The Effect of Probiotics on the Expression of Aβ and the Protective Effect of Neurons in Rats with Cerebral Ischemia-Reperfusion Injury
-
摘要:
目的 探讨益生菌干预对脑缺血再灌注损伤大鼠脑组织Aβ表达的影响及神经元的保护作用。 方法 实验动物为雄性Sprague-Dawley大鼠,随机分为4组,各组n = 12:假手术组、模型组、益生菌组、依达拉奉组。假手术组仅分离颈总动脉,其余各组使用线拴法建立脑缺血再灌注模型。脑损伤程度使用神经行为学评分评估;大鼠脑组织梗死面积使用TTC染色观察;大鼠大脑海马CA1区与皮层区神经元病理形态学改变特征使用HE染色观察;Aβ蛋白的表达使用免疫组化实验检测。 结果 模型组相较假手术组,神经行为学评分升高(P < 0.05),脑梗死面积增加(P < 0.05),海马CA1区与皮层区神经元出现损伤,Aβ蛋白表达显著上调(P < 0.001,P < 0.05);益生菌组、依达拉奉组与模型组相比,神经行为学评分降低(P < 0.05),脑梗死面积减小(P < 0.05),海马CA1区与皮层区神经元损伤减轻,Aβ蛋白表达水平在两组中均显著下调(P < 0.001,P < 0.05);依达拉奉组改善更为明显。 结论 益生菌干预可下调Aβ蛋白的表达,减轻脑缺血再灌注大鼠脑组织损伤、发挥神经元保护作用。 Abstract:Objective To explore the effect of probiotics interventionon of the expression of Aβ in rats with cerebral ischemia-reperfusion and the protective effect of neurons. Methods Male SD rats were selected as the experimental animals and randomly divided into Sham Group, Model Group, Probiotics Group and Edaravone Group. The common carotid artery was separated only to the Sham Group While a cerebral ischemia-reperfusion model was eatablished to the other groups. The degree of brain injury was evaluated using neurobehavioral scoring; The infarct area of rat brain tissue was observed using TTC staining; HE staining was used to observe the pathological and morphological changes of neurons in the CA1 and cortical regions of the hippocampus in rats; The expression of Aβ proteins was detected using immunohistochemistry experiments. Results Compared with the Sham Group, the Model Group showed an increase in neurobehavioral scores (P < 0.05), an increase in cerebral infarction focus (P < 0.05), neuronal damage in the hippocampal CA1 and cortical regions, and a significant upregulation of Aβ protein expression (P < 0.001, P < 0.05); Compared with the Model Group, the Probiotics Group and Edaravone Group showed a decrease in neurobehavioral scores (P < 0.05) and a reduction in cerebral infarction focus (P < 0.05). The Probiotic Group and the Edaravone Group showed the less neuronal damage in the hippocampal CA1 and cortical regions , and the Aβ protein expression levels were significantly down regulated in both groups (P < 0.001, P < 0.05). The improvement was more significant in Edaravone Group. Conclusion Probiotics intervention can downregulate the expression of Aβ protein, alleviate the brain tissue damage and exert neuroprotective effects in rats with cerebral ischemia-reperfusion. -
Key words:
- Probiotics /
- Cerebral ischemia-reperfusion injury /
- β amyloid /
- Edaravone
-
近年来,课题组对姜科姜花属植物萜类成分进行了系统研究[1-6],从滇姜花、圆瓣姜花和毛姜花中分离得到一系列对多种肿瘤细胞具有显著体外细胞毒活性的萜类化合物[7-9]。一些姜科植物中的二萜类成分具有抗菌、抗肿瘤等活性[10-11]。滇姜花(Hedychium yunnanense Gangep)中的呋喃二萜Coronarin E含量较高,该成分没有细胞毒活性和抗菌活性,将其通过光敏氧化反应制备具有生物活性的丁烯酸内酯结构的二萜衍生物[12],有产率较高、选择性高、绿色环保的特点。课题组对Coronarin E经二氧化硒氧化、酰化、光敏氧化三步反应,制备两个衍生物,对其抗菌活性(抑菌圈、MIC和联合用药)及体外抗肿瘤活性进行较为深入的研究,进一步验证了二萜衍生物的生物活性,为寻找较好的药物前体提供了理论基础和科学依据。
1. 材料与方法
1.1 仪器
78-1型磁力加热搅拌器(杭州仪器电机厂);分析天平(上海第二天平仪器厂);AM-500型核磁共振波谱仪((瑞士BRUKER公司);LED灯(上海一恒科学仪器有限公司);OSB-2100 旋转蒸发仪(上海爱朗仪器有限公司);ES-315 高压蒸汽灭菌锅,(TOMY 公司);恒温培养箱(上海一恒科学仪器有限公司);SW-CJ-2FD 超净工作台(AIRTECH公司);电热恒温鼓风干燥箱(上海一恒科技有限公司)。
1.2 试剂
Coronarin E由本课题组从姜科植物滇姜花中分离得到。所用有机试剂(化学纯)和化学试剂均购自昆明市医药公司化学试剂玻璃仪器采供站,柱色谱硅胶均为青岛海洋化工厂产品,培养基配料均购自雅云生物科技有限公司。
1.3 实验方法
1.3.1 Coronarin E的SeO2氧化反应
取400 mg coronarin E、168 mg SeO2溶于5 mL干燥的二氯甲烷中,加入368 mg过氧叔丁醇,在常温下搅拌反应2 h,TLC检测原料反应完全。反应液经200~300目硅胶柱色谱分离纯化,石油醚-乙酸乙酯(80∶1~40∶1)洗脱,得到化合物1(无色油状物,见图1)300 mg,产率为71%。
1.3.2 化合物1的酰化反应
取87 mg 1-萘甲酸、0.12 mL N,N-二异丙基碳二亚胺(DIC),0.5 mg对二甲氨基吡啶(DMAP),溶于5 mL干燥过的二氯甲烷,常温搅拌10 min后,加入79 mg化合物1,搅拌反应2 h后,TLC检测反应完全,加入0.2 mL水及50 mL石油醚继续搅拌10 min,超声30 min。产物用150 mL石油醚与100 mL 70%甲醇分配,后者再用100 mL石油醚萃取,合并两次萃取的石油醚层,浓缩后经200-300目硅胶柱色谱分离,石油醚-氯仿(10∶1~5∶1)洗脱。得到化合物2(白色固体,见图2)52.7 mg,产率为44.1%。
1.3.3 化合物2的光敏氧化反应
取52.7 mg化合物2溶于10 mL吡啶中,加入1.0 mg四苯基卟啉(TPP),通入氧气并搅拌,在LED灯照射下反应2 h,TCL检测原料反应完全。溶剂蒸干,经硅胶色谱分离纯化,石油醚-乙酸乙酯(4∶1~1∶1)洗脱,得到化合物3(白色固体)和4(白色固体),见图3,分别为12 mg 和8 mg,产率分别为21.3%和14.1%。
1.3.4 化合物的抗菌活性筛选
采用滤纸片扩散法测试化合物3和4的抑菌圈直径[13-14];采用微量倍比稀释法[15-16]测定化合物3和4的最小抑菌浓度(MIC);化合物4的联合用药测试采用棋盘法,测试该成分分别与万古霉素、氨苄西林、卡那霉素3种抗生素联合用药的最小抑菌浓度[17]。
1.3.5 体外细胞毒活性测试
采用MTT法[18],测定化合物3和4对五个肿瘤细胞株的体外细胞毒活性(阳性对照采用顺铂)。
2. 结果
2.1 化合物3和4的NMR波谱数据
化合物3:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.98(3H,s,H-19),1.19(3H,s,H-20),3.05(1H,d,J = 10.5 Hz,H-9),5.87(1H,t,J = 3.0 Hz,H-7),4.95(1H,br.s,H-17a),5.34(1H,br.s,H-17b),6.67(1H,dd,J = 16.5,10.5 Hz,H-11),5.94(1H,s,H-14),6.45(1H,d,J = 16.5 Hz,H-12),6.44(1H,s,H-16),8.98(1H,m,H-9′),7.80(1H,m,H-8′),7.36(1H,br.s,H-4′),8.51(1H,d,J = 8.1 Hz,H-3′),8.26(1H,d,J = 8.2 Hz,H-7′),8.23(1H,d,J = 8.2 Hz,H-5′),7.88(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.12(t,C-1),19.38(t,C-2),42.54(t,C-3),33.90(s,C-4),48.99(d,C-5),28.87(t,C-6),76.54(d,C-7),146.57(s,C-8),58.83(d,C-9),40.05(s,C-10),141.53(d,C-11),124.80(d,C-12),162.25(s,C-13),116.77(d,C-14),171.25(s,C-15),98.50(d,C-16),115.57(t,C-17),33.47(q,C-18),21.79(q,C-19),14.56(q,C-20),166.82(s,C-1′),128.88(s,C-2′),130.68(d,C-3′),125.68(d,C-4′),134.30(d,C-5′),132.00(s,C-6′),129.52(d,C-7′),126.39(d,C-8′),128.41(d,C-9′),127.15(d,C-10′),134.86(s,C-11′)。
化合物4:1H-NMR(CD3OCD3,500 MHz)δ(ppm):0.91(3H,s,H-18),0.96(3H,s,H-19),1.19(3H,s,H-20),2.97(1H,d,J = 10.0 Hz,H-9),5.81(1H,t,J = 2.5 Hz,H-7),4.89(1H,br.s,H-17a),5.32(1H,br.s,H-17b),6.97/6.99(1H,dd,J = 15.5,10.0 Hz,H-11),6.23(1H,d,J = 15.5 Hz,H-12),7.15/7.16(1H,s,H-14),6.15/6.16(1H,s,H-15),8.98(1H,m,H-9′),7.16(1H,br.s,H-4′),7.64(1H,m,H-8′),8.01(1H,d,J = 8.2 Hz,H-3′),8.16(1H,d,J = 8.2 Hz,H-7′),8.22(1H,d,J = 8.2 Hz,H-5′),7.87(1H,d,J = 8.6 Hz,H-6′). 13C-NMR(CD3OCD3,500 MHz)δ(ppm):41.18(t,C-1),19.97(t,C-2),42.59(t,C-3),33.81(s,C-4),48.99(d,C-5),29.07(t,C-6),76.95(d,C-7),146.73(s,C-8),58.78(d,C-9),39.98(s,C-10),136.91(d,C-11),122.79(d,C-12),131.00(s,C-13),144.94(d,C-14),97.21(d,C-15),170.67(s,C-16),115.20(t,C-17),33.54(q,C-18),21.94(q,C-19),14.69(q,C-20),166.71(s,C-1′),128.98(s,C-2′),130.64(d,C-3′),125.66(d,C-4′),134.93(d,C-5′),131.97(s,C-6′),129.52(d,C-7′),126.35(d,C-8′),128.29(d,C-9′),127.16(d,C-10′),134.88(s,C-11′)。
2.2 化合物3和4的抗菌活性
化合物3对两种MRSA病原菌具有一定的抗菌活性,化合物4对多种革兰氏阳性菌、革兰氏阴性菌具有明显的抗菌活性,见表1。
表 1 化合物3和4对病原菌株抑菌活性筛选结果(抑菌圈直径:mm)Table 1. The result of antimicrobial activities against pathogens ( diameter of inhibition zone:mm)病原菌株 化合物3 化合物4 金黄色葡萄球菌29213 − 10.1 MRSA 1450 8.2 10.5 MRSA 1505 10.1 10.3 MRSA 2024 − 8.2 MRSA I-20 − 10.0 MRSA I-67 − 9.8 MRSA 1957 − 8.1 MRSA 28299 − 10.5 克雷伯氏菌13883 − 10.8 粪肠球菌29212 − 9.4 白色葡萄球菌1029 − 12.0 铜绿假单胞菌PA01 − 10.2 大肠杆菌25922 − 10.0 鼠伤寒沙门氏菌χ 8956 − 17.0 鲍曼不动杆菌19606 − 13.2 枯草芽孢杆菌6633 − − 注:表中抑菌圈直径为三次测量的平均值;“−”表示无抑菌圈。革兰氏阳性菌:金黄色葡萄球菌(Staphylococcus aureus ATCC 29213),7个耐甲氧西林金黄色葡萄球菌(MRSA 1450、1505、2024、1957、28299、I-20、I-67),白色葡萄球菌(Staphylococcus albus 1029);革兰氏阴性菌:鼠伤寒沙门氏菌(Salmonella typhimurium χ 8956),铜绿假单胞菌(Pseudomonas aeruginosa PA01),大肠杆菌(Escherichia coil ATCC 25922),枯草芽孢杆菌(Bacillus subtilis ATCC 6633),鲍曼不动杆菌(Acinetobacter baumanii ATCC 19606),肺炎克雷伯氏菌(Klebsiella pneumonia ATCC 13883),粪肠球菌(Enterococcus faecalis ATCC 29212)。 2.3 化合物与抗生素的联合用药
化合物4与三种抗生素联合使用时,对MRSA病原菌株抑制活性有不同程度的协同或相加作用,见表2。
表 2 化合物4与三种抗生素的联合用药测试结果Table 2. Combination test of compound 4 with three antibiotics菌株 药物 MIC(μg/ml) 最佳抑菌点(化合物4∶抗生素) FICI 作用方式 鼠伤寒沙门氏菌χ8956 化合物4 0.25 万古霉素 0.25 0.125∶0.0625 0.75 + 氨苄西林 2 0.125∶1 1 + 卡那霉素 2 0.0625∶1 0.75 + 鲍曼不动杆菌19606 化合物4 0.5 万古霉素 0.25 0.125∶0.125 0.75 + 氨苄西林 8 0.25∶8 1.5 − 卡那霉素 4 0.25∶4 1.5 − 白色葡萄球菌1029 化合物4 0.5 万古霉素 0.125 0.125∶0.03125 0.5 ++ 氨苄西林 0.5 0.25∶0.125 0.75 + 卡那霉素 4 0.125∶1 0.5 ++ 注:1、FICI = 甲药MIC联合/甲药MIC单用 + 乙药MIC联合/乙药MIC单用,其中甲药代表化合物4,乙药代表抗生素。FICI > 1,表示两药有无关作用;0.5 < FICI≤1,表示两药有相加作用;FICI≤0.5,表示两药有协同作用。2、以“++”表示协同作用,“+”表示相加作用,“−”表示无关。 2.4 化合物3和4对5种肿瘤细胞株的细胞毒活性
化合物3对5种人类肿瘤细胞株具有显著的体外细胞毒活性;化合物4具有较弱的体外肿瘤生长抑制活性,见表3。
表 3 产物对五种肿瘤细胞株的半数生长抑制浓度IC50(μM)Table 3. The IC50 value of 3 and 4 against five tumor cell lines (μM)化合物编号 白血病HL-60 肝癌SMMC-7721 肺癌A-549 乳腺癌MCF-7 结肠癌SW480 3 2.55 2.77 1.17 2.49 1.37 4 15.71 15.62 26.49 25.13 22.87 顺铂 5.00 4.33 2.17 9.18 13.19 评价标准:无效IC50 > 40 μM;有效IC50 < 40 μM;标示下划线的为活性高于阳性对照顺铂。 3. 讨论
二萜coronarin E经三步衍生化反应,制备具有丁烯酸内酯结构单元的二萜衍生物3和4。对3和4的生物活性测试表明:化合物4对多种革兰氏阳性菌、革兰氏阴性菌有明显的抗菌活性,化合物3对两种MRSA具有一定的抗菌活性。化合物4对鼠伤寒沙门氏菌(Salmonella typhimurium χ8956)、鲍曼不动杆菌(Acinetobacter baumannii ATCC 19606)、白色葡萄球菌(Staphylococcus albus 1029)的抗菌效果显著。化合物4对鼠伤寒沙门氏菌的抗菌活性接近万古霉素,高于氨苄西林、卡那霉素;对鲍曼不动杆菌的抗菌活性高于氨苄西林、卡那霉素;对白色葡萄球菌的抗菌活性接近氨苄西林,高于卡那霉素。化合物4与三种抗生素联用时,对鼠伤寒沙门氏菌抑制活性均具有相加作用。化合物4与万古霉素、卡那霉素联用时对白色葡萄球菌抑制活性具有协同作用,与氨苄西林联用时具有相加作用。化合物4与万古霉素联用时对鲍曼不动杆菌抑制活性具有相加作用。
化合物3对5种人类肿瘤细胞株(白血病细胞株HL-60、肝癌细胞株SMMC-7721、肺癌细胞株A-549、乳腺癌细胞株MCF-7和结肠癌细胞株SW-480)均具有显著的体外细胞毒活性,超过阳性对照顺铂;化合物4具有较弱的体外肿瘤生长抑制活性。
由此可见,以姜科二萜为原料,经结构改造制备丁烯酸内酯结构单元的二萜衍生物,并从中寻找有苗头的抗菌、抗癌活性成分或先导化合物,可作为未来抗菌、抗肿瘤药物研究与开发的一个方向。
-
表 1 Zea Longa 5 级评分法评分标准
Table 1. Zea longa grade 5 scoring method scoring standard
评分 表现 0 无神经损害症状 1 梗死侧对侧前肢和前爪不能完全伸展 2 不能正常行走,行走时向梗死侧旋转 3 行走时向梗死对侧倾倒 4 无法行走和意识障碍 表 2 各组大鼠神经行为学评分[($ \bar x \pm s $),分]
Table 2. Neurobehavioral scores of rats in each group[ ($ \bar x \pm s $),points]
分组 n 神经行为学评分 假手术组 12 0±0* 模型组 12 3.51±0.41 益生菌组 12 2.53±0.41* 依达拉奉组 12 2.27±0.20* 与模型组比较,*P < 0.05。 表 3 各组大鼠脑组织梗死灶体积的比较[($ \bar x \pm s$)%]
Table 3. Comparison of cerebral infarction focus volume of rats in each group [($ \bar x \pm s$)%]
分组 n 脑梗死体积百分比 假手术组 3 0±0* 模型组 3 21.56±2.24 益生菌组 3 9.48±1.43* 依达拉奉组 3 6.99±1.32* 与模型组比较,*P < 0.05。 -
[1] Savitz S I,Baron J C,Yenari M A,et al. Reconsidering neuroprotection in the reperfusion era[J]. Stroke,2017,48(12):3413-3419. doi: 10.1161/STROKEAHA.117.017283 [2] Pawar A,Pardasani K R. Mechanistic insights of neuronal calcium and ip3 signaling system regulating atp release during ischemia in progression of Alzheimer's disease[J]. Eur Biophys J,2023,52(3):153-173. doi: 10.1007/s00249-023-01660-1 [3] Ren H,Ma L,Gong X,et al. Edaravone exerts brain protective function by reducing the expression of aqp4,app and aβ proteins[J]. Open Life Sci,2019,14(1):651-658. doi: 10.1515/biol-2019-0074 [4] Pluta R,Ułamek-Kozioł M,Januszewski S,et al. Participation of amyloid and tau protein in neuronal death and neurodegeneration after brain ischemia[J]. Int J Mol Sci,2020,21(13):4599. doi: 10.3390/ijms21134599 [5] Ułamek-Kozioł M,Czuczwar S J,Januszewski S,et al. Proteomic and genomic changes in tau protein,which are associated with Alzheimer's disease after ischemia-reperfusion brain injury[J]. Int J Mol Sci,2020,21(3):892. doi: 10.3390/ijms21030892 [6] Nguyen T-V V,Hayes M,Zbesko J C,et al. Alzheimer's associated amyloid and tau deposition co-localizes with a homeostatic myelin repair pathway in two mouse models of post-stroke mixed dementia[J]. Acta Neuropathol Commun,2018,6(1):100. doi: 10.1186/s40478-018-0603-4 [7] Wang J,Zhang H,He J,et al. The role of the gut microbiota in the development of ischemic stroke[J]. Front Immunol,2022,28(13):845243. [8] Benakis C,Poon C,Lane D,et al. Distinct commensal bacterial signature in the gut is associated with acute and long-term protection from ischemic stroke[J]. Stroke,2020,51(6):1844-1854. doi: 10.1161/STROKEAHA.120.029262 [9] Li H,Sun J,Du J,et al. Clostridium butyricum exerts a neuroprotective effect in a mouse model of traumatic brain injury via the gut-brain axis[J]. Neurogastroenterol Motil,2018,30(5):e13260. doi: 10.1111/nmo.13260 [10] 冯云,亢君君,方宗平,等. 肠道菌群移植通过降低il-17水平减轻老年小鼠脑缺血再灌注损伤[J]. 细胞与分子免疫学杂志,2019,35(1):52-57. [11] Wang H,Ren S,Lv H,et al. Gut microbiota from mice with cerebral ischemia-reperfusion injury affects the brain in healthy mice[J]. Aging (Albany NY),2021,13(7):10058-10074. doi: 10.18632/aging.202763 [12] Wu H,Chiou J. Potential benefits of probiotics and prebiotics for coronary heart disease and stroke[J]. Nutrients,2021,13(8):2878. doi: 10.3390/nu13082878 [13] Pan F,Zhang L,Li M,et al. Predominant gut lactobacillus murinus strain mediates anti-inflammaging effects in calorie-restricted mice[J]. Microbiome,2018,6(1):54. doi: 10.1186/s40168-018-0440-5 [14] Akhoundzadeh K,Vakili A,Shadnoush M,et al. Effects of the oral ingestion of probiotics on brain damage in a transient model of focal cerebral ischemia in mice[J]. Iran J Med Sci,2018,43(1):32-40. [15] 刘兆兰,刘宝全,李兰花,等. 动态随机分组方法介绍及应用[J]. 中西医结合学报,2011,9(3):246-251. [16] 梁国晶,任海燕,张钰鸽,等. 红茶菌在脑缺血再灌注损伤大鼠模型中的作用研究[J]. 现代生物医学进展,2023,23(3):428-432. [17] 卢小叶,吕倩忆,李棋龙,等. Zea-longa评分与改良Garcia评分应用于针刺治疗CIRI大鼠神经功能缺损评估的研究[J]. 湖南中医药大学学报,2021,41(9):1356-1360. [18] Li Y,Zhang J. Animal models of stroke[J]. Animal Model Exp Med,2021,4(3):204-219. doi: 10.1002/ame2.12179 [19] Maida C D,Norrito R L,Daidone M,et al. Neuroinflammatory mechanisms in ischemic stroke: Focus on cardioembolic stroke,background,and therapeutic approaches[J]. Int J Mol Sci,2020,21(18):6454. doi: 10.3390/ijms21186454 [20] Dai S J,Zhang J Y,Bao Y T,et al. Intracerebroventricular injection of aβ1-42 combined with two-vessel occlusion accelerate Alzheimer's disease development in rats[J]. Pathol Res Pract,2018,214(10):1583-1595. doi: 10.1016/j.prp.2018.07.020 [21] Pluta R,Miziak B,Czuczwar S J. Post-ischemic permeability of the blood-brain barrier to amyloid and platelets as a factor in the maturation of Alzheimer's disease-type brain neurodegeneration[J]. Int J Mol Sci,2023,24(13):10739. doi: 10.3390/ijms241310739 [22] Loh J S, Mak W Q, Tan L K S, et al. Microbiota-gut-brain axis and its therapeutic applications in neurodegenerative diseases[J]. Signal Transduct Target Ther,2024,9(1):37. Published 2024 Feb 16. [23] Abdelhamid M,Zhou C,Ohno K,et al. Probiotic bifidobacterium breve prevents memory impairment through the reduction of both amyloid-β production and microglia activation in app knock-in mouse[J]. J Alzheimers Dis,2022,85(4):1555-1571. doi: 10.3233/JAD-215025 [24] 王亚南, 陈真珍, 王凯华, 等. 补阳壮通饮对脑缺血再灌注损伤大鼠的神经保护作用及其可能机制研究[J]. 医学研究杂志,2024,53(4):40-45. [25] 杨梅芳,程萍,陈治任,等. 肠道菌群代谢产物TMAO激活HMGB1/NLRP3炎症通路促进小鼠脑缺血半暗带损伤的机制研究[J]. 中风与神经疾病杂志,2023,40(12):1096-1100. [26] Gilbert K,Arseneault-Br é ard J,Flores Monaco F,et al. Attenuation of post-myocardial infarction depression in rats by n-3 fatty acids or probiotics starting after the onset of reperfusion[J]. Br J Nutr,2013,109(1):50-56. doi: 10.1017/S0007114512003807 [27] Rezaeiasl Z,Salami M,Sepehri G. The effects of probiotic lactobacillus and bifidobacterium strains on memory and learning behavior,long-term potentiation (ltp),and some biochemical parameters in β-amyloid-induced rat's model of alzheimer's disease[J]. Prev Nutr Food Sci,2019,24(3):265-273. doi: 10.3746/pnf.2019.24.3.265 [28] Kim M S,Kim Y,Choi H,et al. Transfer of a healthy microbiota reduces amyloid and tau pathology in an Alzheimer's disease animal model[J]. Gut,2020,69(2):283-294. doi: 10.1136/gutjnl-2018-317431 [29] 任海燕,赵晖,王蕾,等. 依达拉奉对脑缺血再灌注后aβ及其前体表达干预[J]. 科技导报,2015,33(12):77-82. [30] Ye T,Yuan S,Kong Y,et al. Effect of probiotic fungi against cognitive impairment in mice via regulation of the fungal microbiota-gut-brain axis[J]. J Agric Food Chem,2022,70(29):9026-9038. doi: 10.1021/acs.jafc.2c03142 -