【作者】 王娟；

【导师】 夏中元；

【作者基本信息】 武汉大学 ， 麻醉学， 2021， 博士

【摘要】 第一部分甲氨蝶呤诱导小鼠认知功能障碍及海马铁自噬抑制目的:探讨甲氨蝶呤对小鼠认知功能及海马铁自噬的影响。方法:腹腔注射甲氨蝶呤建立化疗药物诱发的小鼠认知功能障碍模型。SPF级雄性4月龄C57BL/6小鼠24只,体重20-30g,采用随机数字表法,将其分成2组(n=12):对照组(Control组)和甲氨蝶呤处理组(MTX组)。MTX组小鼠每隔7天接受一次甲氨蝶呤处理(100 mg/kg,共三次),Control组小鼠接受同等体积的PBS。最后一次处理后7天进行行为学训练及测试(旷场、新物体识别及条件恐惧实验)。测试结束后,在深麻醉下处死小鼠,Western blot及q RT-PCR检测海马组织中铁自噬相关分子蛋白(HuR、NCOA4、FTH1以及LC3Ⅱ/LC3Ⅰ)和m RNA(HuR、NCOA4及FTH1)表达水平,铁检测试剂盒检测海马组织中总铁浓度。结果:1.旷场实验:Control组和MTX组小鼠总探索路程之间无统计学差异(P>0.05);与Control相比,MTX组小鼠中央格停留时间明显缩短(P<0.01)。新物体识别实验:训练阶段两组小鼠对两个物体探索时间无统计学差异(P>0.05);测试阶段Control组小鼠对新物体探索时间较旧物体显著增加(P<0.01),而MTX组小鼠对新旧物体探索时间无统计学差异(P>0.05)。条件恐惧实验:在场景恐惧实验中,与Control组相比,MTX组小鼠僵直时间明显缩短(P<0.001)。在声音提示实验中,与Control组相比,MTX组小鼠僵直时间无统计学差异(P>0.05)。2.与Control组相比,MTX组小鼠海马组织中HuR蛋白(P<0.001)和m RNA(P<0.001)、NCOA4蛋白(P<0.001)和m RNA(P<0.01)水平以及LC3Ⅱ/LC3Ⅰ(P<0.05)比值显著性降低,FTH1蛋白(P<0.05)和m RNA(P<0.01)水平以及总铁浓度(P<0.001)显著升高。结论:甲氨蝶呤诱导小鼠认知功能障碍及海马铁自噬抑制。第二部分右美托咪定通过NCOA4介导的铁自噬减轻甲氨蝶呤诱导的海马HT22细胞的神经毒性和炎症反应目的:探讨NCOA4介导的铁自噬在右美托咪定减轻甲氨蝶呤诱导海马HT22神经元损伤中的作用机制。方法:选用10M甲氨蝶呤刺激小鼠海马HT22细胞24 h构建体外模型。第一阶段实验将HT22细胞分为5组:对照组(Control组)、甲氨蝶呤处理组(MTX组)、甲氨蝶呤+1 ng/m L右美托咪定组(MTX+DEX1组)、甲氨蝶呤+10 ng/m L右美托咪定组(MTX+DEX10组)和甲氨蝶呤+50 ng/m L右美托咪定组(MTX+DEX50组)。甲氨蝶呤刺激HT22细胞24 h后不同浓度右美托咪定处理1 h。第二阶段实验分为对照组(Control组)、甲氨蝶呤处理组(MTX组)、甲氨蝶呤+50 ng/m L右美托咪定+siRNA对照组(MTX+DEX+NC-siRNA组)以及甲氨蝶呤+50 ng/m L右美托咪定+NCOA4-siRNA组(MTX+DEX+NCOA4-siRNA组)。首先HT22细胞分别转染NC-siRNA或者NCOA4-siRNA 48h,然后甲氨蝶呤刺激24 h,最后使用50 ng/m L右美托咪定处理1 h。CCK-8法检测HT22细胞活力,试剂盒检测LDH活性、TNF-?和IL-1?浓度、ROS水平、细胞内不稳定铁池和总铁水平,流式细胞仪检测HT22细胞凋亡变化,western blot检测NF-?B、NCOA4、FTH1及LC3Ⅱ/LC3Ⅰ变化,免疫荧光检测NCOA4和LC3Ⅱ共标情况。结果:第一阶段实验:10μM的甲氨蝶呤处理HT22细胞24 h能够明显降低其存活率(P<0.05);增加LDH的活性(P<0.05);促进细胞凋亡(P<0.05);上调炎症相关蛋白NF-?B(P<0.05)、TNF-?(P<0.05)、IL-1?(P<0.05)和FTH1(P<0.05)水平;增加ROS(P<0.05)及总铁浓度(P<0.05);降低细胞内不稳定铁池浓度(P<0.05)以及铁自噬相关蛋白NCOA4表达(P<0.05)及LC3Ⅱ/LC3Ⅰ(P<0.05)比值。10及50 ng/m L右美托咪定能够明显减轻上述改变(P<0.05),但1 ng/m L右美托咪定处理无统计学差异(P(29)0.05)。第二阶段实验:转染NCOA4-siRNA能够明显下调NCOA4表达水平(P<0.05)。与MTX+DEX+NC-siRNA组相比,MTX+DEX+NCOA4-siRNA组HT22细胞存活率降低(P<0.05);LDH的活性(P<0.05)和细胞凋亡增加(P<0.05);TNF-?(P<0.05)、IL-1?(P<0.05)、FTH1(P<0.05)、ROS(P<0.05)及总铁浓度(P<0.05)增加;NCOA4表达(P<0.05)、LC3Ⅱ/LC3Ⅰ比值(P<0.05)以及细胞内不稳定铁池浓度(P<0.05)降低。结论:右美托咪定通过NCOA4介导的铁自噬减轻甲氨蝶呤诱导的海马HT22细胞的神经毒性和炎症反应。第三部分右美托咪定通过HuR/NCOA4信号通路介导的铁自噬减轻甲氨蝶呤诱导的海马HT22细胞的神经毒性和炎症反应目的:探讨HuR/NCOA4通路在右美托咪定减轻甲氨蝶呤诱导海马HT22神经元损伤中的作用机制。方法:选用10M甲氨蝶呤刺激小鼠海马HT22细胞24 h构建体外模型。实验分为对照组(Control组)、甲氨蝶呤处理组(MTX组)、甲氨蝶呤+50 ng/m L右美托咪定+siRNA对照组(MTX+DEX+NC-siRNA组)以及甲氨蝶呤+50 ng/m L右美托咪定+HuR-siRNA组(MTX+DEX+HuR-siRNA组)。首先HT22细胞分别转染NC-siRNA或者HuR-siRNA 48 h,然后甲氨蝶呤刺激24 h,最后使用50ng/m L右美托咪定处理1 h。CCK-8法检测HT22细胞活力,试剂盒检测LDH活性、TNF-?和IL-1?浓度、ROS水平、细胞内不稳定铁池和总铁水平,流式细胞仪检测HT22细胞凋亡变化,western blot检测HuR、NCOA4、FTH1及LC3Ⅱ/LC3Ⅰ变化,免疫荧光检测NCOA4和HuR共标情况。结果:转染HuR-siRNA能够明显下调NCOA4表达水平(P<0.05)。与MTX+DEX+NC-siRNA组相比,MTX+DEX+HuR-siRNA组HT22细胞存活率降低(P<0.05);LDH的活性(P<0.05)和细胞凋亡增加(P<0.05);TNF-?(P<0.05)、IL-1?(P<0.05)、FTH1(P<0.05)、ROS(P<0.05)及总铁浓度(P<0.05)增加;铁自噬相关蛋白HuR(P<0.05)、NCOA4(P<0.05)、LC3Ⅱ/LC3Ⅰ(P<0.05)表达以及细胞内不稳定铁池浓度(P<0.05)降低。结论:右美托咪定通过HuR/NCOA4信号通路介导的铁自噬减轻甲氨蝶呤诱导的海马HT22细胞的神经毒性和炎症反应。更多还原

【Abstract】 Part Ⅰ Methotrexate-induced cognitive dysfunction and inhibition of hippocampal ferritinophagy in miceObjective:To investigate the effects of methotrexate on cognitive function and hippocampal ferritinophagy in mice.Methods:Chemotherapeutic drug-induced cognitive dysfunction in mice model was established by intraperitoneal injection of methotrexate.SPF male 4-month-old C57BL/6 mice,weighing 20-30 g,were divided into 2 groups(n=12 per group):Control group and methotrexate treatment group(MTX group)using the random number table method.Mice in the MTX group received methotrexate treatment(100 mg/kg,three times)every 7 days,and mice in the Control group received the same volume of PBS.7 days after the last treatment,behavioral training and testing(open field,new object recognition and fear condition tests)were performed.After the tests,the mice were executed under deep anesthesia,and the expression levels of ferritinophagy-related proteins(HuR,NCOA4,FTH1,and LC3Ⅱ/LC3Ⅰ)and mRNA(HuR,NCOA4,and FTH1)in hippocampus were detected by Western blot and qRT-PCR,respectively.The total iron concentration in hippocampal tissues was measured by the iron assay kit.Results:1.Open field test:there was no statistical difference between the total exploration distance of mice in Control and MTX groups(P>0.05);compared with group control,the time spent in center of mice in MTX group was significantly decreased(P<0.01).New object recognition test:there was no statistical difference between the two groups of mice in the training phase for the exploration time of the two objects(P>0.05);there was a significant increase in the exploration time of the control group mice for the new object compared with the familiar object in the testing phase(P<0.01),while there was no statistical difference between the MTX group mice for the exploration time of the familiar and new objects(P>0.05).Fear condition test:in the context test,the freezing time of mice in the MTX group was significantly decreased compared with the control group(P<0.001).In tone test,there was no significantly difference in the freezing time of mice between MTX group and control group(P>0.05).2.Compared with the control group,protein(P<0.001)and mRNA(P<0.001)levels of HuR,protein(P<0.001)and mRNA(P<0.01)levels of NCOA4,and LC3Ⅱ/LC3Ⅰ protein(P<0.05)levels were significantly decreased,while protein(P<0.05)and mRNA(P<0.01)levels of FTH1 and total iron concentration(P<0.001)were significantly increased in the hippocampus of mice in the MTX group.Conclusions:Methotrexate-induced cognitive dysfunction and inhibition of hippocampal ferritinophagy in mice.Part Ⅱ Dexmedetomidine reverses methotrexate-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via NCOA4-mediated ferritinophagyObjective:To investigate the mechanism of NCOA4 in the improvement of methotrexate-induced hippocampal HT22 neuronal injury by dexmedetomidine.Methods:In vitro model was constructed by stimulating mouse hippocampal HT22 cells with 10 μM methotrexate for 24 h.HT22 cells were divided into five groups in the first phase:Control group,methotrexate treatment group(MTX group),methotrexate+1 ng/mL dexmedetomidine group(MTX+DEX1 group),methotrexate+10 ng/mL dexmedetomidine group(MTX+DEX10 group)and methotrexate+50 ng/mL dexmedetomidine group(MTX+DEX50 group).Methotrexate stimulated HT22 cells for 24 h and then treated with different concentrations of dexmedetomidine for 1 h.HT22 cells were divided into four groups in the second phase:Control group,methotrexate treated group(MTX group),methotrexate+50 ng/mL dexmedetomidine+NC-siRNA group(MTX+DEX+NC-siRNA group)and methotrexate+50 ng/mL dexmedetomidine+NCOA4-siRNA group(MTX+DEX+NCOA4-siRNA group).HT22 cells were first transfected with NC-siRNA or NCOA4-siRNA for 48 h,followed by methotrexate stimulation for 24 h,and finally treated with 50 ng/mL dexmedetomidine for 1 h.HT22 cell viability was measured by CCK-8 assay,and the kit measured LDH activity,TNF-α and IL-1 β concentration,ROS level,intracellular labile iron pool and total iron level,flow cytometry for apoptotic changes in HT22 cells,western blot for NF-κB,NCOA4,FTH1 and LC3Ⅱ/LC3Ⅰ changes,and immunofluorescence for NCOA4 and LC3II co-labeling.Results:Phase Ⅰ:10 M methotrexate treatment for 24 h was able to significantly decreased the HT22 cells viability(P<0.05);increased LDH activity(P<0.05);promoted apoptosis(P<0.05);upregulated inflammation-related proteins NF-κ B(P<0.05),TNF-α(P<0.05),IL-1β(P<0.05)and FTH1(P<0.05)levels;increased ROS(P<0.05)and total iron concentration(P<0.05);decreased intracellular labile iron pool concentration(P<0.05)and ferritinophagy-associated protein NCOA4(P<0.05)and LC3Ⅱ/LC3Ⅰ(P<0.05)expression.10 and 50 ng/mL dexmedetomidine significantly attenuated these changes(P<0.05),but 1 ng/mL dexmedetomidine treatment did not(P<0.05).Phase Ⅱ:transfection of NCOA4-siRNA was able to significantly down-regulated NCOA4 levels(P<0.05).Compared with the MTX+DEX+NC-siRNA group,the HT22 cells viability was reduced(P<0.05),LDH activity(P<0.05)and apoptosis were increased(P<0.05),and TNF-α(P<0.05),IL-1β(P<0.05),FTH1(P<0.05),ROS(P<0.05)and total iron concentration(P<0.05)increased,and the levels of NCOA4(P<0.05),LC3Ⅱ/LC3Ⅰ(P<0.05)and intracellular labile iron pool concentration(P<0.05)decreased in the MTX+DEX+NCOA4-siRNA group.Conclusions:Dexmedetomidine alleviates MTX-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via NCOA4-mediated ferritinophagy.Part Ⅲ Dexmedetomidine reverses methotrexate-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via HuR/NCOA4-mediated ferritinophagyObjective:To investigate the role of HuR/NCOA4 pathway in the improvement of methotrexate-induced hippocampal HT22 neuronal injury by dexmedetomidine.Methods:In vitro model was constructed using 10 M methotrexate to stimulate mouse hippocampal HT22 cells for 24 h.HT22 cells were divided into four groups:control group,methotrexate treatment group(MTX group),methotrexate+50 ng/mL dexmedetomidine+NC-siRNA group(MTX+DEX+NC-siRNA group)and methotrexate+50 ng/mL dexmedetomidine+HuR-siRNA group(MTX+DEX+HuR-siRNA group).HT22 cells were transfected with NC-siRNA or HuR-siRNA for 48 h,then methotrexate was stimulated for 24 h,and finally treated with 50 ng/mL dexmedetomidine for 1 h.The viability of HT22 cells was measured by the CCK-8 assay,and the LDH activity,TNF-α and IL-1β concentration,ROS level,intracellular labile iron pool and total iron level were measured by kit,respectively.Flow cytometry was performed to detect apoptotic changes in HT22 cells,western blot was used to detect HuR,NCOA4,FTH1 and LC3Ⅱ/LC3Ⅰ changes,and NCOA4 and HuR co-labeling was detected by immunofluorescence.Results:Transfection of HuR-siRNA was able to significantly down-regulated NCOA4 levels(P<0.05).Compared with MTX+DEX+NC-siRNA group,HT22 cells in MTX+DEX+HuR-siRNA group showed decreased viability(P<0.05);increased LDH activity(P<0.05)and apoptosis(P<0.05);increased TNF-α(P<0.05),IL-1β(P<0.05),FTH1(P<0.05),ROS(P<0.05)and total iron concentration(P<0.05);decreased levels of HuR(P<0.05),NCOA4(P<0.05),LC3Ⅱ/LC3Ⅰ(P<0.05)and intracellular labile iron pool concentrations(P<0.05).Conclusions:Dexmedetomidine alleviates MTX-induced neurotoxicity and inflammation in hippocampal HT22 cell lines via HuR/NCOA4-mediated ferritinophagy.更多还原