【作者】 张垚；

【导师】 张晖；

【作者基本信息】 江南大学 ， 食品科学与工程， 2022， 博士

【摘要】 阿尔茨海默症（AD）是一种中枢神经退行性病变,主要影响人们的记忆和语言能力,同时伴随认知能力的下降和行为的逐渐变化,由于发病机制尚不明确,目前对此疾病尚无理想的治疗药物。已有大量研究表明,维持大脑内二十二碳六烯酸（DHA）的正常水平有助于延缓AD病程,而通过膳食摄入溶血磷脂型DHA（DHA-Lyso PLs）能够向脑部有效递送DHA,但目前尚缺乏DHA-Lyso PLs延缓AD的机理研究。为此,以甘油磷脂酰丝氨酸（GSP）和DHA为底物,建立了高纯度溶血磷脂酰丝氨酸型DHA（DHA-LPS）的两步合成方法。以游离型DHA为对比,研究了DHA-LPS对AD细胞和AD秀丽线虫神经的保护作用,分析了其作用机制。主要研究内容如下:首先,用自制新型固定化磷脂酶D(OMSC-C₁₈-PLD)为转磷脂酰反应的催化剂,以磷脂酰胆碱和L-丝氨酸为底物制备磷脂酰丝氨酸（PS）。结果表明,OMSC-C₁₈-PLD的酶活是传统的游离磷脂酶D的1.24倍,达到3.18×10⁴U/g_pro,与底物的亲和力增大,Km由2.46m M变为1.51m M,半衰期增加了15天,达到了40天;其在温度40℃、p H 6.0、磷脂酰胆碱与L-丝氨酸的质量比为1:10、加酶量为30%的优化条件下,仅9 h就使PS得率达到91.2%,而游离磷脂酶D催化11 h的得率仅为75.4%,且副反应明显。OMSC-C₁₈-PLD对p H和温度的适应性远优于游离磷脂酶D,重复使用9次后仍能保留31.4%的酶活性,显示出比游离磷脂酶D更高的转磷脂酰活性和更好的底物亲和性,可用于量产DHA-LPS的合成原料高纯度PS,并降低目的产物的后续纯化成本。其次,将PS水解纯化后得到GPS,研究了以DHA和GPS为底物,合成高DHA-LPS的工艺。结果表明,GPS的纯度和回收率分别为96.83%和76.62%;Lipozyme TL IM为优选催化剂,在温度45℃、加酶量400 U/g底物、底物质量比为1:15的条件下,LPS的产率为88.98%;其中,1-酰基-sn-甘油-3-溶血磷脂酰丝氨酸（sn-1-LPS）为产物的主要成分,DHA的插入率为71.63%。再次,研究并对比了DHA-LPS和未酯化DHA（DHA-NEFA）两种剂型对AD细胞的保护作用。结果表明,DHA-LPS和DHA-NEFA处理均可显著逆转AD细胞的活性氧（ROS）、丙二醛（MDA）和乳酸脱氢酶（LDH）水平的升高（p<0.05）,抑制IL-1β、TNF-α和IL-6等炎症因子的显著升高（p<0.05）和细胞凋亡,改善其活力,并呈现浓度依赖关系。虽然两种剂型均能够抑制细胞的炎症和氧化应激（OS）,但仅有DHA-LPS具有改善细胞ATP紊乱和抑制一氧化氮（NO）水平上升的作用,高剂量干预水平的DHA-LPS增加了细胞28%的ATP水平,减少了56%的NO水平（p<0.05）。提示DHA-LPS可能对AD细胞具比DHA-NEFA更有效的AD细胞保护作用。然后,分析了DHA-LPS改善AD细胞OS和炎症的作用机理进。结果表明,与模型组相比,DHA-LPS能够明显降低Bax、Caspase-9、Caspase-3及Cyt-C的表达,高剂量DHA-LPS干预分别使上述基因表达分别下调36%、18%、27%和31%（p<0.05）,上调Bcl-2基因表达29%（p<0.05）,提示其可能通过线粒体介导的通路抑制来改善Aβ_25-35诱导的AD细胞的凋亡情况;另一方面,与模型组相比,DHA-LPS能够降低TNF-α、IL-1β及IL-6等炎症因子m RNA的表达,降低i NOS m RNA的表达,高剂量DHA-LPS干预能够分别下调上述基因表达47%、35%、48%和60%（p<0.05）,下调JNK和p38的25%和33%的蛋白表达（p<0.05）,提示其可能通过MAPK通路抑制来改善细胞的炎症情况。最后,以CL4176秀丽隐杆线虫株系为AD病理模型,研究DHA-LPS对A?_25-35诱导的秀丽线虫麻痹状态的改善作用。结果表明,DHA-LPS能够显著抑制AD秀丽线虫麻痹表型的病理特征,改善AD秀丽线虫的运动能力,相对于对照组,在培养44 h后,高剂量DHA-LPS干预使AD秀丽线虫麻痹率由79%下降至51%（p<0.05）,增加AD秀丽线虫的弯曲次数30%（p<0.05）;高剂量DHA-LPS干预后,秀丽线虫体内的A?沉积现象显著降低,ROS水平降低了38%（p<0.05）,SOD水平上升了84%（p<0.05）;此外,DHA-LPS能够显著上调SKN-1、SOD-3,ctl-1和sir-2.1基因的表达,减轻秀丽线虫麻痹病理特征,提示其可能通过发挥抗氧化活性来改善AD秀丽线虫的病理特征。更多还原

【Abstract】 Alzheimer’s disease（AD）is a degenerative disease of central nervous system,which mainly affects people’s memory and language ability.At the same time,it is accompanied by the decline of cognitive ability and the gradual change of behavior.Due to the unclear pathogenesis,there is no ideal therapeutic drug for this disease at present.A large number of studies have shown that maintaining the normal level of docosahexaenoic acid（DHA）in the brain is helpful to delay the course of AD,and dietary intake of lysophospholipid DHA（DHA-Lyso PLs）can effectively deliver DHA to the brain.However,there is still a lack of research on the mechanism of DHA lysopls delaying AD.Therefore,a two-step synthesis method of high purity lysophosphatidylserine form DHA（DHA-LPS）was established with glycerol phosphatidylserine（GSP）and DHA as substrates;Compared with free DHA,the protective effects of DHA-LPS on AD cells and AD Caenorhabditis elegans were studied,and its mechanism was analyzed.The main research contents are as follows:Firstly,phosphatidylserine（PS）was prepared from phosphatidylcholine and L-serine using a novel immobilized phospholipase D(OMSC-C₁₈-PLD)as the catalyst for the transphosphatidylation reaction.The results showed that the enzyme activity of OMSC-C₁₈-PLD was 1.24 times that of traditional free phospholipase D,reaching 3.18×10⁴ U/g_pro,the affinity with substrate increased,the Km changed from2.46m M to 1.51m M,and the half-life increased by 15 days to 40 days;Under the optimum conditions of temperature 40℃,p H 6.0,mass ratio of phosphatidylcholine to L-serine 1:10 and enzyme dosage 30%,the yield of PS reached 91.2%in only 9 h,while the yield of free phospholipase D was only 75.4%in 11 h,and the side effects were obvious.The adaptability of OMSC-C₁₈-PLD to p H and temperature was much better than that of free phospholipase D,after repeated use for 9 times,it could still retain 31.4%of the enzyme activity,showing higher transphosphatidylation activity and better substrate affinity than free phospholipase D.It can be used as a high-purity PS raw material for mass production of DHA-LPS and reduce the subsequent purification cost of the target product.Secondly,PS was hydrolyzed and purified to obtain GPS.The process of synthesizing high DHA-LPS with DHA and GPS as substrates was studied.The results showed that the purity and recovery of GPS were 96.83%and 76.62%,respectively;Lipozyme TLIM was the preferred catalyst.Under the conditions of temperature 45℃,enzyme dosage 400 U/g substrate and substrate mass ratio 1:15,theyieldofLPSwas88.98%;Amongthem,1-acyl-sn-glycerol-3-lysophosphatidylserine（sn-1-LPS）was the main component of the product,and the insertion rate of DHA was 71.63%.Thirdly,the protective effects of DHA-LPS and nonesterified DHA（DHA-NEFA）on AD cells were studied and compared.The results showed that DHA-LPS and DHA-NEFA treatment could significantly reverse the increase of reactive oxygen species（ROS）,malondialdehyde（MDA）and lactate dehydrogenase（LDH）levels in AD cells（p<0.05）,inhibit IL-1β、TNF-αand IL-6 and other inflammatory factors increased significantly（p<0.05）,inhibit apoptosis,improved the activity of cells,and showed a concentration dependent relationship.Although both formulations could inhibit cell inflammation and oxidative stress（OS）,only DHA-LPS could improve cell ATP disorder and inhibit the increase of nitric oxide（NO）level.DHA-LPS with high dose intervention level increased cell ATP level by 28%and reduced NO level by56%（p<0.05）.It is suggested that DHA-LPS may have more effective protective effect on AD cells than DHA-NEFA.Then,the mechanism of DHA-LPS improving OS and inflammation in AD cells was analyzed.The results showed that compared with the model group,DHA-LPS could significantly reduce the expression of Bax,Caspase-9,Caspase-3 and Cyt-c.the intervention of high-dose DHA-LPS reduced the expression of the above genes by36%,18%,27%and 31%（p<0.05）,and increased the expression of Bcl-2 gene by29%（p<0.05）,suggesting that it may improved Aβ_25-35-induced apoptosis of AD cells through mitochondrial mediated pathway inhibition;On the other hand,compared with the model group,DHA-LPS could reduce the m RNA expression of TNF-α、IL-1β、IL-6 and other inflammatory factor,reduce the expression of i NOS m RNA.High dose DHA-LPS intervention could down regulate the above gene expression by 47%,35%,48%and 60%（p<0.05）,and down regulate the protein expression of JNK and p38 by 25%and 33%（p<0.05）,suggesting that it may improve the inflammation condition of cells through the inhibition of MAPK pathway.Finally,taking cl4176 Caenorhabditis elegans strain as the pathological model of AD,the effect of DHA-LPS on the paralysis of Caenorhabditis elegans induced by Aβ_25-35 was discussed.The results showed that DHA-LPS could significantly inhibit the pathological characteristics of the paralysis phenotype of Caenorhabditis elegans and improve the motor ability of Caenorhabditis elegans.Compared with the control group,the intervention of high-dose DHA-LPS reduced the paralysis rate of Caenorhabditis elegans from 79%to 51%（p<0.05）and increased the bending times of Caenorhabditis elegans by 30%（p<0.05）.After the intervention of AD Caenorhabditis elegans model with high-dose DHA-LPS,the Aβdeposition in Caenorhabditis elegans decreased significantly,the level of ROS decreased by 38%（p<0.05）,and the level of SOD increased by 84%（p<0.05）.In addition,DHA-LPS could significantly up regulate the expression of SKN-1,SOD-3,ctl-1 and sir-2.1genes and reduce the pathological characteristics of Caenorhabditis elegans paralysis,suggesting that DHA-LPS could improve the pathological characteristics of AD Caenorhabditis elegans by exerting its antioxidant biological activity.更多还原