NF-κB、MAPKs等信号通路在TMT致神经元损害中的作用与机制研究
发布时间：2018-07-25 06:52
【摘要】：第一部分TMT致细胞凋亡模型的建立及其对NF-ΚB、MAPKs等信号通路的影响目的：建立TMT中毒的体外细胞模型,研究TMT对细胞的毒性作用及NF-ΚB、MAPKs等信号通路的影响。方法：MTT法测定细胞存活率；流式细胞术检测细胞凋亡；免疫细胞化学、免疫荧光技术、WB检测NF-ΚB信号通路的激活；免疫细胞化学检测Ub的变化；WB检测MAPKs、p-Akt、Bcl-2、XIAP等蛋白的变化。结果：(1)TMT对SH-SY5Y细胞存活率与凋亡的影响：与正常对照组相比,5、10、20、40μmol/LTMT分别作用24h、48h,细胞存活率明显下降(P0.05),有较好的剂量-时间-反应关系；2.5、5、10μmol/LTMT能使早期凋亡率明显增加,10μmol/LTMT能增加晚期凋亡率,TMT可诱导SH-SY5Y细胞凋亡,并以早期凋亡为主；(2)TMT对PC12细胞存活率与凋亡率的影响：与正常对照组相比,2.5、5、10、20、40μmol/L TMT分别作用24h、48h,细胞存活率明显下降(P0.05),有较好的剂量-时间-反应关系；1.25、2.5、5、10μmol/LTMT能使早期凋亡率和晚期凋亡率明显增加,并以晚期凋亡为主；因此将选用SH-SY5Y细胞作为研究模型,染毒剂量为2.5、5、10μmol/L,作用时间为24h；(3)NF-ΚB信号通路激活：免疫细胞化学、免疫荧光技术对NF-ΚB的分布进行定位,随着染毒剂量增加,胞浆中的NF-ΚB p65逐渐向细胞核转移,WB检测核蛋白NF-ΚB p65,中、高剂量组核蛋白NF-ΚB p65相对表达量分别是对照组的2.87、4.69倍(P0.05),中、高剂量组IΚBa水平明显下降(P0.05)；(4)Ub表达变化：5、10μmol/LTMT使细胞胞体变小,核轻微皱缩,胞浆中棕色加深,Ub表达增加；(5) MAPKs: 10μmol/LTMT可使p-JNK、p-p38增加(P0.05),2.5、5、10μmol/LTMT可使p-ERK1/2增加(P0.05),但各剂量组TMT对总MAPKs表达变化无明显影响；(6) p-Akt、Bcl-2、XIAP:随着TMT剂量增加,p-Akt、Bcl-2、XIAP水平均明显下降(P0.05)。结论：在本实验室条件下,选择两种细胞株为模型,发现2.5、5、10μmol/L TMT可诱导SH-SY5Y细胞凋亡,并以早期凋亡为主,而1.25、2.5、5、10μmol/L TMT均可诱导PC12细胞出现凋亡,以晚期凋亡,即坏死为主。因此,本研究将选用SH-SY5Y细胞作为研究对象,染毒剂量为2.5、5、10μmol/L,染毒时间为24h。TMT引起SH-SY5Y细胞泛素表达增加,IΚB；α降解,NF-ΚB p65核转位增加,p-ERX1/2、p-JNK、p-p38增加,p-Akt降低,提示NF-ΚB、UPPs、MAPKs、Akt信号通路可能参与TMT的毒作用机制。第二部分TMT致SH-SY5Y凋亡模型中NF-ΚB与MAPKs等其他信号通路的'Cross-talk"目的：以不同信号通路的特异性抑制剂为工具,从蛋白表达层面研究TMT致细胞凋亡模型中NF-ΚB与MAPKs等其他信号通路的"Cross-talk",并观察各抑制剂对TMT致细胞凋亡的影响,试图探明其毒性途径。方法：MG132、BAY11-7082、SP600125、U0126、LY294002分别预处理SH-SY5Y细胞2h后,TMT染毒24h,提取蛋白,WB检测蛋白表达变化；流式细胞术检测各抑制剂对TMT导致的细胞凋亡的影响。结果：(1)蛋白酶体抑制剂MG132与NF-ΚB抑制剂BAY11-7082预处理后再进行TMT染毒,早期凋亡率明显高于单独的TMT染毒(P0.05),对TMT诱导的细胞凋亡有促进作用；而SP600125、U0126、LY294002作用后TMT诱导的早期凋亡率明显降低(P0.05),可拮抗TMT诱导的细胞凋亡。(2)泛素-蛋白酶体途径：与单独的TMT染毒组相比,蛋白酶体抑制剂MG132预处理使核蛋白NF-ΚB减少,总蛋白IΚBa增加(P0.05)；磷酸化ERK/总ERK比值降低(P0.05)；磷酸化JNK/总JNK比值升高(P0.05)；磷酸化p38/总p38比值降低(P0.05)；Bcl-2表达减少(P0.05)。(3) NF-ΚB信号通路：与单独的TMT染毒组相比,NF-ΚB特异性抑制剂BAY11-7082预处理可使核蛋白NF-ΚB p65减少(P0.05)；总蛋白IΚBα增加(P0.05)；磷酸化ERK/总ERK比值减小(P0.05)；磷酸化JNK/总JNK比值增加(P0.05)；磷酸化p38/总p38比值减小(P0.05)；Bcl-2表达减少(P0.05)；XIAP表达减少(P0.05)。(4) JNK/SAPK信号通路：与单独的TMT染毒组相比,20μM JNK特异性抑制剂SP600125预处理可使核蛋白NF-ΚB p65增加(P0.05)；总蛋白IΚBa减少(P0.05)；磷酸化ERK/总ERK比值增加(P0.05)；磷酸化JNK/总JNK比值降低(P0.05)；磷酸化p38/总p38比值降低(P0.05)；磷酸化Akt水平降低(P0.05)；Bcl-2表达增加(P0.05)；XIAP表达增加(P0.05)。(5)ERK1/2信号通路：与单独的TMT染毒组相比,10μM ERK1/2特异性抑制剂U0126预处理可使核蛋白NF-ΚB p65增加(P0.05)；总蛋白IΚBa减少(P0.05)；磷酸化ERK/总ERK比值降低(P0.05)：磷酸化JNK/总JNK比值增加(P0.05)；磷酸化p38/总p38比值降低(P0.05)；Bcl-2表达增加(P0.05)；XIAP表达增加(P0.05)。(6) P13K/Akt信号通路：与单独的TMT染毒组相比,20μM Akt特异性抑制剂LY294002预处理可使磷酸化ERK/总ERK比值降低(P0.05)；磷酸化JNK/总JNK比值升高(P0.05)：磷酸化p38/总p38比值升高(P0.05)；XIAP表(?)增加(P0.05)。结论：在TMT致细胞凋亡模型中,NF-ΚB、泛素蛋白酶体信号通路的激活使Bcl-2、XIAP表达增加,具有拮抗细胞凋亡的作用,泛素蛋白酶体信号通路(?)NF-ΚB的激活；而JNK、ERK1/2信号通路的激活使Bcl-2、XIAP表达减少,进细胞凋亡。途径研究表明各信号通路间存在“Cross-talk":NF-ΚB与JNK信号通路相互抑制,JNK与ERK1/2信号通路间相互抑制,JNK促进p38信号通路的激活,而ERK1/2与NF-kB则抑制p38信号通路,ERK1/2对NF-kB信号通路也有抑制作用。这些信号通路问相互调节,最终结局为TMT使Bcl-2、XIAP蛋白表达减少,凋亡发生。第三部分TMT中毒整体动物模型的建立目的：建立TMT中毒的体内模型,研究TMT对大鼠学习记忆能力的影响。方法：40只雄性SD大鼠随机分为4组,每组10只；TMT染毒剂量分别为0.75、1.5、3mg/kg以及溶剂对照组(双蒸水)；每日上午8点经口灌胃染毒,下午两点进行水迷宫实验,晚上8点进行一般行为学评分与癫痫评分,共6天；末次染毒24h后断头处死动物,收集血液,分离血清,测定血液生化指标及血清钾离子浓度；分离脏器,计算脏器系数；每只动物大脑一分为二,一半用于制备石蜡切片,一半分离海马、皮层等,冻存备用。结果：(1)一般行为改变：TMT可引起大鼠出现震颤、尖叫、易激惹、攻击性、阵挛等神经系统中毒症状,高剂量组从第4天开始,出现中毒症状,症状评分分值增加,第5、6、7天症状评分分值持续升高,明显高于对照组(P0.05)；并可诱发癫痫,各剂量组在染毒初期均无明显癫痫症状,均处于癫痫分级0,染毒结束后(第7天),对照组90%动物仍然处于0级,低剂量组90%动物也处于0级,中剂量组60%动物为0级,40%动物为1级,高剂量组所有动物均达到5级；(2)大鼠体重变化：与对照组相比,3mg/kg组第6、7天体重明显下降(P0.05)；(3)生化指标改变：与对照组相比高剂量组AST、ALT明显升高(P0.05),中、高剂量组BUN、Cr明显升高(P0.05),高剂量组血清钾离子浓度明显降低(P0.05)；(4)学习记忆能力改变：训练第四天,各组大鼠逃避潜伏期明显缩短(P0.05),随着染毒继续,低剂量组与对照组持续缩短,而中、高剂量组逃避潜伏期反而增加(P0.05),第6天训练结束后撤掉平台,中、高剂量组在目标区域停留时间比例较少(P0.05),穿越平台次数较少(P0.05)。结论：本室成功建立TMT中毒的整体动物模型,3mg/kgTMT可引起SD大鼠中毒,观察到震颤、易激惹、攻击性、癫痫发作等症状,以及血清钾离子浓度明显下降等特异性中毒特征,TMT对学习记忆能力也有显著的影响。第四部分TMT对大鼠海马神经元的损伤及机制研究目的：观察TMT对海马神经元的损伤及分子生物学机制研究。方法：尼氏染色观察神经元损伤；流式细胞术测定大鼠海马神经元凋亡率；免疫组织化学法检测泛素水平变化；Western blot检测NF-ΚB、MAPKs等信号通路蛋白变化。结果：(1)神经细胞损伤：尼氏染色表明中、高剂量组海马各区神经细胞排列紊乱、尼氏小体减少、细胞间隙变大、细胞肿胀、数量减少；(2)TMT对大鼠海马神经元凋亡的影响：与对照组相比,各剂量组海马神经元早期凋亡率明显增加(P0.05),并且有剂量-反应关系；(3)泛素表达变化：中、高剂量组海马各区泛素蛋白沉积明显增加,表现为棕黄色颗粒物增加；(4)蛋白免疫印迹检测海马蛋白表达变化：与对照组相比,高剂量组核蛋白NF-ΚB水平增加,总蛋白IΚBα减少,p-JNK增加,低、中、高剂量组p-p38均增加,中、高剂量组p-ERK1/2增加,差异均有统计学意义(P0.05)。结论：在整体动物模型中,TMT可导致神经元尼氏体丢失,神经细胞凋亡,其分子生物学机制涉及NF-ΚB、MAPKs、泛素-蛋白酶体信号通路的激活,与体外细胞模型较为吻合。
[Abstract]:The first part of the TMT induced apoptosis model and its effect on the signal pathways such as NF- B, MAPKs and other signal pathways: to establish the cell model of TMT poisoning in vitro, to study the toxicity of TMT to the cells and the effect of NF- B, MAPKs and other signaling pathways. Methods: MTT method was used to determine cell survival rate; flow cytometry was used to detect cell apoptosis; immunization was used. Study, immunofluorescence technology, WB detection of the activation of NF- B signaling pathway; immunocytochemical detection of Ub changes; WB detection of MAPKs, p-Akt, Bcl-2, XIAP and other proteins. Results: (1) TMT on the survival rate and apoptosis of SH-SY5Y cells: compared with the normal control group Down (P0.05), there is a good dose time response relationship; 2.5,5,10 micron mol/LTMT can increase the early apoptosis rate obviously, 10 mu mol/LTMT can increase the late apoptosis rate, TMT can induce apoptosis of SH-SY5Y cells and early apoptosis; (2) the effect of TMT on the survival rate and apoptosis rate of PC12 cells: 2.5,5,10,20,40 mu mol/L compared with the normal control group The survival rate of 24h and 48h decreased significantly (P0.05), and there was a good dose time response relationship; 1.25,2.5,5,10 micron mol/LTMT could increase the early apoptosis rate and the late apoptosis rate, and was mainly in the late stage of apoptosis. Therefore, SH-SY5Y cells were selected as the research model, the dose was 2.5,5,10 u mol/L, and the action time was 24h. The action time was 24h. (3) the activation of NF- B signaling pathway: immunocytochemistry and immunofluorescence technique to locate the distribution of NF- B, with the increase of the dose, the NF- B p65 in the cytoplasm gradually transferred to the nucleus, WB detection of the nucleoprotein NF- B p65, and the high dose of nucleoprotein NF- The level of I Ba in the dose group decreased (P0.05), and (4) the changes in Ub expression: 5,10 micron mol/LTMT made the cell body smaller, the nucleus slightly crinkled, the cytoplasm brown deepened, and the Ub expression increased; (5) MAPKs: 10 micron could increase p-JNK, p-p38 increase (P0.05). Obviously, (6) p-Akt, Bcl-2, XIAP: decreased significantly with the increase of TMT dose, p-Akt, Bcl-2 and XIAP (P0.05). Conclusion: in the laboratory conditions, two cell lines were selected as models, and 2.5,5,10 micron mol/L TMT could induce apoptosis in SH-SY5Y cells. In this study, SH-SY5Y cells are selected as the research object, and the dose of SH-SY5Y cells is 2.5,5,10 Mu and 24h.TMT causes SH-SY5Y cells to increase the expression of ubiquitin, I B, alpha degradation, NF- B p65 nuclear transposition, p-ERX1/ 2, p-JNK, p-JNK, p-JNK, decreasing, decreasing Signaling pathway may be involved in the toxic mechanism of TMT. Second part TMT induces'Cross-talk in other signaling pathways, such as NF-, B and MAPKs in SH-SY5Y apoptosis model, "objective: To study NF- B and MAPKs and other signaling pathways in TMT induced apoptosis models from the protein expression level by the specific inhibitors of different signaling pathways. K "and observe the effect of each inhibitor on apoptosis induced by TMT, and try to explore the toxic pathway. Methods: MG132, BAY11-7082, SP600125, U0126, LY294002 were pretreated with SH-SY5Y cells 2h, TMT was poisoned 24h, protein extraction, protein expression changes; flow cytometry was used to detect the effect of each inhibitor on apoptosis. (1) the proteasome inhibitor MG132 and the NF- B inhibitor BAY11-7082 were pretreated with TMT, and the early apoptosis rate was significantly higher than that of TMT (P0.05), which could promote the apoptosis induced by TMT, while SP600125, U0126 and LY294002 acted on the apoptosis rate of the early stage induced by TMT, and could antagonize the cells induced by TMT. Apoptosis. (2) the ubiquitin proteasome pathway: compared with the single TMT, proteasome inhibitor MG132 preconditioning reduces the nuclear protein NF- B, the total protein I Ba increases (P0.05); the total ERK ratio of the phosphorylated ERK/ is reduced (P0.05); the JNK/ total JNK ratio of phosphorylation is increased; the expression of phosphorylated ERK/ is reduced; the expression decreases (P0.05) (3) NF- B signaling pathway: compared with a single TMT exposure group, the NF- B specific inhibitor BAY11-7082 preconditioning can reduce the nucleoprotein NF- B p65 (P0.05); the total protein I decreases; Bcl-2 expression decreased (P0.05); XIAP expression decreased (P0.05). (4) JNK/SAPK signaling pathway: compared with a single TMT exposure group, the 20 M JNK specific inhibitor SP600125 preconditioning can increase the nuclear protein NF- B p65; (P0.05); the total p38 ratio of phosphorylated p38/ decreased (P0.05); the level of phosphorylated Akt decreased (P0.05); Bcl-2 expression increased (P0.05); XIAP expression increased (P0.05). (5) ERK1/2 signaling pathway .05); the total ERK ratio of phosphorylated ERK/ decreased (P0.05): the total JNK ratio of phosphorylated JNK/ increased (P0.05), the total p38 ratio of phosphorylated p38/ decreased (P0.05), Bcl-2 expression increased (P0.05); (6) The total ERK ratio of RK/ decreased (P0.05); the total JNK ratio of phosphorylated JNK/ increased (P0.05): the total p38 ratio of phosphorylated p38/ increased (P0.05); XIAP table (?) increased (P0.05). Conclusion: in the apoptosis model of the TMT cell, the activation of the ubiquitin proteasome signaling pathway increases the expression of ubiquitin proteasome, and has the effect of antagonistic apoptosis, ubiquitin protease The activation of the body signal pathway (?) NF- B; while the activation of JNK, ERK1/2 signaling pathway reduces the expression of Bcl-2 and XIAP and enters the cell apoptosis. The pathway study indicates that there is a "Cross-talk" between the signaling pathways, and that the NF- B and JNK signaling pathways are mutually inhibited, and that JNK and ERK1/2 signal pathways are interdependent and promote the activation of the signaling pathway. Inhibition of the p38 signaling pathway and the inhibitory effect of ERK1/2 on the NF-kB signaling pathway. These signaling pathways are mutually regulated, and the final outcome is that TMT causes Bcl-2, XIAP protein expression to decrease, and apoptosis occurs. Third part of the whole animal model of TMT poisoning: establish an intracellular model of TMT poisoning and study the shadow of TMT on learning and memory ability in rats. Methods: 40 male SD rats were randomly divided into 4 groups, with 10 rats in each group; the dose of TMT was 0.75,1.5,3mg/kg and the control group (double water), at 8 p.m. daily, the water maze experiment was carried out at two o'clock in the afternoon, the general behavior score and the epileptic score were 6 days at 8 o'clock in the evening, and the last time after 24h was executed. Animals, collect blood, separate serum, determine blood biochemical index and serum potassium ion concentration, separate organs, calculate viscera coefficient; each animal brain is divided into two, half used to prepare paraffin section, half of hippocampus, cortex, etc., and frozen in reserve. Results: (1) general behavior changes: TMT can cause tremor, screaming, irritability in rats. Intoxication symptoms such as provoking, aggression, clonus and other nervous system poisoning, the high dose group started from fourth days, the symptom of poisoning, the score of symptom increased, the score of symptom score increased continuously on day 5,6,7, obviously higher than that of the control group (P0.05), and could induce epilepsy. After seventh days (seventh days), 90% animals in the control group were still in grade 0, 90% in low dose group were 0, 60% in middle dose group were 0, 40% animals were 1, and all animals in high dose group reached 5. (2) body weight changes in rats: Group 6,7 days weight decreased significantly compared with control group (P0.05); (3) biochemical index changes: and control group Compared to high dose group AST, ALT significantly increased (P0.05), high dose group BUN, Cr significantly increased (P0.05), high dose group potassium ion concentration decreased significantly (P0.05); (4) learning and memory ability changes: training fourth days, rats escape latency significantly shortened (P0.05), with continued exposure, low dose group and the control group continued to shorten, and middle, In high dose group, the escape latency increased (P0.05). After sixth days of training, the platform was withdrawn. In the high dose group, the stay time in the target area was less (P0.05), and the number of crossing platforms was less (P0.05). Conclusion: the whole animal model of TMT poisoning was established successfully in this room. 3mg/kgTMT could cause the poisoning of SD rats, and observed the tremor, irritability and attack. The characteristics of specific poisoning, such as striking, epileptic seizures, and a significant decrease in the concentration of serum potassium ions, and the significant effects of TMT on learning and memory. Fourth the damage and mechanism of TMT on the hippocampus neurons in rats: the study of the damage and molecular biological mechanism of the hippocampal neurons by TMT. Methods: Nissl staining The apoptosis rate of hippocampal neurons in rats was measured by flow cytometry; changes in ubiquitin level were detected by immunohistochemistry; Western blot was used to detect the changes in signal pathway proteins such as NF- B and MAPKs. Results: (1) nerve cell damage: Nissl staining showed that the nerve cells in the hippocampus of the high dose group were in disorder, Nissl was small The effect of TMT on the apoptosis of hippocampal neurons in rats: the early apoptosis rate of hippocampal neurons in each dose group increased significantly (P0.05), and there was a dose-response relationship with the control group. (3) the ubiquitin protein deposition in the hippocampus of the high dose group increased significantly in the high dose group. The expression of brown yellow particles increased; (4) protein immunoblotting detected the changes in the expression of hippocampal protein: compared with the control group, the level of nuclear protein NF- B increased in the high dose group, the total protein I B alpha decreased, the p-JNK increased, and the high dose group p-p38 increased, and the high dose group p-ERK1/2 increased, the difference was statistically significant (P0.05). Conclusion: in the high dose group, the difference is statistically significant (P0.05). Conclusion: in the group (P0.05). In the whole animal model, TMT can lead to the loss of Nissl body and apoptosis of neurons, and its molecular mechanism involves the activation of NF- B, MAPKs, and ubiquitin proteasome signaling pathway, which is more consistent with the cell model in vitro.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
本文编号：2142993
[Abstract]:The first part of the TMT induced apoptosis model and its effect on the signal pathways such as NF- B, MAPKs and other signal pathways: to establish the cell model of TMT poisoning in vitro, to study the toxicity of TMT to the cells and the effect of NF- B, MAPKs and other signaling pathways. Methods: MTT method was used to determine cell survival rate; flow cytometry was used to detect cell apoptosis; immunization was used. Study, immunofluorescence technology, WB detection of the activation of NF- B signaling pathway; immunocytochemical detection of Ub changes; WB detection of MAPKs, p-Akt, Bcl-2, XIAP and other proteins. Results: (1) TMT on the survival rate and apoptosis of SH-SY5Y cells: compared with the normal control group Down (P0.05), there is a good dose time response relationship; 2.5,5,10 micron mol/LTMT can increase the early apoptosis rate obviously, 10 mu mol/LTMT can increase the late apoptosis rate, TMT can induce apoptosis of SH-SY5Y cells and early apoptosis; (2) the effect of TMT on the survival rate and apoptosis rate of PC12 cells: 2.5,5,10,20,40 mu mol/L compared with the normal control group The survival rate of 24h and 48h decreased significantly (P0.05), and there was a good dose time response relationship; 1.25,2.5,5,10 micron mol/LTMT could increase the early apoptosis rate and the late apoptosis rate, and was mainly in the late stage of apoptosis. Therefore, SH-SY5Y cells were selected as the research model, the dose was 2.5,5,10 u mol/L, and the action time was 24h. The action time was 24h. (3) the activation of NF- B signaling pathway: immunocytochemistry and immunofluorescence technique to locate the distribution of NF- B, with the increase of the dose, the NF- B p65 in the cytoplasm gradually transferred to the nucleus, WB detection of the nucleoprotein NF- B p65, and the high dose of nucleoprotein NF- The level of I Ba in the dose group decreased (P0.05), and (4) the changes in Ub expression: 5,10 micron mol/LTMT made the cell body smaller, the nucleus slightly crinkled, the cytoplasm brown deepened, and the Ub expression increased; (5) MAPKs: 10 micron could increase p-JNK, p-p38 increase (P0.05). Obviously, (6) p-Akt, Bcl-2, XIAP: decreased significantly with the increase of TMT dose, p-Akt, Bcl-2 and XIAP (P0.05). Conclusion: in the laboratory conditions, two cell lines were selected as models, and 2.5,5,10 micron mol/L TMT could induce apoptosis in SH-SY5Y cells. In this study, SH-SY5Y cells are selected as the research object, and the dose of SH-SY5Y cells is 2.5,5,10 Mu and 24h.TMT causes SH-SY5Y cells to increase the expression of ubiquitin, I B, alpha degradation, NF- B p65 nuclear transposition, p-ERX1/ 2, p-JNK, p-JNK, p-JNK, decreasing, decreasing Signaling pathway may be involved in the toxic mechanism of TMT. Second part TMT induces'Cross-talk in other signaling pathways, such as NF-, B and MAPKs in SH-SY5Y apoptosis model, "objective: To study NF- B and MAPKs and other signaling pathways in TMT induced apoptosis models from the protein expression level by the specific inhibitors of different signaling pathways. K "and observe the effect of each inhibitor on apoptosis induced by TMT, and try to explore the toxic pathway. Methods: MG132, BAY11-7082, SP600125, U0126, LY294002 were pretreated with SH-SY5Y cells 2h, TMT was poisoned 24h, protein extraction, protein expression changes; flow cytometry was used to detect the effect of each inhibitor on apoptosis. (1) the proteasome inhibitor MG132 and the NF- B inhibitor BAY11-7082 were pretreated with TMT, and the early apoptosis rate was significantly higher than that of TMT (P0.05), which could promote the apoptosis induced by TMT, while SP600125, U0126 and LY294002 acted on the apoptosis rate of the early stage induced by TMT, and could antagonize the cells induced by TMT. Apoptosis. (2) the ubiquitin proteasome pathway: compared with the single TMT, proteasome inhibitor MG132 preconditioning reduces the nuclear protein NF- B, the total protein I Ba increases (P0.05); the total ERK ratio of the phosphorylated ERK/ is reduced (P0.05); the JNK/ total JNK ratio of phosphorylation is increased; the expression of phosphorylated ERK/ is reduced; the expression decreases (P0.05) (3) NF- B signaling pathway: compared with a single TMT exposure group, the NF- B specific inhibitor BAY11-7082 preconditioning can reduce the nucleoprotein NF- B p65 (P0.05); the total protein I decreases; Bcl-2 expression decreased (P0.05); XIAP expression decreased (P0.05). (4) JNK/SAPK signaling pathway: compared with a single TMT exposure group, the 20 M JNK specific inhibitor SP600125 preconditioning can increase the nuclear protein NF- B p65; (P0.05); the total p38 ratio of phosphorylated p38/ decreased (P0.05); the level of phosphorylated Akt decreased (P0.05); Bcl-2 expression increased (P0.05); XIAP expression increased (P0.05). (5) ERK1/2 signaling pathway .05); the total ERK ratio of phosphorylated ERK/ decreased (P0.05): the total JNK ratio of phosphorylated JNK/ increased (P0.05), the total p38 ratio of phosphorylated p38/ decreased (P0.05), Bcl-2 expression increased (P0.05); (6) The total ERK ratio of RK/ decreased (P0.05); the total JNK ratio of phosphorylated JNK/ increased (P0.05): the total p38 ratio of phosphorylated p38/ increased (P0.05); XIAP table (?) increased (P0.05). Conclusion: in the apoptosis model of the TMT cell, the activation of the ubiquitin proteasome signaling pathway increases the expression of ubiquitin proteasome, and has the effect of antagonistic apoptosis, ubiquitin protease The activation of the body signal pathway (?) NF- B; while the activation of JNK, ERK1/2 signaling pathway reduces the expression of Bcl-2 and XIAP and enters the cell apoptosis. The pathway study indicates that there is a "Cross-talk" between the signaling pathways, and that the NF- B and JNK signaling pathways are mutually inhibited, and that JNK and ERK1/2 signal pathways are interdependent and promote the activation of the signaling pathway. Inhibition of the p38 signaling pathway and the inhibitory effect of ERK1/2 on the NF-kB signaling pathway. These signaling pathways are mutually regulated, and the final outcome is that TMT causes Bcl-2, XIAP protein expression to decrease, and apoptosis occurs. Third part of the whole animal model of TMT poisoning: establish an intracellular model of TMT poisoning and study the shadow of TMT on learning and memory ability in rats. Methods: 40 male SD rats were randomly divided into 4 groups, with 10 rats in each group; the dose of TMT was 0.75,1.5,3mg/kg and the control group (double water), at 8 p.m. daily, the water maze experiment was carried out at two o'clock in the afternoon, the general behavior score and the epileptic score were 6 days at 8 o'clock in the evening, and the last time after 24h was executed. Animals, collect blood, separate serum, determine blood biochemical index and serum potassium ion concentration, separate organs, calculate viscera coefficient; each animal brain is divided into two, half used to prepare paraffin section, half of hippocampus, cortex, etc., and frozen in reserve. Results: (1) general behavior changes: TMT can cause tremor, screaming, irritability in rats. Intoxication symptoms such as provoking, aggression, clonus and other nervous system poisoning, the high dose group started from fourth days, the symptom of poisoning, the score of symptom increased, the score of symptom score increased continuously on day 5,6,7, obviously higher than that of the control group (P0.05), and could induce epilepsy. After seventh days (seventh days), 90% animals in the control group were still in grade 0, 90% in low dose group were 0, 60% in middle dose group were 0, 40% animals were 1, and all animals in high dose group reached 5. (2) body weight changes in rats: Group 6,7 days weight decreased significantly compared with control group (P0.05); (3) biochemical index changes: and control group Compared to high dose group AST, ALT significantly increased (P0.05), high dose group BUN, Cr significantly increased (P0.05), high dose group potassium ion concentration decreased significantly (P0.05); (4) learning and memory ability changes: training fourth days, rats escape latency significantly shortened (P0.05), with continued exposure, low dose group and the control group continued to shorten, and middle, In high dose group, the escape latency increased (P0.05). After sixth days of training, the platform was withdrawn. In the high dose group, the stay time in the target area was less (P0.05), and the number of crossing platforms was less (P0.05). Conclusion: the whole animal model of TMT poisoning was established successfully in this room. 3mg/kgTMT could cause the poisoning of SD rats, and observed the tremor, irritability and attack. The characteristics of specific poisoning, such as striking, epileptic seizures, and a significant decrease in the concentration of serum potassium ions, and the significant effects of TMT on learning and memory. Fourth the damage and mechanism of TMT on the hippocampus neurons in rats: the study of the damage and molecular biological mechanism of the hippocampal neurons by TMT. Methods: Nissl staining The apoptosis rate of hippocampal neurons in rats was measured by flow cytometry; changes in ubiquitin level were detected by immunohistochemistry; Western blot was used to detect the changes in signal pathway proteins such as NF- B and MAPKs. Results: (1) nerve cell damage: Nissl staining showed that the nerve cells in the hippocampus of the high dose group were in disorder, Nissl was small The effect of TMT on the apoptosis of hippocampal neurons in rats: the early apoptosis rate of hippocampal neurons in each dose group increased significantly (P0.05), and there was a dose-response relationship with the control group. (3) the ubiquitin protein deposition in the hippocampus of the high dose group increased significantly in the high dose group. The expression of brown yellow particles increased; (4) protein immunoblotting detected the changes in the expression of hippocampal protein: compared with the control group, the level of nuclear protein NF- B increased in the high dose group, the total protein I B alpha decreased, the p-JNK increased, and the high dose group p-p38 increased, and the high dose group p-ERK1/2 increased, the difference was statistically significant (P0.05). Conclusion: in the high dose group, the difference is statistically significant (P0.05). Conclusion: in the group (P0.05). In the whole animal model, TMT can lead to the loss of Nissl body and apoptosis of neurons, and its molecular mechanism involves the activation of NF- B, MAPKs, and ubiquitin proteasome signaling pathway, which is more consistent with the cell model in vitro.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363
【引证文献】
相关硕士学位论文 前2条
1 付留中;IL-4、IL-6、TNF-a、NF-κB在椎间盘源性下腰痛发病中的作用[D];新乡医学院;2013年
2 杜清清;溴苯腈对SH-SY5Y细胞毒性及NF-κB、MAPKs通路的影响[D];华中科技大学;2013年
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