Study On The Mechanism Of Ubiquitin-proteasome Pathway (UPP) In Pyrethroids Neurotoxicity

Pyrethroid (Py) pesticides, the major class of insecticides, are commonly used in agriculture and urban settings due to their high potency and selectivity as nerve poisons and low persistent residues compared with other classes of insecticides. However, the pyrethroids can elicit neurotoxicity, characterized by hyperexcitation, incoordination, tremor and paralysis. A large amount of researches demonstrate that typeⅡpyrethroids such as deltamethrin (DM) can induce the activity of caspase-3 and apoptosis in nerve cells, but the exact mechanism of apoptosis induced by DM is not known yet.The ubiquitin-proteasome pathway (UPP) is the major extra-lysosomal pathway responsible for intracellular protein degradation in eukaryotes. It is composed of the ubiquitin-conjugating system and the 26S proteasome. Degradation of proteins through this pathway involves two distinct and successive steps. Firstly, target proteins, usually abnormal proteins, are conjugated to polyubiquitin, called ubiquitinated proteins. Secondly, they are recognized and recruited to the 26S proteasome where they are rapidly degraded. The UPP is important in controlling intracellular levels of a variety of short-lived proteins and in maintaining cellular growth and metabolism. The UPP plays a role in many cellular processes, including cell cycle controlling, signal transduction, DNA damage and repair, apoptosis, but there are few reports about how the UPP play a role in toxic mechanism.Based on departed researches on Py neurotoxic mechanism, the aims of this study were to determine how the Py poisoning was associated with the ubiquitin-proteasome pathway. Taking advantage of proteasome inhibitor MG-132, to test the hypothesis that if the UPP induces neurocyte toxicity. The present study will provide new insights to molecular mechanisms of the UPP in the toxic mechanism of pesticide.Objective:To explore the effects of DM on the expression of ubiquitinated proteins in hippocampus of rats.Methods:Male Wistar rats weighing 180-220g were divided into 4 groups:control group, 12.5 mg/kg body weight DM were administered by intraperitoneal injection. The animals were decapitated after treatment of DM for 5 h,24 h and 48 h. Immunohistochemical method was applied to measure the expression of ubiquitinated proteins in CA1, CA2, CA3, CA4 area of hippocampus of rat brain. The expression of ubiquitinated proteins in hippocampus were detected by western blot.Results:The results of immunohistochemistry showed that after treatment of DM, the expression of ubiquitinated proteins enhanced in all areas of hippocampus in 5 h,24 h and 48 h groups. Compared to control group, there was significant difference of optical density (P<0.05). Western blot experiment indicated that after treatment of DM on different time points, the expression of ubiquitinated proteins increased in hippocampus. Compared to control group, there was significant difference of relative band intensity (P<0.05).Conclusions:In this condition, DM could enhance the expression of ubiquitinated proteins, which indicated that UPP was activated.Objective:To observe the effect of DM on apoptosis in hippocampus cell of rat.Methods:The animal model was the same as sectionⅠ. The utilization of flow cytometry (FCM), the comet assay and western blot was aim to analyze apoptosis of hippocampus of rat from many aspects.Results:FCM analysis indicated that in all DM treated group, the early apoptotic rate were increased (P<0.05). The results of the comet assay showed that in all DM treated group, DNA damage was worse compared to the control group (P＜0.05). Western blot indicated that bcl-2 was decreased in 24h and 48h of DM-treated group (P<0.01), but didn't show significant change in 5h group. Procaspase-3 was decreased in 5h of DM-treated group (P<0.05), there was significant difference (P<0.01) in 24h and 48h group.Conclusions:The apoptosis of hippocampus cell could be induced after treatment of DM.Objective:To study the effect of UPP on DM-induced apoptosis in hippocampus cell of rat.Methods:Male wistar rats were divided into 4 groups:(1) control group (salad oil); (2) DM 12.5mg/kg BW; (3) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 2 h before DM (12.5mg/kg BW), both chemicals were intraperitoneal injected; (4) MG-132 (0.5mg/kg BW) then 2 h later coin oil. After treatment rats were sacrificed by decapitation and the hippocampus were immediately removed for later use. FCM, the comet assay, western blot and the caspase-3 activity kit were utilized to measure the potential protetive effect of MG-132.Results:FCM showed that compared to DM group, the early apoptotic rate of hippocampus cells were decreased in MG-132+DM group (P＜0.05). The comet assay indicated that compared to DM group, the DNA damage were decreased in MG-132+DM group (P<0.05). The bcl-2 western blot showed that compared to DM group, the expression of bcl-2 was increased in MG-132+DM group (P＜0.01). The activity of caspase-3 in MG-132+DM group was lower than DM group (P<0.05). Compared to control group, MG-132 treated alone group didn't show any apoptotic change (P>0.05).Conclusions:In this experimental condition, using MG-132 at the dose of 0.5mg/kg and pre-treated for 2h could attenuate the apoptotic effect induced by DM. Objective:To observe the mechanism of UPP on DM-induced apoptosis in hippocampus cell of rat.Methods:The animal model was the same as sectionⅢ. Co-immunoprecipitation was utilized to detect the interaction between bcl-2 and ubiquitin.Results:The co-immunoprecipitation showed that compared to control group, the bcl-2 ubiquitination was significantly increased (P＜0.01). Pre-treatment with MG-132 almost completely blocked the increase of bcl-2 ubiquitination (P＜0.01). MG-132 treated alone group didn't show any change compared to control group (P>0.05).Conclusions:In this experimental condition, DM could induce bcl-2 ubiquitination which could be inhibited by MG-132.Objective:To study the effect of UPP on BF-induced apoptosis in hippocampus cell of rat.Methods:Male wistar rats were divided into 7 groups:(1) control group (salad oil); (2) BF 7mg/kg BW for 24h; (3) rats were treated daily with BF in the dose of 7mg/kg BW for 5d; (4) MG-132 (0.5mg/kg BW, dissolved in DMSO) administered 1 h, then treated with BF (12.5mg/kg BW) for 24h, both chemicals were intraperitoneal injected; (5) MG-132 administered 1 h, then treated with BF, five successive days (6) MG-132+salad oil for 24h; (7) MG-132+salad oil for five successive days. After treatment, rats were sacrificed by decapitation and the hippocampus were immediately removed to detect apoptotic rate through FCM.Results:Compared to control group, the early apoptosis rate was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the early apoptosis rate was decreased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). Compared to control group, the early apoptosis rate was increased in MG-132 treated alone for 24h and 5d group (P<0.05). Conclusions:In this experimental condition, BF could induce apoptosis of hippocampus cell, and this induction could be blocked by MG-132, which indicated that UPP may play a role in BF-induced apoptosis.Objective:To study the effect of UPP on BF-induced oxidative stress in hippocampus of rat.Methods:The animal model was the same as sectionⅠ. SOD kit, MDA kit and GSH kit were utilized to detect oxidative stress.Results:(1) MDA level:compared to control group, the level of MDA was increased in BF 24h and 5d group (P<0.05). Compared to BF 24h group, the level of MDA was decreased in MG-132+BF for 24d group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group (P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (2) SOD activity unit:compared to control group, the SOD activity unit was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 5d group, the SOD activity unit was increased in MG-132+BF for 5d group (P<0.05). But there wasn't significant change between BF 24h group and MG-132+BF for 24h group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05). (3) GSH level:compared to control group, the level of GSH was decreased in BF 5d group (P<0.05) but not in 24h group (P>0.05). Compared to BF 24h group, the level of GSH was increased in MG-132+BF for 24h group (P<0.05). But there wasn't significant change between BF 5d group and MG-132+BF for 5d group(P>0.05). However, compared to control group, MG-132 treated alone for 24h and 5d group didn't show any change (P>0.05).Conclusions:In this experimental condition, BF could induce oxidative stress of hippocampus cell, and this induction could be inhibited by MG-132, which indicated that UPP may take part in BF-induced oxidative damage. Objective:To study the effect of UPP on cytotoxicity induced by Py in SH-SY5Y cell.Methods:Cells were divided into 7 groups:blank control group, vehiche control group (1‰DMSO), DM group, MG-132+DM group, BF group, MG-132+BF group, MG-132 group. The dose of DM and BF was the same:1×10-9,1×10-8,1×10-7,1×10-6,1×10-5,1×10-4,1×10-3 mol/L. The final concentration of MG-132 was 1×10-6μmol/L (DM, BF, MG-132 were all dissolved in DMSO). MG-132 was treated 2h before DM and 1h before BF. After DM or BF treated for 24h, the cell survival rate was measured by the method of MTT.Results:(1) The cell survival rate of DM group was 105.48%,95.32%,98.15%,82.09%, 74.79%,72.06%,80.45%, there were significantly lower than blank control group in the dose of 1 10-6,1×10-5,1×10-4,1×10-3 mol/L (P<0.01). Pretreated with 1×10-6μmol/L MG-132, the cell survival rate of DM all groups were 109.35%,97.14%,95.67%,91.86%,82.53%, 75.49%,78.74%, there were significantly higher than DM group in the dose of 1×10-6,1×10-5 mol/L (P＜0.05). (2) The cell survival rate of BF group was 101.12%,94.24%,92.52%,92.61 %,87.67%,76.22%,66.95%, there were significantly lower than blank control group in the dose of 1×10-5,1×10-4,1×10-3 mol/L (P＜0.01). After 1×10-6μmol/L MG-132 intervention, the cell survival rate of BF all groups were 105.64%,98.32%,94.75%,90.68%,95.24%, 86.24%,70.84%, there were significantly higher than BF group in the dose of 1×10-5,1×10-4 mol/L (P<0.05).Conclusions:In this research, DM and BF under certain concentration could induce cytotoxicity of SH-SY5Y cell. MG-132 can inhibit the adverse effect induced by DM and BF, which implied that UPP may play a role in the induction of cytotoxicity.Taken together, these findings demonstrate that DM can induce neuron apoptosis through stimulating ubiquitin-proteasome pathway (UPP). DM induces an activation of ubiquitinated proteins and caspase-3, as well as the DNA damage. Meanwhile DM lowers the expression of bcl-2. The inhibitor of UPP, MG-132, may block the apoptosis in a certain degree induced by DM. Furthermore, bcl-2 ubiquitination was inhibited after MG-132 treated, which suggested that bcl-2 ubiquitination may be a mechanism of DM neurotoxicity. Besides, UPP could play a role in BF-induced neuron cytotoxicity, including apoptosis and oxidative stress. At the same time, MG-132 had a certain protection against cytotoxicity induced by DM and BF in vitro. These findings imply that UPP play a role in the toxic mechanism of pesticide.