxic effects on human beings of pyrethroids (Py) are paid more and more attentions. A large amount of researches indicate that pyrethroid is a kind of neural toxin and mainly displays exciting toxicity. However the exact mechanism of its neural toxicity is not known yet. The departed studies on the neural toxicity of typeâ…¡pyrethroids such as deltamethrin(DM) in our laboratory showed that after treatment of DM, the cellular Ca2+ stepped up obviously, the activity of Caspase-3 changed and the apoptosis in nerve cells were induced in rat brain.Apoptosis is cell independent orderly death controlled by genes in order to maintain the homeostasis and it is one of normal physiologic adjustments in the progress of cell growth whose mechanism of apoptosis expresses highly conservatism. Therefore the mistakes appearing in this progress may lead to cancers, disharmony of autoimmunity and neural degenerative diseases and so on.Mitochondria is the centre of energy and metabolism in eukaryotic organism, and it is also the key cell organ in the apoptosis transmission. More and more researches show that stimulated by apoptosis signals mitochondria swell, the outer membrane potential decreases and many apoptosis related factors are released from intermembrance space which act as the master switch. The long-term opening of mitochondria permeability transition pore results in the release of cytochrome C, apoptosis inducing factors, Ca2+ and other apoptosis factors into cytoplasm, which can destruct the whole cell structure and function by activating the main member of apoptosis protein family——caspase, or destroying the intranuclear chromoplasm independently, or affecting other protein relying on Ca2+, and finally lead to the formation of apoptotic body.Based on departed researches on DM neurotoxic mechanism, our study intensively investigated the mechanism of apoptotic signal transmission mediated by mitochondria from two aspects of in vivo and in vitro studies by modern methods and techologies of biochemstry and molecular biology, further consummated the neurotoxic mechanism of DM and then referred the theory basis to the precaution and therapy of toxicosis.Partâ… Effect of DM on mitochondria appearance and function in neurocyteObjective: To obverse the effect of DM on mitochondria appearance and function in neurocyteMethods: Male Wistar rats weighing 180-220g were used after administered with 12.5 mg/kg body weight DM solved in salad oil by intraperitoneal injection once and then executed after 5 hours. Transmission electron microscope was applied to observe the changes of mitochondri-a appearance in rat brain cortex and hippocampus. The animals were decapitated after treatment of DM for 5 h, 24 h, 48 h and 72 h and the activities of SDH, Na+-Ka+-ATPase and Ca2+-Mg²⁺-ATPase were measured by spectrophotometry. And the membrane fluidity of mitochon-drial was detected by fluorospectrophotometry.Results: After treatment of DM, under the electron microscope the mitochondria appearance in treated groups changed obviously which displayed decrease of mitochondria numbers, swell of volume, dissolve and break of cristae but those of control group was normal. Compared with the control, in 5 h, 24 h, 48 h and 72 h groups, the activities of SDH, Na+-Ka+-ATPase and Ca²⁺-Mg²⁺-ATPase were inhibited. In cortex, the activity of SDH decreased respectively 40%, 42%, 55%, 37%, that of Na+-Ka+-ATPase decreased respectively 23%, 59%, 68%, 54%, and as to Ca²⁺-Mg²⁺-ATPase it was 33%, 51%, 53%, 49%. Compared with control, the coefficient of viscosity accordingly increased 40%, 50%, 50%, 34%. In hippocampus, the activity of SDH decreased respectively 23%, 15%, 29%, 19%, that of Na+-Ka+-ATPase decreased respectively to 24%, 39%, 73%, 66%, and as to Ca²⁺-Mg²⁺-ATPase it was 45%, 62%, 71%, 65%. The coefficient of viscosity accordingly increased 48%, 69%, 75%, 72%. There was a significant difference (P <0.01).Conclusions: Under the conditions of this experiment, from the angle of morphology, biochemstry and biophysics, DM may change the appearance of neurocyte mitochondria, inhibit the activity of SDH, Na+-Ka+-ATPase and Ca²⁺-Mg²⁺-ATPase, decrease the fluidity of membrane. The injury of cortex is similar to that of hippocampus after treatment of DM. Partâ…¡Effect of DM on mitochondria membrane permeability and potential in neurocytesObjective: To analysis the influence of DM on the mitochondria membrane permeability and potential in neurocytes.Methods: In vivo, Male Wistar rats weighing 180-220g were used after administered with 12.5 mg/kg body weight DM solved in salad oil by intraperitoneal injection once and then executed after 5 h, 24 h, 48 h and 72 h. The activity of cytochrome C oxidase in the mitochondria extracted from the cortex and hippocampus and the membrane permeability were detected by spectrophotometry. FCM was used to measure the mitochondria membrane potential of cortex and hippocampus in rat brain; In vitro, set up the model of rat glial cell. After treatments of DM ranged from 10-8-10-3 mol/L and 1μmol/L CsA, the survival rate of glial cells was measured by MTT. FCM was used to measure the membrane potential of the rat glial cells which were treated by DM and CsA.Results: In vivo, after treatment of DM on rats, in different time group compared with control, the activity of cytochrome C oxidase was inhibited significantly (P < 0.01), and the number of OD₅₄₀ also decreased obviously. Compared with control, in cortex, the number of OD₅₄₀ most decreased in 5 h group and in hippocampus was in 48 h group. Respectively treated by 25μmol/L CaCl₂, 50μmol/L CaCl2, and 2μmol/L CsA + 50μmol/L CaCl₂, the numbers of OD₅₄₀ in the groups changed. Compared with untreated group, OD₅₄₀ decreased apparently in simple CaCl₂ and the difference was significant (P < 0.01). But between the two groups of different doses of CaCl₂, there was no significant difference on the numbers of OD₅₄₀. After exposed to CsA, the number of OD₅₄₀ in 50μmol/L CaCl2 group stepped up obviously and compared with control group, the difference was not significant ( P > 0.05 ). The mitochondria potential in cortex and hippocampus of rat brain decreased apparently and compared with control group there was significant difference. In vitro, after treatment of DM, in 5 h group, the survival rates of DM doses of 1×10^-7)～10^-3 mol/L treated glial cells were respectively 148.18%, 140.77%, 124.15%, 92.25% and 77.12%; in 24 h group, the survival rates of such doses were 100%, 97.89%, 91.76%, 86.39% and 51.34%; in 48 h group, those were 69.11%, 53.96%, 44.55%, 13.37% and 10.38%; and in 72 h group, those rates were only 13.76%, 23.34%, 8.62%, 3.17% and 0.78%. After treatment of DM, the mitochondria potential of glial cells in middle and high groups obviously decreased but there was no transparent change in low dose group. And after exposed to CsA in advance, that potential in middle and high dose groups did not change obviously.Conclusions: In vivo, after treatment of DM, the mitochondria membrane potential decreased and the permeability increased. Ca²⁺ might induce the increase of mitochondria membrane permeability, which would be blocked by CsA. In vitro, DM could lead to the decrease of mitochondria membrane potential in glial cells, which there was dose-effect relationship. It could also be blocked by CsA.The result indicated that DM could affect the mitochondria pathway of apoptosis by enlarged the mitochondria membrane permeability and decreased the membrane potential.Partâ…¢Effect of DM on release of cytochrome C in neurocyte mitochondriaObjectivity: To study the effect of DM on release of cytochrome C in neurocyte mitochondria.Methods: In vivo experiments, western blot was used to determine the expression of cytochrome C in mitochondria and cytoplasm of rat brain cortex and hippocampus. Immunohistochemical method was applied to measure the expression of cytochrome C in cortex and CA1, CA2, CA3, CA4 area of hippocampus of rat brain. In vitro experiments, the expression of cytochrome C in glial cells was detected by western blot and immunofluorescence cytochemistry.Results: In vivo western blot experiment indicated that after treatment of DM, on different time points, the expression of cytochrome C decreased in mitochondria of rat brain cortex and hippocampus but that in cytoplasm increased, compared with control there was significant difference ( P < 0.05 or P < 0.01 ). The results of immunohistochemical showed that after treatment of DM, the expression of cytochrome C enhanced in cortex and CA1,CA2 area of hippocampus in 5 h, 24 h and 48 h groups; and the same effect was found in CA4 area in 24 h group. There was significant difference of optical density ( P< 0.05 or P < 0.01). However, there was no significant difference in CA2 area of 72 h group and CA3, CA4 area of 5 h, 48 h and 72 h groups ( P > 0.05). In vitro western blot and immunofluorescence cytochemistry experiments showed that the expression of cytochrome C enhanced in middle and high dose groups and compared with control group the difference was significant. The expression in low dose group did not change obviously. When treated in advance by CsA, DM did not induce higher expression of cytochrome C.Conclusions: DM might heighten the release of cytochrome C which showed that the expression of cytochrome C decreased in mitochondria and increased in cytoplasm.In vitro, there was the same effects of DM which displayed dose-effect relationship, and CsA would block the effect. All those results indicated that DM increased the realease of cytochrome C through the mitochondria membrane permeability pore and further affected neurcyte apoptosis by mitochondria-mediated pathway.Partâ…£Effect of DM on expression of Caspase-3 in neurocytesObjective: To study the effect of DM on expression of Caspase-3 in neurocytes.Methods: After treatment of 1×10^-6 mol/L DM,1×10^-5 mol/L DM,1×10^-4 mol/L DM,1×10^-5 mol/L DM + 1μmol/L CsA,1×10^-4 mol/L DM + 1μmol/L CsA, the activity and mRNA expression of caspase-3 were detected respectively by chromometry and RT-PCR.Results: Compared with control, after treatment of DM, the activity and mRNA expression of caspase-3 in high-dose group enhanced significantly ( P < 0.01), but there was no obvious change in low and middle groups. After allied treatment of DM+CsA, the activity and mRNA expression of caspase-3 did not change apparently.Conclusions: In the model of glial cell culture, DM could lead to enhance of the activity of caspase-3 possibly by the way of higher expression of mRNA. After treatment of CsA, such effects disappeared. All the above indicated that cytochrome C released from mitochrondria induced by DM could lead to neurocyte apoptosis through Caspase pathway.Partâ…¤Effect of DM on apoptosis in neurocytesObjective: To observe the effect of DM on apoptosis in neurocytes. Methods: After treatment of 1×10-6 mol/L DM,1×10-5 mol/L DM,1×10-4 mol/L DM,1×10-5 mol/L DM + 1μmol/L CsA,1×10-4 mol/L DM + 1μmol/L CsA, the apoptotic rate of glial cell was measured by FCM.Results: Compared with control, after treatment of DM, the apoptotic rate in high group increased significantly ( P < 0.01), but there was no obvious change in low and middle groups. After allied treatment of DM+CsA, the apoptotic rate did not change apparently.Conclusions: CsA could against neurocytes apoptosis induced by DM, which indicated that DM could induce neurocyte apoptosis through mitochrondria-mediated pathway.