针药并用对ApoE基因敲除小鼠自噬水平及学习记忆能力的影响  

Effect of Acupuncture Combined with Traditional Chinese Medicine on Autophagy Level and Learning and Memory Ability of ApoE Knockout Mice

作　　者：吕美娟[1] 王玉 马贤德[1] 任路[1] LYU Meijuan;WANG Yu;MA Xiande;REN Lu(Liaoning University of Traditional Chinese Medicine,Shenyang 110032,Liaoning,China)

机构地区：[1]辽宁中医药大学,辽宁沈阳110032

出　　处：《辽宁中医杂志》2022年第5期180-185,224,共7页Liaoning Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81373718);辽宁省特聘教授项目;国家重点研发计划(2108YFC1704300)

摘　　要：目的研究ApoE^(-/-)小鼠,采用针刺与中药相结合共同干预,观察其对ApoE^(-/-)小鼠海马自噬功能的作用以及对β-淀粉样蛋白(Aβ_(1-42))表达的影响,探寻针药并举、联合治疗能否提高ApoE^(-/-)小鼠的认知能力及其可能的作用机制。方法选用同龄ApoE^(-/-)小鼠及C57BL/6L小鼠,均8周龄,各50只及10只,分别为喂养高脂饲料及常规饲料。C57BL/6L小鼠作为空白组。16周后全程进食高脂饲料的ApoE^(-/-)小鼠分成模型组、低药组、高药组、针药组、针刺组5组,均为随机选取,每组10只。17周起,除外空白组的各组ApoE^(-/-)小鼠各自给予相应干预8周,检测行为学,并对数据进行记录。结束后,研究小鼠海马区的变化,包括自噬体的形成情况,Aβ_(1-42)及微管相关蛋白轻链3、p62蛋白的表达水平,分别进行透射电镜观察,免疫组化法及Western blot法检测。结果在水迷宫测验中,模型组小鼠比照空白组需花更长时间找到平台,即逃避潜伏期延长(P<0.05);其余各治疗组相比模型组,逃避潜伏期呈有不同程度缩短趋势,但只有针药组比较之下,差异有统计学意义(P<0.05)。透射电镜下,模型组海马区神经元扭曲畸形,甚至萎缩,细胞核膜不光滑,完整度下降,双层膜结构不清晰,自噬泡鲜见于胞质内;针药组仍有神经元局部变形、一定程度上出现萎缩,自噬泡在胞质内可见。免疫组织化学结果显示,模型组比照空白组,海马区更多的Aβ_(1-42)阳性细胞表达数量(P<0.05),伴随出现一定数量的老年斑;针药组比照模型组,小鼠海马区减少的Aβ_(1-42)阳性细胞表达(P<0.05)。Western blot结果显示,模型组比照空白组,海马区中有明显增高的LC3-I、p62蛋白表达(P<0.05),显著降低的LC3-Ⅱ蛋白表达及LC3-Ⅱ/I比值(P<0.05);比照模型组,针药组LC3-I、p62蛋白表达水平在海马区中下调明显(P<0.05),相反,LC3-Ⅱ蛋白表达及LC3-Ⅱ/I显然升高(P<0.05)。结论ApoE^(-/-)基因敲除Objective To study the effect of acupuncture combined with traditional Chinese medicine on hippocampal autophagy of ApoE^(-/-)knockout(ApoE^(-/-))mice and its influence on the expression ofβ-amyloid protein(Aβ_(1-42))to explore whether the combination of acupuncture and medicine can improve the cognitive ability of ApoE^(-/-)mice and its possible mechanism.Methods ApoE^(-/-)mice(n=50)and C57 BL/6 L(n=10)mice at the same age were selected,all of which were 8 weeks old.They were fed high-fat diet and conventional diet respectively.C57 BL/6 L mice were used as the blank group.After 16 weeks,ApoE^(-/-)mice which were fed high-fat diet were divided into 5 groups:model group,low dose drug group,high dose drug group,acupuncture and drug group and acupuncture group,10 mice in each group.From the 17 th week on,ApoE^(-/-)mice in each group except the blank group were given the corresponding intervention for 8 weeks.The behavior was detected,and the data was recorded.After that,the changes of mouse hippocampus,including the formation of autophagy,the expression levels of Aβ_(1-42)and microtubule associated protein light chain 3 and p62 were observed by transmission electron microscope,immunohistochemistry and Western blot,respectively.Results In the water maze test,the mice in the model group took longer time to find the platform than those in the blank group,that is to say the escape latency was prolonged(P<0.05).Compared with that of the model group,the escape latency of the other treatment groups showed a trend of shortening in varying degrees,but the difference was only obvious in the acupuncture and drug group(P<0.05).Under the transmission electron microscope,the neurons in the hippocampus of the model group were distorted and deformed,and even atrophied,the cell nuclear membrane was not smooth,the integrity decreased,the bilayer membrane structure was not clear,and autophagic vesicles were rarely seen in the cytoplasm.In the acupuncture and drug group,there were still local deformation and atrophy of neurons to a

关 键 词：APOE基因敲除小鼠 针药联合 细胞自噬 β-淀粉样蛋白1-42 学习记忆能力 

分 类 号：R285.5[医药卫生—中药学]