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慢性脑缺血(CCI)是指各种原因引发的长期大脑血灌流量不足,在血管性痴呆和阿尔茨海默病等神经系统疾病的发展过程发挥重要作用[1-2]。长期慢性脑缺血会导致慢性神经炎症、海马自噬异常、脑部神经元凋亡[3-4],为此,CCI已经严重威胁人类的健康。肠道被称为人类的“第二大脑”,肠道微生物参与周围神经系统和中枢神经系统的双向调节,并通过脑-肠互动(即脑-肠轴)与机体的神经系统密切相关[5]。研究证明中枢系统疾病会导致肠道菌群失调[6],患者发生脑卒中后,不仅会出现胃肠道并发症,还会发生肠道菌群失调,例如肠道菌群中的阿克曼菌属(Akkermansia)会发生变化[7]。同时,肠道菌群失调也推动了脑卒中等疾病的发展,文献报道厚壁菌门增加与认知功能损伤有一定的关联[8-9]。因此,肠道菌群参与了脑部神经系统疾病的进展。中药复方虎杖清脉饮是上海市著名中医脉管病专家奚九一教授数十年临床诊治经验的总结,临床上对辩证属“热郁毒聚,脉滞络痹”的脑小血管损伤疗效显著。有报道虎杖清脉饮通过调节p38和NF-κB信号通路,抑制高糖诱导的人视网膜毛细血管内皮细胞的损伤[10],提示虎杖清脉饮具有抗炎和抗凋亡作用。然而,虎杖清脉饮对慢性脑缺血的治疗作用缺乏临床前研究,作用机制尚不清楚。因此,本实验研究虎杖清脉饮对慢性脑缺血小鼠认知功能的影响,并基于肠道菌群角度初步探讨其作用机制,为防治慢性脑缺血提供新的思路。
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如图1A所示,假手术组小鼠从水迷宫实验第1 d开始到第5 d,逃避潜伏期在不断减少。而模型组小鼠随天数的增加,找到平台的时长并没有明显缩短,并且与假手术组相比,第4天和第5天逃避潜伏期有显著性差异(P<0.05),各给药组小鼠逃避潜伏期在逐渐减少,第5天阳性药组、虎杖清脉饮组与模型组之间的逃避潜伏期差异有统计学意义(P<0.05)。如图1B所示,模型组小鼠的跨越平台次数显著少于假手术组(P<0.01)。而虎杖清脉饮组的跨越平台次数显著大于模型组(P<0.01)。如图1C所示,模型组小鼠的目标象限百分比显著少于假手术组(P<0.05),阳性药组和虎杖清脉饮组的目标象限百分比均显著大于模型组(P<0.05),提示虎杖清脉饮可以改善慢性脑缺血小鼠的学习记忆能力。如图1D所示,假手术组中,小鼠胼胝体的LFB染色,髓鞘排列整齐,无水肿、碎裂及空泡的形成。模型组,LFB着色浅,髓鞘崩解,部分髓鞘空泡化。经银杏叶提取物和虎杖清脉饮治疗,髓鞘病变减轻,染色加深,提示虎杖清脉饮可以改善慢性脑缺血小鼠的白质损伤。
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各组小鼠肠道菌群优化后序列数目为964830,碱基数目为398083175,平均长度为412 bp,序列长度分布在400~440 bp内。图2A为3组小鼠肠道微生物等级丰度曲线,从丰度曲线可以看到各组在缓慢平稳下降,说明各组小鼠肠道菌群物种丰富度高并且分布均匀。图2B、2C所示,总物种曲线可分析样本包含的物种总和,共享物种曲线可分析样本共享物种数目,本研究各组总数目和共享项目增加或减少趋势均逐步减缓并趋于平坦,表明各组样本量满足评估物种丰富度和核心物种数的要求。
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如图3A、3B、3C所示,丰富度实际观测值指数、物种丰富度ACE指数和物种丰富度Chao1指数可解释生物群落丰富度。相较于假手术组,模型组小鼠肠道菌群丰富度显著下降(P<0.05);相较于模型组,虎杖清脉饮组肠道菌群丰富度显著升高(P<0.05),说明虎杖清脉饮可以提升模型组小鼠肠道菌群的丰富度。如图3D所示,3组小鼠肠道菌群的Shannon均匀度测量指数无明显差异。综上,模型组慢性脑缺血小鼠菌群的物种丰富度明显下降,虎杖清脉饮可提升慢性脑缺血小鼠菌群的物种丰富度。
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通过主成分分析(PCA)各组小鼠样本菌群群落组成差异,运用反差分解,在图中通过距离反映不同组之间的组成差异,如样本物种组成越相似,反映在主成分分析图中的距离越近。假手术组和模型组聚集在一起,说明模型组对肠道菌群的影响较小。而虎杖清脉饮组与模型组距离较远,说明慢性脑缺血小鼠肠道菌群改变较小,虎杖清脉饮具有调控模型组小鼠肠道菌群组成的作用(图4)。
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门水平上,3组小鼠肠道中占主导地位的微生物主要有厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、脱硫菌门(Desulfobacterota)、髌骨细菌门(Patescibacteria)、放线菌门(Actinobacteriota),其中后壁菌门和拟杆菌门为优势门类(图5A)。由图5B所示,各组小鼠肠道菌群中髌骨细菌门
和弯曲菌门丰度有显著性差异(P<0.05)。如图5C所示,相较于假手术组,模型组疣微菌门丰度显著升高(P<0.05)。如图5D所示,相较于模型组,虎杖清脉饮组小鼠的疣微菌门丰度显著降低(P<0.05),髌骨细菌门丰度显著增加(P<0.05)。门水平差异性分析显示,慢性脑缺血小鼠疣微菌门丰富度增加;虎杖清脉饮降低慢性脑缺血小鼠疣微菌门和增加髌骨细菌门的群落丰度。 -
属水平上,3组小鼠肠道中优势菌为毛螺菌科_NK4A136_group、norank_f_Muribaculaceae和乳杆菌属(Lactobacillus)等(图6A)。如图6B所示,3组小鼠的肠道菌群中,阿克曼菌属(Akkermansia)、绿脓杆菌(Turicibacter)、嗜木聚糖真杆菌属(Eubacterium_xylanoPhilum_group)、异杆菌属(Allobaculum)、丹毒荚膜菌属(Erysipelatoclostridium)、回肠杆菌(Ileibacterium)、乳酸球菌属(Lactococcus)群落丰度具有显著性差异(P<0.05,P<0.01)。如图6C所示,与假手术组相比,模型组阿克曼菌属、丹毒荚膜菌属丰度显著上升(P<0.05);嗜木聚糖真杆菌属、异杆菌属、绿脓杆菌、回肠杆菌和乳酸球菌属丰度显著下降(P<0.05,P<0.01)。如图6D所示,与模型组相比,虎杖清脉饮组的阿克曼菌属、丹毒荚膜菌属丰度显著下降(P<0.05);嗜木聚糖真杆菌属、异杆菌属、绿脓杆菌、回肠杆菌和乳酸球菌属丰度显著上升(P<0.05,P<0.01)。
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如表1所示,LEfSe可以检测各组之间存在显著丰度差异特征。相较于假手术组,模型组疣微菌门(P_Verrucomicrobiota)、疣微菌纲(c_Verrucomicrobiae)、疣微菌目(o_Verrucomicrobiales)、阿克曼菌科(f_Akkermansiaceae)等相对丰度显著上调;嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group)、罗氏菌属(g_Roseburia)、回肠杆菌属(g_Ileibacterium)等相对丰度显著下调,表明慢性脑缺血小鼠上述肠道菌群丰度发生显著性变化。
表 1 各组小鼠肠道菌群LEfSe多级物种差异判别分析
菌群 模型组vs
假手术组虎杖清脉饮组vs
模型组疣微菌门(P_Verrucomicrobiota) ↑ ↓ 疣微菌纲(c_Verrucomicrobiae) ↑ ↓ 疣微菌目(o_Verrucomicrobiales) ↑ ↓ 克里斯滕菌目(o_Christensenellales) ↑ 阿克曼菌科(f_Akkermansiaceae) ↑ ↓ 克里斯滕菌科(f_Christensenellaceae) ↑ 粪芽孢菌属(g_Coprobacillus) ↑ ↓ 阿克曼菌属(g_Akkermansia) ↑ ↓ 嗜胆菌属(g_Bilophila) ↑ 克里斯滕菌属
(g_Christensenellaceae_R-7_group)↑ 丹毒荚膜菌属(g_Erysipelatoclostridium) ↑ ↓ 嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group) ↓ 罗氏菌属(g_Roseburia) ↓ 未分类克里斯滕森菌科(g_unclassified_f_Christensenellaceae) ↓ 异杆菌属(g_Allobaculum) ↓ ↑ 理研菌属(g_Rikenella) ↓ 绿脓杆菌属(g_Turicibacter) ↓ ↑ g_Tyzzerella ↓ 回肠杆菌属(g_Ileibacterium) ↓ 蓝细菌纲(c_Cyanobacteriia) ↓ f_Tannerellaceae ↓ f_norank_o_Chloroplas ↓ g_norank_f_norank_o_Chloroplast ↓ 副杆菌属(g_Parabacteroides) ↓ o_PeptostrePtococcales-Tissierellales ↑ 髌骨细菌门(P_Patescibacteria) ↑ c_Saccharimonadia ↑ o_Saccharimonadales ↑ f_Saccharimonadaceae ↑ 丹毒丝菌科(f_Erysipelotrichaceae) ↑ 消化链球菌科(f_Peptostreptococcaceae) ↑ g_Candidatus_Saccharimonas ↑ g_UCG-005 ↑ g_Romboutsia ↑ 嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group) ↑ g_norank_f_Atopobiaceae ↑ 安德克氏菌属(g_Adlercreutzia) ↑ 分节丝状菌属(g_Candidatus_Arthromitus) ↑ g_Lachnospiraceae_UCG-006 ↑ g_NK4A214_group ↑ 注:↑表示相对丰度增加,↓表示相对丰度降低。 相较于模型组,虎杖清脉饮组疣微菌纲(c_Verrucomicrobiae)、蓝细菌纲(c_Cyanobacteriia)、疣微菌门(P_Verrucomicrobiota)、阿克曼菌科(f_Akkermansiaceae)、阿克曼菌属(g_Akkermansia)、粪芽孢菌属(g_Coprobacillus)、丹毒荚膜菌属(g_Erysipelatoclostridium)、副杆菌属(g_Parabacteroides)等相对丰度显著下调;丹毒丝菌科(f_Erysipelotrichaceae)、消化链球菌科(f_PeptostrePtococcaceae)、绿脓杆菌属(g_Turicibacter)、异杆菌属(g_Allobaculum)、嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group)等相对丰度显著上调。说明虎杖清脉饮通过调节嗜木聚糖真杆菌属、异杆菌属、绿脓杆菌、疣微菌、阿克曼菌和丹毒荚膜菌的丰度,达到治疗慢性脑缺血的治疗作用。
Effects of Huzhang Qingmai decoction on cognitive function and intestinal flora in mice with chronic cerebral ischemia
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摘要:
目的 从肠道菌群角度探讨虎杖清脉饮对慢性脑缺血小鼠认知功能改善的作用机制。 方法 放置微弹簧圈于双侧颈总动脉致狭窄制备慢性脑缺血小鼠模型。灌胃给药12 w后,采用Morris水迷宫和LFB染色测定小鼠的认知功能和白质的损伤程度;提取各组小鼠盲肠内容物,进行16S rRNA基因V3+V4去扩增子信息采集与分析。 结果 水迷宫实验结果显示,虎杖清脉饮组小鼠逃避潜伏期显著缩短,跨越平台次数和目标象限百分比增加,学习记忆功能明显改善。LFB染色显示,模型组白质损伤严重;虎杖清脉饮组白质损伤程度较轻。16S rRNA测序结果显示,与模型组相比,虎杖清脉饮组小鼠肠道菌群中疣微菌门、阿克曼菌属和丹毒荚膜菌属的丰度显著降低(P<0.05),而嗜木聚糖真杆菌属和异杆菌属的丰度显著增加(P<0.05)。 结论 虎杖清脉饮具有改善慢性脑缺血小鼠认知功能的作用,其保护作用可能与调节慢性脑缺血小鼠肠道菌群有关。 Abstract:Objective To investigate the mechanism of action of Huzhang Qingmai decoction (HZQMY) on the improvement of cognitive function in mice with chronic cerebral ischemia from the perspective of intestinal flora. Methods A mouse model of chronic cerebral ischemia was established by placing microcoils around the bilateral common carotid arteries to induce bilateral carotid artery stenosis (BCAS). After 12 weeks of intragastric administration, the cognitive function of the mice was measured by the Morris water maze; the myelin damage was analyzed by LFB staining; The contents of the cecum of the mice in each group were extracted and analyzed by 16S rRNA sequencing. Results The results of the water maze experiment showed that the mice in the HZQMY group had a significantly shorter escape latency, increased the number of crossings platform and the percentage of target quadrants. LFB staining showed that the white matter damage in the model group was severe; the white matter damage in the HZQMY group was milder. The results of 16S rRNA sequencing showed that compared with the model group, the abundance of Verrucomicrobiota, Akkermansia, and ErysiPelatoclostridium capsulatum in the intestinal flora in HZQMY group was significantly reduced (P<0.05), while the abundances of Eubacterium_xylanoPhilum and Allobaculum were significantly increased (P<0.05). Conclusion The protective effect of HZQMY, which has the effect of improving cognitive function in mice with chronic cerebral ischemia, may be related to the regulation of intestinal flora in mice with chronic cerebral ischemia. -
Key words:
- HZQMY /
- chronic cerebral ischemia /
- cognitive function /
- 16S rRNA /
- intestinal flora
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表 1 各组小鼠肠道菌群LEfSe多级物种差异判别分析
菌群 模型组vs
假手术组虎杖清脉饮组vs
模型组疣微菌门(P_Verrucomicrobiota) ↑ ↓ 疣微菌纲(c_Verrucomicrobiae) ↑ ↓ 疣微菌目(o_Verrucomicrobiales) ↑ ↓ 克里斯滕菌目(o_Christensenellales) ↑ 阿克曼菌科(f_Akkermansiaceae) ↑ ↓ 克里斯滕菌科(f_Christensenellaceae) ↑ 粪芽孢菌属(g_Coprobacillus) ↑ ↓ 阿克曼菌属(g_Akkermansia) ↑ ↓ 嗜胆菌属(g_Bilophila) ↑ 克里斯滕菌属
(g_Christensenellaceae_R-7_group)↑ 丹毒荚膜菌属(g_Erysipelatoclostridium) ↑ ↓ 嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group) ↓ 罗氏菌属(g_Roseburia) ↓ 未分类克里斯滕森菌科(g_unclassified_f_Christensenellaceae) ↓ 异杆菌属(g_Allobaculum) ↓ ↑ 理研菌属(g_Rikenella) ↓ 绿脓杆菌属(g_Turicibacter) ↓ ↑ g_Tyzzerella ↓ 回肠杆菌属(g_Ileibacterium) ↓ 蓝细菌纲(c_Cyanobacteriia) ↓ f_Tannerellaceae ↓ f_norank_o_Chloroplas ↓ g_norank_f_norank_o_Chloroplast ↓ 副杆菌属(g_Parabacteroides) ↓ o_PeptostrePtococcales-Tissierellales ↑ 髌骨细菌门(P_Patescibacteria) ↑ c_Saccharimonadia ↑ o_Saccharimonadales ↑ f_Saccharimonadaceae ↑ 丹毒丝菌科(f_Erysipelotrichaceae) ↑ 消化链球菌科(f_Peptostreptococcaceae) ↑ g_Candidatus_Saccharimonas ↑ g_UCG-005 ↑ g_Romboutsia ↑ 嗜木聚糖真杆菌属(g_Eubacterium_xylanophilum_group) ↑ g_norank_f_Atopobiaceae ↑ 安德克氏菌属(g_Adlercreutzia) ↑ 分节丝状菌属(g_Candidatus_Arthromitus) ↑ g_Lachnospiraceae_UCG-006 ↑ g_NK4A214_group ↑ 注:↑表示相对丰度增加,↓表示相对丰度降低。 -
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