| Part I Identification of neurotoxicity of TMT using mice and patch clamp modelObjective:To investigate the influences of TMT on general toxicity, behavior, learning and memory on TMT poisoning animal model and patch clamp model.Methods:48 male Kunming mice were randomLy divided into 6 groups,8 mice per group. The groups were including three TMT exposure groups (0.25,0.75,1.25 mg/kg), control group (saline group), LBP group (2 g/kg), and treatment group (TMT 1.25mg/kg+LBP 2 g/kg).The control group, treatment group and TMT groups were administrated with saline or TMT by intraperitoneal injection at 8:00 in the morning for 5 days. The LBP group and treatment group were Iavaged with LBP three days in advance. The general behavior changes were observed at 8:00 in the injection evening. All animals were decapitated, the brain were separated and calculated the quotient of brain. Took 4 animals of the brain in two in each group, half for paraffin section and other brain were separated into hippocampus. All of the tissues were collected and preserved in-80℃. The patch clamp model was including the control group (saline group) and TMT group (1.25 mg/kg),3 male KM mice per group. Intraperitoneal injection for 5 days, free the hippocampus after anesthesia, then detected the change of LTP in hippocampus CA1 area.Results:(1) Weight and the quotient of brain:Compared with the control group, the body weight of 1.25 mg/kg TMT group significantly decreased started at 3 th day (P<0.05). Compared with TMT group, the body weight of treatment group was increased at 8th day (P<0.05). Compared with control group, the quotient of brain of 1.25 mg/kg TMT and treatment groups was increased (P<0.01). (2) The general behavior changes:The TMT can cause nervous system symptoms of poisoning in mice.0.75 mg/kg and 1.25 mg/kg TMT groups began to have symptoms at the 3th day. The scores of symptoms continued to increase after the 4th day (P<0.01). TMT can induce seizures each dose group had no obvious symptoms of epilepsy at the beginning of exposure. All of the animals of 1.25 mg/kg TMT group broke out of three grade of epilepsy according to the epilepsy standards, and treatment group was decreased compared with 1.25 mg/kg TMT group (P<0.01). (3) The ability of learning and memory:Compared with the control group, TMT can decreased LTP of hippocampus CA1 area (P<0.05).Conclusions:The animal model of TMT neurotoxicity was established successfully. 0.75mg/kg TMT can cause KM mouse poisoning. The symptoms included attack behavior, irritability, clonus, tremors screaming.1.25 mg/kg TMT can induce continuous tremors, seizure, and the decrease of body weight. There was a neuron protect effect of LBP,1.25 mg/kg TMT can decrease the LTP level of hippocampus CA1 area. TMT had a significant impact on learning and memory abilities.Part ⅡI Molecular biological mechanisms of mice hippocampus neurons caused by TMTObjective:To observe the neuron damage and molecular biological mechanism of TMT on hippocampus neurons in mice.Methods:Nissl’s staning and immunofluorescence technique were used to detect the neuron injury. Thiobarbituric acid method was used to detect the change of MDA. WST-1 method was used to detect SOD. Immunohistochemical method was used to observe the change of Shh protein. Western blot was used to detect the protein of Sonic Hedgehog, PI3K/Akt, and apoptosis-related protein Bcl-2/Bax.Results:(1) Neuron injury:Nissl’s staining showed that hippocampus neurons arranged in disorder in 0.75 mg/kg and 1.25 mg/kg TMT, the number of neurons reduced, cell gap became larger and Nissl’s body reduced. The immunofluorescence result showed the damage and neuron missing at 0.75 mg/kg and 1.25 mg/kg TMT in mice hippocampus DG area. LBP reduced the neuron apoptosis induced by TMT. (2) The effect of apoptosis proteins in neurons:Compared with the control group, Bcl-2 expression was decreased in TMT groups, and the Bax was increased. Compared with the 1.25 mg/kg TMT group, Bcl-2 expression was increased and Bax was decreased in treatment group (P<0.05). (3) MDA and SOD changes:With the increase of the concentration of TMT, the MDA level was increased in hippocampus, especially in the dose of 0.75 mg/kg and 1.25 mg/kg TMT groups (P<0.05). Compared with the control, SOD was decreased within the increase of the concentration of TMT. Compared with 1.25 mg/kg TMT group, SOD level was increased (P<0.05). (4) Sonic Hedgehog protein changes:Compared with control group the Shh protein was decreased in CA areas, showing a brown particulate matter decreasing. (5) Changes of Sonic Hedgehog and PI3K/Akt pathways:Compared with the control group, Shh was decreased in 0.25 mg/kg and 0.75 mg/kg TMT, and increased in 1.25 mg/kg TMT. There were no significant changes in total Akt and total GSK-3p, but p-Akt and p-GSK-3β were decreased following the increase of the TMT concentration. Compared with the 1.25 mg/kg TMT, the Shh, p-Akt, and p-GSK-3β expression were significant increased in treatment group (P<0.05).Conclusions:In the vivo animal model, TMT can lead Nissl’s body lose, neuron damage. Nissl’s staining and immunofluorescence methods are confirmed the material basis for the TMT nerves damage. TMT can induce the abnormal of apoptosis and oxidative stress. The molecular biological mechanisms involve the inhibition of Sonic Hedgehog, which showed the Shh and Gli1 decreasing; PI3K/Akt pathway inhibition, which show the decrease of p-Akt and activity of GSK-3β; lipid peroxidation reaction.Part III Effect of Sonic Hedgehog and PI3K/Akt signaling pathways on apoptosis induced by TMT in N2a cellsObjectives:To establish TMT neurotoxicity model in vitro cell lines; whether the Sonic Hedgehog and P13K/Akt pathways are concerned with the apoptosis induced by TMT in N2a cells, and discuss the protective effect of LBP.Methods:The intoxication dose was determined by MTT assay. Cell apoptosis was observed by Hoechst33258 staining. The rate of cell apoptosis was determined by Flow cytometry. ELISA method was used to detect the change of caspase-3. The change of ROS was determined by DCFH-DA fluorescence probe. The change of MDA was determined by TBA assay. The change of SOD was determined by WST-1 assay. Sonic Hedgehog, PI3K/Akt signaling pathways and Bcl-2/Bax changes were detected by Western blot. The mRNA expression of Sonic Hedgehog and PI3K/Akt pathways were detected by RT-PCR.Results:(1) The impacts of TMT on N2a cell survival:Compared with the control,3.75 μmol/L and 7.5μmol/L of TMT can decrease the cell viability significantly after 24 h exposure, and it presented good dose-response relationship (P<0.01). Morphological observation showed that, the number of N2a cell was reduced, synapses retract, and cell adherent ability was weakened when cell was treated with 3.75 and 7.5μmol/L TMT after 24 h. (2) The impacts of TMT on N2a cell apoptosis:Compared with the normal control group,3.75μmol/L and 7.5μmol/L of TMT can increase both early apoptosis rate and later apoptosis rate significantly after 24 h exposure (P<0.01). Hoechst33258 result showed 3.75 μmol/L and 7.5μmol/L of TMT can induce chromosome gathered and DNA breakage. Western blot showed the Bcl-2 decreasing and Bax increasing (P<0.01). (3) Caspase cascade reaction activity:Compared with the normal control group,3.75μmol/L and 7.5 μmol/L of TMT can increase both the mRNA of caspase-9 and the activity of caspase-3 significantly after 24 h exposure (P<0.05). LBP can reduce the activity of caspase. (4) Oxidative stress:Compared with the control, the ROS and MDA level were increased, and the SOD level was decreased after TMT treatment. Compared with the 3.75μmol/L of TMT group, the ROS and MDA level was decreased, and the SOD level was increased after LBP was used. (5) The inhibition of Sonic Hedgehog and PI3/Akt signaling pathways: After intoxication by TMT, the Shh and Gli1 proteins were decreased, and the mRNA of Ptch and Sufu were increased (P<0.05); the level of p-Akt, p-GSK-3β,and the mRNA of PDK1 were decreased (P<0.05). There was no significant change of total Akt and GSK-3β. The downstream proteins of two pathways, C-Myc and CyclinD were decreased. Compared with the 3.75μmol/L of TMT group, LBP had a protect effect on TMT-induced changes.Conclusions:In our laboratory conditions,1.875,3.75,7.5μmol/L TMT and 300μg/mL LBP were used. It presented good dose-response relationship. Apoptosis and oxidative stress were used as toxicity index. The inhibition of Sonic Hedgehog and PI3K/Akt signaling pathways showed the Shh and Gli1 expression decreased in Shh pathway, p-Akt and p-GSK-3p decreased in PI3K/Akt pathway. LBP has a protect effect on TMT-induced neuron injury. This revealed that apoptosis, oxidation damage, and the inhibition of Sonic Hedgehog and PI3K/Akt pathways may participate in TMT-induced neurotoxic mechanism.Part IV "Cross-talk" between Sonic Hedgehog and PI3K/Akt signaling pathways in TMT-induced cell injuryObjectives:Specific inhibitors of two pathways were used to learn the "Cross-talk" between Sonic Hedgehog and PI3K/Akt signaling pathways at protein expression and mRNA level in TMT-induced cell apoptosis model, and to research the role of GSK-3β in Sonic Hedgehog and PI3K/Akt pathways. To observe the effects of inhibitors on the TMT caused apoptosis, and try to discuss the mechanism.Methods:N2a cells were pretreated with LY294002 for 1 h and GDC-0449 for 2 h before intoxication with TMT for 24 h. Cell viability in N2a cells were determined by MTT assay. The rate of cell apoptosis was determined by Flow cytometry. ELISA method was used to detect the change of caspase-3, and RT-PCR was used to analyze the change of caspase-9. ROS generation was determined using DCFH-DA fluorescence probe. MDA content was determined by TBA assay. The change of SOD was determined by WST-1 assay. Sonic Hedgehog, PI3K/Akt signaling pathways changes were detected by Western blot and RT-PCR.Results:(1) Cell viability:Compared with the 3.75μmol/L TMT group, pretreatment before TMT exposure with Sonic Hedgehog inhibitor GDC-0449 and PI3K/Akt inhibitor LY294002 significantly decreased cell viability, and decreased cell number, synapses shrink, and ball float.(2) Cell apoptosis:Compared with the 3.75μmol/L TMT group, pretreatment before TMT exposure with Sonic Hedgehog inhibitor GDC-0449 and PI3K/Akt inhibitor LY294002 significantly increased the rate of total apoptosis (P<0.01). Pretreatment with LY294002 enhanced the rate of later apoptosis. The mRNA level of caspase-9 and activity of caspase-3 were increased after inhibitors were used (P<0.01).(3) Oxidative stress:Pretreated with GDC-0449 and LY294002 significantly increased ROS and MDA levels and decreased SOD level compared with 3.75μmol/L TMT (P<0.01).(4) PI3K/Akt signaling pathway:Compared with 3.75μmol/L TMT, pretreatment with LY294002 for 1 h reduced p-GSK-3β, Shh, and Glil protein levels (P<0.05). The mRNA expression of Ptch and Sufu were increased. However, the mRNA expression of Smo and PDK1 were decreased (P<0.05).(5) Sonic Hedgehog pathway:Compared with 3.75μmol/L TMT, pretreatment with GDC-0449 for 2 h reduced p-GSK-3β and p-Akt protein levels (P<0.01). The mRNA expression of Sufu was increased. However, the mRNA expression of PDK1 was decreased (P<0.01).Conclusions:The study showed that the inhibition of Sonic Hedgehog and PI3K/Akt signaling pathways can increase the rate of apoptosis in TMT-induced apoptosis model. Activity of Caspase-9 and caspase-3 can accelerate the apoptosis and oxidative stress. Compared with the single signaling pathway research, it is more realistic to study the association between signaling pathways. The study indicated that there was "Cross-talk" among those two pathways:Sonic Hedgehog and PI3K/Akt pathways were suppressed, and GSK-3β may play a bridge-like role. There was an interaction between two pathways and oxidative stress. This kind of interaction may lead to the caspase family activation and cause apoptosis. |
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