【作者】 张文杰；

【导师】 金其贯；

【作者基本信息】 扬州大学 ， 运动人体科学， 2024， 硕士

【摘要】 目的:本研究旨在探讨有氧运动和牡蛎肽（Oyster Peptide,OP）对D-半乳糖和亚硝酸钠（Na NO₂）联合诱导的阿尔茨海默病（Alzheimer’s disease,AD）样大鼠神经精神症状（Neuropsychiatric Symptoms,NPS）的干预效应和交互效应。并基于Sirt1-PGC-1α-NRF1-TFAM信号通路,从前额叶皮质（PFC）线粒体生物发生探讨有氧运动联合补充牡蛎肽干预AD样大鼠NPS产生的生物学机制,为有氧运动联合OP在预防和治疗AD患者NPS提供实验依据。方法:在本研究中,50只雄性12周龄的SD大鼠被随机分为5个组:正常对照组（NC,n=10）、AD样大鼠对照组（ADC,n=10）、AD样大鼠有氧运动干预组（ADE,n=10）、AD样大鼠OP干预组（ADOP,n=10）以及AD样大鼠有氧运动加OP干预组（ADEOP,n=10）。NC组大鼠正常饮水和摄食,不接受干预。而ADC、ADE、ADOP和ADEOP组大鼠通过腹腔注射D-半乳糖和饮用Na NO₂溶液的方式诱导AD样状态。在此基础上,ADOP和ADEOP组每天按1.0g/kg体重的剂量灌服OP溶液。ADE和ADEOP组每天下午进行60min无负重游泳训练,每周5次。所有干预持续8周。在取材前一周通过旷场试验和三箱试验来评估各组大鼠的焦虑抑郁状况和社交互动倾向,以测定焦虑抑郁样情绪和社交淡漠状态。利用HE染色观察PFC内神经细胞的形态结构;利用镀银染色观察PFC中神经纤维缠结产生状况;利用ELISA法检测PFC内Aβ42、线粒体呼吸复合体IV含量;利用RT-PCR和Western blot检测PFC内mt DNA相对含量、Sirt1、PGC-1α、NRF1、TFAM的mRNA和蛋白表达。结果:（1）与NC组相比,ADC组在旷场中移动的总距离和平均速度以及在中央区的进入次数、移动距离、移动时间均显著降低（P=0.001,P=0.001,P=0.000,P=0.002,P=0.000）。与ADC组相比,ADE、ADOP、ADEOP组的移动总距离显著增加（P=0.013,P=0.009,P=0.006）,移动平均速度显著增加（P=0.004,P=0.000,P=0.000）,进入中央次数显著增加（P=0.019,P=0.029,P=0.000）,中央移动时间显著增加（P=0.002,P=0.037,P=0.000）,ADE和ADEOP组的中央移动距离显著增加（P=0.049,P=0.000）,但ADOP组无显著性差异（P=0.111）。与ADE组相比,ADEOP组进入中央区域活动的次数显著增加（P=0.012）,其他旷场试验指标虽有进一步改善趋势,但无显著性差异（P=0.991,P=0.851,P=0.289,P=0.129）。在三箱试验第二阶段:NC组大鼠对陌生大鼠1的嗅探时间显著大于空笼（P=0.000）。而ADC组大鼠对两者的嗅探时间无显著差异（P=0.359）,且与NC组相比,其对陌生大鼠1的嗅探时间显著缩短（P=0.003）;与ADC组相比,ADE、ADOP、ADEOP组对陌生大鼠1的嗅探时间显著延长（P=0.044,P=0.040,P=0.002）;与ADE组相比,ADEOP组有延长陌生大鼠1嗅探时间的趋势,但无显著性差异（P=0.635,P=0.835）;通过组内比较表明,ADE、ADOP、ADEOP组陌生大鼠1的嗅探时间显著大于空笼（P=0.004,P=0.033,P=0.000）。在第三阶段:NC组对陌生大鼠2的嗅探时间显著大于陌生大鼠1（P=0.000）,而ADC组大鼠对两者的嗅探时间无显著差异（P=0.972）,且与NC组相比,ADC组对陌生大鼠2的嗅探时间显著缩短（P=0.001）;与ADC组相比,ADE、ADOP、ADEOP组对陌生大鼠2的嗅探时间显著延长（P=0.002,P=0.004,P=0.001）。与ADE组相比,ADEOP组有对延长陌生大鼠2嗅探时间的趋势,但无显著性差异（P=0.996）。通过组内比较表明,ADE、ADOP、ADEOP组陌生大鼠2的嗅探时间显著大于陌生大鼠1（P=0.000,P=0.006,P=0.000）。（2）与NC组比较,ADC组PFC中部分神经细胞缺少、空泡增多,并可见细胞核固缩、变性,存在较多的NFTs聚集,Aβ42含量显著增高（P=0.000）。ADOP、ADE、ADEOP组相较于ADC组,结构清晰的神经细胞数量明显增多,神经细胞变性明显减少,正常的神经细胞数量明显增多,NFTs情况有所改善,Aβ42含量显著降低（P=0.046,P=0.013,P=0.002）。与ADE相比,ADEOP组神经细胞状态明显进一步改善,有更加清晰的细胞结构和更多的正常神经细胞,神经纤维的缠结情况改善更加明显,Aβ42含量虽无显著性差异（P=0.559）,但呈现出进一步下降的趋势。（3）与NC组相比,ADC组PFC中mt DNA的相对含量以及COX IV含量显著下降（P=0.000,P=0.000）。与ADC组相比,ADE、ADEOP组mt DNA的相对含量均显著升高（P=0.016,P=0.000）,COX IV含量显著升高（P=0.040,P=0.000）,ADOP组mt DNA的相对含量显著升高（P=0.038）,但COX IV含量无显著性差异（P=0.738）。与ADE组相比,ADEOP组PFC中COX IV含量显著升高（P=0.035）,mt DNA的相对含量有进一步提高趋势,但无显著性差异（P=0.071）。（4）与NC组相比,ADC组PFC中Sirt1、PGC-1α、NRF1、TFAM mRNA和蛋白表达均显著降低（P=0.000,P=0.000;P=0.000,P=0.005;P=0.007,P=0.003;P=0.000,P=0.000）。与ADC组相比,ADE、ADOP、ADEOP组Sirt1、TFAM mRNA和蛋白表达均显著升高（P=0.000,P=0.000,P=0.000;P=0.043,P=0.048,P=0.002;P=0.014,P=0.023,P=0.001;P=0.003,P=0.009,P=0.006）;ADE、ADEOP组PFC内PGC-1αmRNA表达显著增加（P=0.022,P=0.000）,ADEOP组PFC内PGC-1α蛋白显著增加（P=0.000）;ADEOP组PFC内NRF1 mRNA,ADOP、ADEOP组NRF1蛋白显著增加（P=0.022,P=0.035,P=0.001）;ADE组PGC-1α蛋白、NRF1 mRNA和蛋白的增加无显著差异（P=0.397,P=0.090,P=0.226）,ADOP组PGC-1αmRNA和蛋白、NRF1 mRNA表达的增加无显著性差异（P=0.687,P=0.201,P=0.299）。与ADE组相比,ADEOP组PGC-1αmRNA和蛋白、NRF1和TFAM mRNA表达显著增加（P=0.013,P=0.028,P=0.003,P=0.000）;ADEOP组Sirt1 mRNA和蛋白、NRF1和TFAM蛋白表达有进一步增加趋势,但无显著性差异（P=0.190,P=0.555,P=0.102,P=0.629）。结论:（1）D-gal联合Na NO₂可以下调PFC的Sirt1-PGC-1α-NRF1-TFAM通路,降低PFC的线粒体生物发生和功能,诱导AD的NPS发生。（2）在D-gal与Na NO₂共同作用诱导AD样大鼠模型形成过程中,采用有氧运动或OP干预可以有效抑制PFC中Aβ的产生,改善PFC中的Sirt1-PGC-1α-NRF1-TFAM信号通路,促进线粒体生物发生,并维持线粒体的呼吸功能,对预防AD的NPS具有重要的价值。此外,有氧运动与OP补充的联合干预效应在一定程度上比单纯有氧运动更好。更多还原

【Abstract】 Objective:This study examines the impact and interaction of aerobic exercise combined with Oyster Peptide（OP）on neuropsychiatric symptoms（NPS）in Alzheimer’s disease（AD）model rats,induced by D-galactose and sodium nitrite（Na NO₂）.Focusing on the Sirt1-PGC-1α-NRF1-TFAM signaling pathway,the research delves into the mechanisms through which mitochondrial biogenesis in the PFC occurs during NPS intervention in AD model rats using aerobic exercise and OP supplementation.The findings aim to elucidate the influence of mitochondrial biogenesis on NPS management and provide a scientific basis for employing aerobic exercise and OP in the prevention and treatment of NPS among AD patients.Methods:In this research,50 male SD rats aged 12 weeks were divided randomly into five experimental groups:Normal Control（NC,n=10）,AD Model Control（ADC,n=10）,AD Model with Aerobic Exercise（ADE,n=10）,AD Model with Oyster Peptide Supplementation（ADOP,n=10）,and AD Model with Combined Aerobic Exercise and Oyster Peptide Supplementation（ADEOP,n=10）.The NC group was provided with standard food and water without any interventions.The AD phenotype in ADC,ADE,ADOP,and ADEOP groups was induced using intraperitoneal D-galactose injections and Na NO₂ingestion.Additionally,ADOP and ADEOP groups received daily OP solution at 1.0 g/kg body weight.The ADE and ADEOP groups engaged in 60 minutes of non-weighted swimming each afternoon,five times a week for eight weeks.Behavioral assessments for anxiety,depression,and social interaction were conducted using open field and three-chamber tests one week prior to tissue collection.The study included HE staining for neuronal cell condition,silver staining for neurofibrillary tangles,ELISA forβ-amyloid 42 and mitochondrial respiratory complex IV levels,and RT-PCR and Western blot for mt DNA,Sirt1,PGC-1α,NRF1,and TFAM expression in the prefrontal cortex.Results:（1）Compared to the NC group,the ADC group exhibited significant reductions in total distance moved,average speed,and the number of entries into the central area,as well as the distance and time moved in the center during the open field test（P=0.001,P=0.001,P=0.000,P=0.002,P=0.000）.Relative to the ADC group,the ADE,ADOP,and ADEOP groups showed significant increases in total distance moved（P=0.013,P=0.009,P=0.006）,average speed（P=0.004,P=0.000,P=0.000）,frequency of central area entries（P=0.019,P=0.029,P=0.000）,and time spent moving in the central area（P=0.002,P=0.037,P=0.000）.The ADE and ADEOP groups also demonstrated significant increases in the distance moved in the central area（P=0.049,P=0.000）,whereas the ADOP group did not show a significant difference（P=0.111）.Compared to the ADE group,the ADEOP group significantly increased their entries into the central area（P=0.012）,although other open field test indicators showed trends for improvement without significant differences（P=0.991,P=0.851,P=0.289,P=0.129）.In the second phase of the three-chamber test:The NC group rats significantly increased their sniffing time towards stranger rat 1 compared to the empty cage（P=0.000）.In contrast,the ADC group rats showed no significant difference in sniffing time between the stranger rat1 and the empty cage（P=0.359）,and their sniffing time towards stranger rat 1 was significantly shorter compared to the NC group（P=0.003）.Compared to the ADC group,the ADE,ADOP,and ADEOP groups showed significantly extended sniffing times towards stranger rat 1（P=0.044,P=0.040,P=0.002）.When compared to the ADE group,the ADEOP group exhibited a trend towards longer sniffing times towards stranger rat 1,but the difference was not significant（P=0.635,P=0.835）.Within-group comparisons revealed that the sniffing times for stranger rat 1 in the ADE,ADOP,and ADEOP groups were significantly longer than those for the empty cage（P=0.004,P=0.033,P=0.000）.In the third phase:The NC group’s sniffing time towards stranger rat 2 was significantly longer than towards stranger rat 1（P=0.000）,whereas the ADC group rats showed no significant difference in sniffing times between the two stranger rats（P=0.972）,and their sniffing time towards stranger rat 2 was significantly shorter compared to the NC group（P=0.001）.Compared to the ADC group,the ADE,ADOP,and ADEOP groups exhibited significantly prolonged sniffing times towards stranger rat 2（P=0.002,P=0.004,P=0.001）.Relative to the ADE group,the ADEOP group showed a trend towards extended sniffing time towards stranger rat 2,but the difference was not significant（P=0.996）.Within-group comparisons indicated that the sniffing times for stranger rat 2 in the ADE,ADOP,and ADEOP groups were significantly greater than those for stranger rat 1（P=0.000,P=0.006,P=0.000）.（2）When compared to the NC group,the ADC group exhibited a notable decrease in the quantity of neurons in the PFC,alongside increased vacuolization and observable signs of nuclear condensation and neuronal degeneration.There was also a substantial accumulation of neurofibrillary tangles and a significant elevation in Aβ42 concentration（P=0.000）.On the other hand,the ADOP,ADE,and ADEOP groups demonstrated marked improvements relative to the ADC group.These included a noticeable increase in the count of structurally intact neurons,a reduction in neuronal degeneration,an enhancement in the quantity of healthy neurons,betterment in the condition of NFTs,and a significant decrease in Aβ42 levels（P=0.046,P=0.013,P=0.002）.Relative to the ADE group,the ADEOP group showed additional enhancements in neuronal health,with more defined cellular structures and a higher count of normal neurons.Improvements in neurofibrillary tangle conditions were more pronounced.Although there was no significant difference in Aβ42 levels（P=0.559）,a downward trend was observed.（3）In the PFC of the ADC group,both the relative content of mt DNA and COX IV levels were significantly lower compared to the NC group（P=0.000,P=0.000）.When compared with the ADC group,the ADE and ADEOP groups displayed significantly higher levels of mt DNA（P=0.016,P=0.000）and COX IV（P=0.040,P=0.000）.The ADOP group also showed a significant increase in mt DNA（P=0.038）,although the COX IV levels did not differ significantly（P=0.738）.Additionally,compared to the ADE group,the ADEOP group had significantly higher COX IV levels in the PFC（P=0.035）,and while the relative content of mt DNA showed an upward trend,the difference was not statistically significant（P=0.071）.（4）In the ADC group,the expression levels of Sirt1,PGC-1α,NRF1,and TFAM mRNA and protein in the PFC were significantly lower compared to the NC group（P=0.000,P=0.000;P=0.000,P=0.005;P=0.007,P=0.003;P=0.000,P=0.000）.In contrast,the ADE,ADOP,and ADEOP groups showed significantly increased expressions of Sirt1 and TFAM mRNA and protein compared with the ADC group（P=0.000,P=0.000,P=0.000;P=0.043,P=0.048,P=0.002;P=0.014,P=0.023,P=0.001;P=0.003,P=0.009,P=0.006）.The expressions of PGC-1αmRNA were significantly elevated in the ADE and ADEOP groups（P=0.022,P=0.000）,and PGC-1αprotein levels were significantly higher in the ADEOP group（P=0.000）.NRF1mRNA in the PFC of the ADEOP group and NRF1 protein in the ADOP and ADEOP groups also showed significant increases（P=0.022,P=0.035,P=0.001）.There were no significant changes in PGC-1αprotein,NRF1 mRNA,and protein levels in the ADE group（P=0.397,P=0.090,P=0.226）,and no significant differences in the increases of PGC-1αmRNA and protein,and NRF1 mRNA in the ADOP group（P=0.687,P=0.201,P=0.299）.Compared with the ADE group,the ADEOP group exhibited significant increases in PGC-1αmRNA and protein,NRF1,and TFAM mRNA levels（P=0.013,P=0.028,P=0.003,P=0.000）.Additionally,there was a further trend toward increased expression of Sirt1 mRNA and protein,NRF1,and TFAM proteins in the ADEOP group,though these changes were not statistically significant（P=0.190,P=0.555,P=0.102,P=0.629）.Conclusions:（1）The combination of D-gal and Na NO?can downregulate the Sirt1-PGC-1α-NRF1-TFAM pathway in the PFC,inhibit mitochondrial biogenesis,and induce the onset of NPS in AD.（2）During the induction of an AD-like rat model with D-gal and Na NO?,interventions such as aerobic exercise or OP can effectively inhibit the production of Aβin the PFC.These interventions also improve the Sirt1-PGC-1α-NRF1-TFAM signaling pathway,activate mitochondrial biogenesis,and maintain mitochondrial respiratory function,thereby offering significant value in preventing NPS in AD.Furthermore,the combined intervention of aerobic exercise and OP supplementation shows a superior effect compared to aerobic exercise alone.更多还原