The Roles Of NF-κB And MAPKs Singaling Pathways In Trimethyltin Chloride Neurotoxicity And Mechanism Exploration

Part I Establishment of the apoptosis model induced by TMT in vitro and effects of TMT on NF-ΚB and MAPKs and otherObjective:To establish TMT neurotoxicity model in vitro cell lines and observe proteins expressions of NF-ΚB and MAPKs signaling pathways.Methods:Different doses of TMT were administered to SH-SY5Y and PC 12. MTT assay was used to determine the intoxication dose and time. Then apoptosis was detected by flow cytometry (FCM). NF-ΚB signaling pathway activation was detected by immunocytochemistry, immunofluorescence and western blot technique. The levels of MAPKs, p-Akt, Bcl-2 and XIAP were detected by western blot.Results:(1) The impacts of TMT on SH-SY5Y cell survival and apoptosis:compared with normal control group 5,10,20,40μmol/L of TMT can decrease cell viability significantly after intoxication 24h and 48h. It presented good dose-time-response relationship.2.5,5,10μmol/L of TMT induced early apoptosis significantly,10μmol/L of TMT can increase the rate of late apoptosis. (2) The impacts of TMT on PC 12 cell survival and apoptosis:compared with normal control group,2.5,5,10,20, 40μmol/L of TMT can decrease cell viability significantly after intoxication 24h and 48h. It presented good dose-time-response relationship.1.25,2.5,5,10μmol/L of the TMT induced cell apoptosis significantly, and mainly the late apoptosis. So the following experiments chosen SH-SY5Y cells as a vitro model, and the intoxication doses were 2.5,5,10μmol/L, reaction time was 24h. (3) After intoxication by TMT, the NF-ΚB p65 transferred from the cytoplasm into the nucleus significantly. Nuclear NF-ΚB p65 in 5μmol/L and 10μmol/L groups were 2.87,4.69 times respectively,equal of the control group (P<0.05).The highest dose of TMT caused IΚBαdecreasing significantly. (4) 5μmol/L and 10μmol/L of TMT caused the cell body smaller, slight shrinkage of the nuclear, cytoplasm brown. These indicate the Ub expression increased. (5) MAPKs:The levels of p-JNK and p-p38 were increased significantly in 10μmol/L TMT group,2.5,5,10μmol/L TMT group can induced the p-ERK1/2 activation, but all the TMT groups had no significant influence on the expression of total MAPKs; (6) p-Akt and Bc1-2, XIAP levels were significantly decreased in all TMT groups.Conclusion:In our laboratory conditions,2.5,5,10μmol/L TMT induced SH-SY5Y cell apoptosis, and mainly early apoptosis,1.25,2.5,5,10μmol/L TMT induced PC 12 cells apoptosis, and mainly late apoptosis, therefore, SH-SY5Y cells was selected as the vitro model, the exposure doses were 2.5,5,10μmol/L, the exposure time was 24h. TMT can activate the NF-ΚB and MAPKs signaling pathways and decrease the level of PI3k/Akt and the anti-apoptosis proteins Bcl-2 and XIAP.Part II The "Cross-talk" among NF-ΚB and MAPKs and other signaling pathways in the TMT-induced apoptosis modelObjective:To learn the "Cross-talk" among the NF-ΚB and MAPKs and other signaling pathways at protein expression level in TMT-induced apoptosis model, specific inhibitors of different signaling pathways were used. To observe the effects of various inhibitors on the TMT caused cell apoptosis, trying to ascertain the toxicity pathway. Methods:SH-SY5Y cells were pretreated with MG132, BAY11-7082, SP600125, U0126, LY294002 for 2h before intoxication with TMT for 24h, then extracted the protein. Western blot was used to detect the changes of the protein. Flow cytometry(FCM) asssy was used to detect the effects of inhibitors on TMT induced apoptosis.Results:(1) Pretreatment before TMT exposure with proteasome inhibitor MG132 and NF-κB inhibitor BAY 11-7082 can increase early apoptosis rate significantly. Both inhibitors promoted TMT-induced apoptosis. SP600125, U0126, LY294002 can decrease early apoptosis rate significantly, which can antagonize apoptosis induced by TMT.(2) The ubiquitin-proteasome pathway:Pretreatment with the proteasome inhibitor MG132 reduced NF-κB p65 in nuclear significantly but IκBαin total protein was increased significantly. The ratio of p-ERK/ERK ratio was decreased (P<0.05); p-JNK/JNK was increased (P<0.05); p-p38/p38 ratio was decreased (P<0.05); Expression of Bcl-2 was decreased (P<0.05).(3) NF-κB signaling pathway:Compared with the TMT exposure group, the specificity inhibitor of NF-κB BAY11-7082 pretreatment reduced nucleus NF-κB p65(P<0.05); increased IκBα(P<0.05); reduced the ratio of p-ERK/ERK (P<0.05); phosphorylation of JNK was increased (P<0.05); phosphorylation of p38 was reduced (P<0.05); the expression of Bcl-2 and XIAP was lower(P<0.05).(4) JNK/SAPK signaling pathway:Pretreatment with 20μM of JNK specific inhibitor SP600125 increased the nucleus NF-κB p65 (P<0.05); total protein IκBαwas reduced (P<0.05); Phosphorylation of ERK was increased (P<0.05); Phosphorylation of JNK was decreased (P<0.05); Phosphorylation of p38 was decreased (P<0.05);The level of phosphorylated Akt was reduced(P<0.05); Bcl-2 and XIAP were increased (P<0.05).(5) ERK1/2 signaling pathway:10μM of ERK1/2 specific inhibitor U0126 pretreatment can increase the nucleus NF-ΚB p65 (P<0.05), reduce total protein IΚBa(P<0.05), decrease the phosphorylation of ERK(P<0.05), increase the p-JNK(P<0.05), decrease the phosphorylation of p38 (P<0.05), Bcl-2 expression increase the Bcl-2 and XIAP expression (P<0.05).(6) PI3K/Akt signaling pathways:20μM of Akt specific inhibitor LY294002 can decrease the phosphorylation of ERK (P<0.05); increase the phosphorylation of JNK and p38(P<0.05).Conclusion:(1) In the vitro apoptosis model, the activation of NF-ΚB ubiquitin-proteasome signaling pathway can increase the Bcl-2 of XIAP expression, that can antagonise the apoptosis induced by TMT. The ubiquitin-proteasome signaling pathway was involved in the NF-ΚB activation. However the activation of the JNK and ERK 1/2 signaling pathways lead Bcl-2 XIAP expression reduction, promote apoptosis.(2) The results indicated that there was "Cross-talk" among signaling pathways. NF-ΚB and JNK signaling pathways inhibited each other. JNK and ERK 1/2 signal pathways inhibited each other, too. JNK activation promoted the activation of p38 signaling pathway, but ERK 1/2 and NF-ΚB inhibited p38 signaling pathway. ERK 1/2 signaling pathway inhibited NF-ΚB activation. Because of the mutual adjustment of these signaling pathways, the final outcome for TMT exposure was Bcl-2 of XIAP decreasing, cell apoptosis.PartⅢEstablishment of TMT neurotoxicity model in vivoObjective:To establish the TMT poisoning animal model and study the influences of TMT on learning and memory.Methods:40 male SD rats were randomly divided into 4 groups (n=10). The TMT exposure doses were 0.75,1.5,3 mg/kg, the solvent control group were given equal volume distilled water. The rats were administrated with TMT by oral gavage at 8:00 in the morning for 6 days. The water maze test were done at 2:00 in the afternoon.24h after the last exposure 24h all the animals were decapitated, collected the blood and the blood serum was separated, then detected the biochemical parameters and serum potassium concentration. The organs were carefully separated and calculated the organ coefficient. One half of the brain of each animal was used to make paraffin sections, another half of the hippocampus and cortex were frozen for spare.Results:(1) The general behavior changes:The TMT can cause tremors screaming, irritability, attack behavior, clonus, and other nervous system symptoms of poisoning in rats. The 3mg/kg TMT group began to have symptoms at the 4 day. The scores of symptoms continued to increase after the 4 day. TMT can induce seizures, each dose group had no obvious symptoms of epilepsy at the beginning of exposure. All the animals of 3mg/kg TMT group broke out of five grade of epilepsy according to the epilepsy standards. (2) Weight:Compared with the control group, the body weight of 3mg/kg TMT group decreased significantly at 6th and 7th days(P<0.05). (3) Biochemical parameters:The 3mg/kg TMT can induce AST and ALT significantly. Compared with the control group, BUN and Cr increased significantly in 1.5mg/kg and 3mg/kg TMT groups. The serum potassium concentration decreased significantly in 3mg/kg TMT group (P<0.05). (4)The ability of learning and memory:the escape latency of all rats decreased significantly until the 4 day (P<0.05). After the 4th day, the escape latency of Omg/kg and 0.75mg/kg TMT groups continued to decrease, yet the escape latency of 1.5mg/kg and 3mg/kg TMT groups increased (P<0.05). After six days of training the platform was removed. The space exploration test was carried out. The numbers of across the original platform and the time spending in the target quadrant decreased significantly in 3mg/kg TMT groups, compared with the control group.Conclusions:In our laboratory we established a vivo animal model of TMT intoxication successfully.3mg/kg TMT can cause SD rat poisoning. The symptoms included tremor, irritability, aggression, seizures. The serum potassium concentration decreased significantly, which was poisoning characteristics of TMT. TMT also had significant impact on learning and memory abilities.Part IV Injuries of rat hippocampal neurons caused by TMT and mechanisms studyObjective:To observe the damages and molecular biological mechanism of TMT on hippocampal neurons.Methods:Nissl's staining was used to detect the neuron injury. Flow cytometry was used to measure the apoptosis rate of the rats hippocampal neurons. Ubiquitin level was determined by immunohistochemistry assay Western blot was carried out to analysis the protein expression of NF-ΚB and MAPKs and other signaling pathways.Results:(1)Nerve cell damage:Nissl's staining showed that hippocampus nerve cells arranged in disorder in 3mg/kg TMT group, Nissl's body reduced, cell gap became larger and the number of the cells reduced. (2) Neuronal apoptosis:Compared with the control group, each dose group of hippocampal neurons had significantly early apoptosis(P<0.05). (3)Ubiquitin expression:Ubiquitin increased significantly in 3mg/kg TMT group, showing a brown particulate matter increasing. (4) Protein expression:Compared with the control group, the nucleus NF-ΚB p65 increase significantly in 3mg/kg TMT group. IΚBαreduced significantly and p-JNK, p-p38, p-ERK1/2 were all activated by TMT.Conclusions:In the vivo animal model, TMT can lead neuron losing and apoptosis in hippocamp. The molecular biological mechanisms involved in included activation of NF-ΚB,MAPKs and other signaling pathway, along with the vitro results.