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Rhubarb ameliorates cognitive dysfunction in a rat model of Alzheimer's disease through regulation of the intestinal microbiome
Journal of Traditional Chinese Medical Sciences (2019) 6, 234e243
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.elsevier.com/locate/jtcms
Rhubarb ameliorates cognitive dysfunction in a rat model of Alzheimer’s disease through regulation of the intestinal microbiome Huizhen Zhao, Demin Gao, Xiaoyan Gao* School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China Received 26 June 2019; received in revised form 10 August 2019; accepted 10 August 2019
Available online 16 August 2019
KEYWORDS Rhubarb; Intestinal microbiome; Alzheimer’s disease; Cognitive function; 5-hydroxytryptamine
Abstract Objective: To determine the mechanism whereby rhubarb (Rheum tanguticum MAXIM. Ex BALF.) may ameliorate cognitive dysfunction through regulation of the intestinal microbiome. Methods: We used a rat model of human microbiome-associated (HMA)-AD to characterize the therapeutic effect of rhubarb on cognitive dysfunction by assessing learning and spatial memory, tissue pathology, and neurotransmitter expression in brain tissue. Then, 16S rRNA gene sequencing was used to analyze the intestinal microbial composition of the rats before and after rhubarb intervention, to determine whether changes in the intestinal microbiome might underpin the beneficial effect of rhubarb on cognitive dysfunction. Results: Morris water maze experiments showed that the learning and spatial memory of HMAAD rats were improved after rhubarb administration. Examination of brain sections showed that rhubarb had a protective effect on neurons in the brain tissue of HMA-AD rats. Brain tissue neurotransmitter analysis showed that rhubarb significantly reduces the 5-hydorxytryptamine concentration in the hippocampus of HMA-AD rats (P Z .0013). Furthermore, rhubarb affected the abundance of the Lachnospiraceae_NK4A136_group and Lactobacillus in the large intestine. Conclusion: This study suggests that rhubarb ameliorates cognitive dysfunction in rats with HMA-AD by regulating the abundance of beneficial bacteria, which likely affects the concentration of 5-hydorxytryptamine in the hippocampus. ª 2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
* Corresponding author. E-mail address: [email protected] (X. Gao). Peer review under responsibility of Beijing University of Chinese Medicine. https://doi.org/10.1016/j.jtcms.2019.08.004 2095-7548/ª 2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction
Materials
Alzheimer’s disease (AD), the most common form of dementia, is a degenerative disease of the central nervous system characterized by progressive memory loss, cognitive impairment, and personality changes. According to the ‘World 2018 AD Report’ issued by the International Alzheimer’s Association, there were about 50 million patients with AD worldwide in 2018, and the incidence of AD was rapidly increasing,1 such that it represents a serious threat to human health and social development. Microbial dysbiosis may mediate or affect the pathogenesis of AD, because the microbiome and its metabolites might affect signaling pathways2e4 and the production of proinflammatory cytokines associated with the pathogenesis of AD.5e8 Therefore, targeting of the intestinal microbiome may provide a means of preventing and/or treating AD. Rhubarb (Rheum tanguticum MAXIM. Ex BALF.) is obtained from the roots and rhizomes of Rheum officinale Baills., R Palmatum L., and R. tanguticum Maxim. ex Baill., which is a laxative with actions of unblocking the bowels and directing the turbid downward, clearing heat and purging fire, detoxifying, activating blood circulation and dissolving blood stasis, as recorded in the Pharmacopoeia of the People’s Republic of China.9 It is also indicated for the treatment of dementia as a traditional Chinese medicine (TCM), targeting phlegm and blood stasis obstructing collaterals and poisoning brain collaterals.10 It is one of the most common TCMs used for the treatment of AD. However, although a therapeutic effect is reported to have been achieved in the clinic,11 the mechanism involved in the amelioration of AD remains to be established. A limited number of studies in vitro have shown that anthraquinones, which are found in rhubarb, can reduce the deposition of Ab and Tau proteins,12,13 can reduce the inflammatory response of nerve cells,14 and are neuroprotective.15 There has been recent interest in the interactions between TCMs and the intestinal microbiome, and accumulating evidence suggests that rhubarb, and especially anthraquinones, affect the composition and function of the intestinal microbiome.16,17 Therefore, we hypothesized that rhubarb may ameliorate the symptoms of AD by correcting the associated intestinal dysbiosis. To address this hypothesis, we established a human microbiome-associated rat model of AD (HMA-AD), in which the intestinal environment of the rats would mirror the human intestinal environment as closely as possible. The effects of rhubarb were investigated in this model with respect to learning and spatial memory, tissue pathology, and neurotransmitter expression in brain tissue. The structure of the intestinal microbiome before and after rhubarb administration, and the effect of rhubarb on the intestinal microbiome of the rat model of AD were analyzed.
We used a high performance liquid chromatograph (Alliance 2695, Waters, Milford, MA) with an electrochemical detector (ECD2465, Waters), a Morris water maze (Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, China), centrifuges (N13462C, 5430R, and 5424R, Eppendorf, Hamburg, Germany), an ultramicrospectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA), an electrophoresis instrument (DYY-6C, Beijing Liuyi Biotechnology, Beijing, China), a PCR cycler (ABI GeneAmp 9700, Thermo Fisher Scientific), MISEQ and HISEQ sequencers (Illumina hiseq, Illumina, San Diego, CA), a microplate reader (BioTek ELx800, Biotek, Winooski, Vermont), a microfluorometer (TBS380, TurnerBioSystems, Sunnyvale, CA), a focused-ultrasonicator (Covaris M220, Gene, Hong Kong, China), vortex mixers (QL-901, Kylin-Bell Lab Instruments, Haimen, China), a grinding instrument (TL-48R, Wonbio, Shanghai, China), and DNA extraction kits (OMEGA-soil DNA Kit, Omega Bio-Tek, Norcross, GA). Donepezil hydrochloride was supplied by Eisai (Suzhou, China). Rhubarb herbal medicine was provided by the Modern Research Center for TCMs (Peking University, Beijing, China) and had been authenticated by Professor Pengfei Tu (Peking University) and tested using high-performance liquid chromatography (HPLC; Supplementary Fig. 1). Imipenem and cilastatin sodium were obtained from Merck Sharp & Dohme (Kenilworth, NJ), Ab25e35 (907532V) and 5hydorxytryptamine (5-HT; 091M5163V) from Sigma (Burlington, MA), dopamine (DA; 100070e201507) from the National Institutes for Food and Drug Control (Beijing, China), 2% agarose (50164) from Biowest (Madrid, Spain), Fast Pfu (120428) from TransGen (Beijing, China), and AxyPrep DNA Gel Extraction Kits (AP-GX-250) from Axygen (Tewksbury, MA).
Materials and methods
Experimental animals Three-week-old male specific pathogen-free Sprague-Dawley rats, weighing 70 5 g, were kept in the barrier facility of Beijing University of Chinese Medicine. The rats were maintained at 20 2 C, with a relative humidity of 60% 5%, and under a 12-hour light/dark cycle, and were acclimated for one week prior to the commencement of the study. Standard chow and Milli-Q water were provided ad libitum. All rats were fasted overnight, with free access to water, before drug administration. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals. Rats were allocated randomly to a conventional group, a control group, a model group, a rhubarb-treated group, and a positive control group (n Z 10 per group).
Establishment of a human microbiome-associated rat model of AD
Ethical approval The study protocol was approved by the Ethics Committee for Animal Care and Treatment of Beijing University of Chinese Medicine (BUCM-4-2018122801-4066).
Conventional group rats were maintained without any treatment. Microbe-free rats were obtained by gavaging the control, model, rhubarb-treated, and positive control groups with imipenem and cilastatin sodium (5 mg$mL1).
236 Humanization of the gut microbiome was then performed using human fecal samples collected from two healthy male donors. Fresh feces were diluted in nine volumes of anaerobic medium and 1.5 mL of fecal suspension was administered to the four groups of rats by gavage, once per day for 3 weeks. These rats were then fed a standard diet for 1 week, by which time a stable HMA-AD rat model had been established. After this, rats in the model, rhubarbtreated, and positive control groups were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1), prior to the attachment of a stereotaxic apparatus and exposure of the anterior fontanelle. Each rat was injected with 2.5 mL of an Ab25e35 (2 mg$mL1) into the right lateral ventricle, according to the rat brain stereotaxic map. The coordinates used were 1.2 mm caudal to the anterior fontanelle and 1.2 mm to the right side of the sagittal suture. The needle was inserted 3.2 mm deep to the surface of skull, remained in place for 8 min, and was extracted at 1 mm$min1. The rats were kept in their cage to recover. The rats in the control group were similarly injected with the same volume of saline.
Preparation of drug solutions Small pieces of rhubarb herbal medicine (w30 g) were weighed and soaked in 300 mL distilled water for 1 hour and decocted twice in boiling water for 30 minutes on each occasion. The combined products of this water decoction were filtered and dried in a 60 C water bath, then made up to 50 mL with ultrapure water, to yield a 0.6 g$mL1 solution. Donepezil hydrochloride solution was prepared by crushing one tablet, containing 5 mg donepezil hydrochloride, and dissolving it in 100 mL of normal saline by ultrasonication for 30 minutes, to obtain a 0.05 mg$mL1 solution.
Administration of treatments After the intracranial administration of Ab25e35, rhubarb (2.1 g$kg1) or donepezil hydrochloride solution (0.6 mg$kg1) were orally administered to the rats in the rhubarb-treated and positive control groups, respectively, for 12 days. Normal saline (1 mL) was similarly administered to rats in the control and model groups.
Morris water maze test Morris water maze testing was conducted as previously described,18 for rats in the conventional, control, model, rhubarb-treated, and positive control groups from 8 days after intracranial injection of Ab25e35. Navigation training was conducted on days 8e11 of the interventions. Each trial started 30 minutes after drug administration, with the rat being placed against the edge of the pool, which was divided into four virtual quadrants. Each rat underwent a trial every half-day for eight successive half-days, making a total of eight trials over 4 successive days. The starting positions for the eight trials were evenly distributed among the four quadrants. Each rat was given 60 seconds to locate the platform during each trial. The platform locating latency (PLL) was
H. Zhao et al. defined as the time taken for the experimental animal to locate and ascend the platform. If the rat had failed to ascend the platform within 60 seconds, it was guided to the platform and allowed to stay here for 30 seconds, which should have assisted with knowledge acquisition and memory formation, and in these instances the PLL was recorded as 60 seconds. After navigation training, spatial training was performed on day 12 of the intervention, in which the platform was removed and the rats were placed against the edge of the pool, as before. Then, the swimming time and the number of times the rat crossed the quadrant where the original platform was located, over 60 seconds were recorded for each rat.
Hematoxylin and eosin staining of brain tissue After behavioral testing, three rats were selected randomly from the control, model, rhubarb-treated, and positive control groups for hematoxylin and eosin (H&E) staining of brain sections. Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1), then their thoracic cavities were opened to expose the heart. A needle was inserted from the apex of the heart, into the left ventricle, and from there into the ascending aorta, and the right atrium was incised to collect blood. Simultaneously, normal saline was quickly perfused. When the liquid emerging from the right atrium became clear and the liver turned white, pre-cooled 10% neutral formalin was infused until the neck and upper limbs became stiff. Then, the brain was quickly removed and fixed in 10% neutral formalin for subsequent sectioning and H&E staining. The histology of the nerve cells in each rat was then characterized by microscopic examination.
Quantification of neurotransmitters in brain tissue Preparation of brain tissue samples After 12 days of treatment, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1) and then decapitated. After craniotomy on ice, the whole brain was removed and the hippocampus quickly separated, weighed, and frozen at 80 C for subsequent analysis.2.9.2 Preparation of brain homogenates. Brain tissue was placed in test tubes and 0.1 mol$L1 perchloric acid containing 100 mmol$L1 ethylenediamine tetraacetic acid disodium salt was added at a volume ratio of 1:3 to deproteinize each sample. Whole brain and hippocampal homogenates were prepared in homogenizers, and then centrifuged twice at 13 523.2g at 4 C for 10 minutes, and the supernatant was collected. Twenty microliters of each supernatant were separated by HPLC. The neurotransmitter concentrations in each sample were calculated using a standard curve. Neurotransmitter standard curve construction Precise quantities of 5-HT and DA were weighed out and placed into a 5-mL volumetric bottle, in which they were dissolved in citric acid-sodium acetate, to create a 1 mg$mL1 standard solution for each neurotransmitter. This was diluted to yield 150, 300, 600, 1200, 2400, and
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4800 ng$mL1 solutions of the two neurotransmitters, which were refrigerated at 4 C.
indices. Rank-abundance curves directly reflected species abundance and evenness. The composition of the intestinal microbiome in the fecal samples of rats in the control, model, and rhubarb-treated groups were analyzed on the basis of genus taxonomy. The R tool was used to construct community pie charts to visually characterize the species of intestinal bacteria in the feces of rats in each group, and the proportion of each bacterial taxon in each group was calculated according to the relative abundance of each.
HPLC conditions for brain tissue samples An Acquity HPLC C18 column (4.6 mm 150 mm, 5 mm, Waters) was used to separate the brain samples. Mobile phase A comprised 50 mM citric acid-sodium acetate (pH 3.5), 1 mM B8 ion-pair reagent, 1.8 mM dibutylamine, and 0.3 mM ethylenediamine tetraacetic acid disodium salt, and mobile phase B was methanol. The volumes of mobile phases A and B were always kept at 97% and 3%, respectively in each elution. The flow rate was 0.8 mL$min1 and the detection voltage was þ0.75 V.
454 pyrosequencing of the V3eV4 regions of the 16S rRNA genes of the gut microbiome Fecal samples from three groups of rats were collected in 5mL sterilized tubes and snap-frozen in liquid nitrogen. Samples were stored at 80 C until further processed. The total genomic DNA of the gut microbiome was extracted from the fecal samples using an Omega-stool DNA Kit in accordance with the manufacturer’s protocol. The concentrated and purified DNA samples were quantified using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific), and the DNA quality was verified by 2% agarose gel electrophoresis. The V3eV4 regions of the 16S rRNA genes in the fecal samples were amplified using barcoded primers (ID-P2 [50 -sampleIDtag-ATTACCGCGGCTGCT3’] and ID-P3 [50 -sampleIDtag-CCTACGGGAGG CAGCAG-3’]) and a GeneAmp 9700 thermocycler (ABI). Purified amplicons were pooled in equimolar quantities and paired-end sequenced (2 300) on an Illumina MiSeq platform (Illumina), using the standard protocol of Majorbio Bio-Pharm Technology (Shanghai, China).
Analysis of the microbial population of fecal samples The sequences of DNA fragments obtained by paired-end sequencing on the Illumina platform were spliced into long reads within the highly variable region (V3eV4) on the basis of the overlaps between reads using PANDAseq. Low-quality data were filtered to remove reads with a mean mass value < 20 and those with more than three N bases, leaving only clean reads of 220e500 nucleotides in length. Alpha analysis was used to analyze the richness and diversity of microbial communities in fecal samples, and quantitative insights into microbial ecology software was used to evaluate the diversity of microbial communities with a series of Table 1
Statistical analysis The results were expressed as mean (standard deviation (SD)). One-way analysis of variance (ANOVA) was used to analyze the Morris water maze data, the levels of neurotransmitters, and the abundance of intestinal microbiota from 16S rRNA gene sequencing among the control, model, and rhubarb-treated groups. P < .05 was considered statistically significant. Analyses were carried out using SPSS18.0 software (IBM SPSS, Armonk, NY).
Results The learning and memory of HMA-AD rats was improved by rhubarb treatment Navigation training in a Morris water maze was used to evaluate the learning and memory of the rats with regard to spatial location and direction. The PLLs in each group gradually shortened with training time. To determine the effect of colonization with a human intestinal microbial population on cognitive function, the PLLs were compared between the conventional and control group rats. This showed that there was no significant difference in cognitive function between conventional rats and HMA-AD rats, indicating that colonization with human intestinal microbes had no effect on the learning or memory of the rats. The PLLs of rats in the model group were higher than those of the control group, and there were significant reductions in the PLLs from the third day of the experiment (P Z .018). The injection of Ab25e35 into the hippocampus causes cognitive impairment in rats.19 After drug intervention, the PLLs of the rhubarb-treated and positive control groups were also shorter than that of the model group, with significant differences appearing on the fourth day (P Z .0211 and .0003, respectively) (Table 1). Here, there was no significant difference in the PLL after 4 days of training between the positive control and rhubarb-treated groups, implying that rhubarb and donepezil were similarly effective.
Changes in the mean escape latency in each group during the 4 days of training.
Group
1st day (s)
2nd day (s)
3rd day (s)
4th day (s)
Conventional Group Control Group Model Group Rhubarb-treated Group Positive control Group
50.54 53.38 57.32 56.73 44.87
39.45 41.71 45.42 37.71 43.16
21.34 20.02 38.22 29.31 31.38
22.15 22.56 34.92 16.07 10.36
(0.89) (10.68) (0.68) (2.92) (20.05)
(11.54) (15.83) (17.38) (18.63) (15.62)
Note: Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; #P < .05 and
###
(7.65) (14.27) (20.91)* (18.71) (20.06)
(12.65) (15.01) (15.02)** (15.55)# (3.86)###
P < .001 vs the model group.
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In the spatial training in the Morris water maze, the frequency of crossing and the mean swimming speed were used to assess the formation and application of spatial memory of the rats. There was no significant difference in the frequency of the rats crossing the target quadrant between the conventional and control groups (P > .05), implying that colonization with healthy human intestinal microbes does not affect the cognitive function of normal rats. Compared with the control group, the frequency with which the rats crossed the target quadrant was significantly lower in the model group (P Z .0265), showing that the spatial memory of model rats was relatively poor. Compared with the model group, the frequencies with which the rats crossed the platform in the rhubarb-treated and positive control groups were significantly higher (P Z .005 and .0097, respectively), implying that administration of rhubarb ameliorates the Ab25e35-induced defect in spatial memory. However, the mean swimming speeds of rats in the rhubarb-treated and positive control groups were not significantly different from those of rats in the model group. Moreover, there were no significant differences in the frequency of rats crossing the platform or the mean swimming speeds between the rhubarb-treated and positive control groups (P > .05), implying that rhubarb and donepezil have similar effects to ameliorate the defect in spatial learning in rats with AD (Table 2).
Effects of rhubarb administration on the brain histology of HMA-AD rats The ‘Ab cascade hypothesis’ holds that the loss of neurons induced by high levels of Ab in brain tissue is one of the main factors leading to cognitive impairment. Therefore, microscopic inspection of H&E-stained sections was used to characterize the histology of brain neurons and to determine the effect of rhubarb on the apoptosis of neurons induced by Ab in the brains of rats with AD. In the control group, the brain tissue structure was clear and intact, the nerve cells were densely packed and orderly, and the glial cells were intact and well organized. There was no edema in the pericellular space and the neurons contained abundant lightly-stained cytoplasm. The neuronal nuclei were centrally located and obvious, with a uniform light blue and purple color. In comparison, rats in the model group demonstrated many fewer neurons in all the regions of the hippocampus. Moreover, the neuronal cytoplasm was
Table 2 Crossing frequency and mean swimming speed of each group of rats during the spatial training. Group
Frequency of Average swimming rats crossing speed (cm$sL1) the platform
Conventional Group Control Group Model Group Rhubarb-treated Group Positive control Group
7.34 7.11 5.50 7.44 7.67
(0.95) (1.37) (1.36)* (1.17)## (1.70)##
22.64 24.02 27.92 26.41 22.67
(1.87) (4.37) (5.61) (2.11) (2.03)
Note: Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; ##P < .01 vs the model group.
concentrated, the nucleus was pyknotic, and the cells were stained deep red. The neurons were also more disordered than those of rats in the control group, such that there were wider gaps between neurons, and some of them demonstrated cytoplasmic vacuolation. In rats that had been rhubarb-treated, the structure of parts of the brain was more normal, with the nerve cells showing greater organization. There were no observable differences between the positive control and model groups with regard to the neuronal histology of the brain (Fig. 1).
Effects of rhubarb on 5-HT and DA concentrations in the brains of HMA-AD rats 5-HT and DA are the major neurotransmitters involved in cognitive function. Clinical studies have shown that the levels of 5-HT and DA are low in the brain of AD patients.20 We measured the 5-HT and DA concentrations in the whole brain and hippocampus of rats in each group to delineate the mechanism whereby rhubarb might ameliorate ADassociated cognitive impairment. The concentrations of 5-HT and DA in the brain tissue of the model and control groups were similar (P > .05). After treatment with rhubarb or donepezil, the whole-brain concentrations of 5-HT tended to be higher (P > .05), but there were no effects of these substances on the wholebrain concentrations of DA (P > .05). The hippocampus plays a key role in learning and memory formation; specifically, it is responsible for the consolidation of short-term memory and its transformation into long-term memory. We measured the concentrations of 5-HT and DA as key mediators of learning and memory formation in the hippocampus. The results showed that the concentration of 5-HT in the hippocampus of the model group was significantly lower than that of the control group (P Z .0212). In rhubarb-treated rats, the concentration of 5-HT in the hippocampus was significantly higher compared with that in the model group (P Z .0013), as was that of the positive control group, but this difference did not reach significance (P > .05). Moreover, there was no significant difference in hippocampal 5-HT concentration between the positive control and rhubarb-treated groups (P > .05). The concentrations of DA in the hippocampi of each group were similar (P > .05) (Fig. 2).
Rhubarb administration influences the intestinal microbial structure in HMA-AD rats After high-throughput screening of the V3eV4 variable region sequence of the 16S rRNA genes in the fecal samples of rats in each group, 666 285 valid 16S rRNA gene sequences were obtained, with a mean of 39 193 (4589) per sample. The mean length of each sequence was 439 bp (range 423e486 bp). These sequences were classified as 270 operational taxonomic units (OTUs) at the 97% similarity level. Alpha diversity analysis of the sequencing data from the fecal samples showed that the Shannon diversity curves of bacterial species evenness and richness for each group were flattened (Fig. 3), indicating that the sequencing data adequately reflected the diversity of the major components of the bacterial population in each group.
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Figure 1 Histopathology of the rat brains (light microscope 40). (a) control group; (b) model group; (c) rhubarb-treated group; (d) positive control group.
Figure 2 Whole-brain and hippocampal concentrations of 5-HT and DA in each group. (a) The concentrations of 5-HT in the whole brain; (b) The concentrations of DA in the whole brain; (c) The concentrations of 5-HT in the hippocampus; (d) The concentrations of DA in the hippocampus. Note: 5-HT: 5-hydorxytryptamine; DA: dopamine. Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; ##P < .01 vs the model group.
Previous studies have shown that the intestinal microbial structure is disordered in AD.21 In the present study, norank_f_Bacteroidales_S24-7_group, Alloprevotella, Prevotella_1, Lactobacillus, and Prevotellaceae_UCG-001 were the dominant intestinal bacterial taxa in fecal
samples from rats in the control group, with respective percentages of 28.80%, 7.80%, 7.78%, 6.63%, and 6.15% (Fig. 4A). Norank_f_Bacteroidales_S24-7_group, Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, Prevotella_1, and Alloprevotella were the dominant intestinal
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Figure 3 Rarefaction curves for fecal samples from rats in each group. Abbreviations: CG: control group; Rhu_G: rhubarb-treated group; MG: model group.
H. Zhao et al. bacterial taxa in fecal samples from rats in the model group, with respective percentages of 24.91%, 9.00%, 6.86%, 6.05%, and 5.39% (Fig. 4B). Thus, the intestinal microbiome differed in the HMA-AD and control rats. However, Norank_f_Bacteroidales_S24-7_group, Lactobacillus, Prevotella_9, and unclassified_f_Lachnospiraceae were the dominant intestinal bacterial taxa in fecal samples from rats in the rhubarb-treated group, with percentages of 33.00%, 7.86%, 7.08%, and 5.40%, respectively (Fig. 4C), implying that rhubarb may be able to modify the intestinal microbial structure in AD. We next determined the intestinal microbes that influenced AD pathology and the bacterial genera that were modified by rhubarb administration. First, the differences in abundance of specific microbial taxa among the control, model, and rhubarb-treated groups were analyzed using the ANOVA test. We found that the Lachnospiraceae_NK4A136_group was more abundant, while Lactobacillus was less abundant in the model than in the control group (Fig. 5), which was consistent with previously reported findings.22 Thus, the pathologic changes associated with AD may be the result of changes in the abundance of
Figure 4 Intestinal microbial structure of three groups of rats, at the genus level. (a) control group; (b) model group; (c) rhubarbtreated group.
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the Lachnospiraceae_NK4A136_group and Lactobacillus in the intestinal tract of the host. Lactobacillus is known to be able to metabolize and synthesize neurotransmitters, such as 5-HT and g-aminobutyric acid, which affect the function of the central nervous system via neuroendocrine or vagal pathways that comprise the brain-intestinal axis.23 Rhubarb treatment was associated with lower abundance of the Lachnospiraceae_NK4A136_group and greater abundance of Lactobacillus in fecal samples from HMA-AD rats (Fig. 5). Thus, rhubarb may affect the metabolic behavior of intestinal bacteria and the cognitive function of the host by regulating the abundance of the Lachnospiraceae_NK4A136_group and Lactobacillus.
smaller number of hippocampal C3 neurons in the model group. Furthermore, the rats showed impaired learning and spatial memory, all of which are consistent with the successful establishment of a model of AD that can be used for mechanistic research. Rhubarb administration ameliorated the neuron loss in the AD rats, suggesting that it could significantly improve their learning and spatial memory, and it also had a protective effect on hippocampal neurons, suggesting that it might alleviate cognitive dysfunction. Rhubarb is used as a TCM with the action of unblocking the bowels and directing the turbid downward and can ameliorate cognitive impairment in AD, as shown in the present study, but the mechanism involved remains to be established. Because of accumulating evidence of a relationship between AD and the microbiome, many researchers are now assessing the influence of the intestinal microbiome on brain function. This interaction between intestinal microorganisms and the central nervous system is referred to as the ‘microbial-intestinal-brain’ axis. Researchers are now exploring the relationships between intestinal microbial diversity and the pathology of AD,28e30 and the intestinal microbiome has become a potential new target for the treatment of AD. It has been shown that rhubarb ingestion affects the composition and function of the intestinal microbiome. It can directly promote intestinal motility, facilitate the excretion of various toxins, prevent intestinal microbial translocation, reduce endotoxin absorption,31 and protect intestinal barrier function. It has also been shown to preserve the intestinal microbial balance,32 and thus the local microecological environment. Therefore, in the present study we determined whether rhubarb influences the intestinal microbial structure and whether this might be linked to amelioration in cognitive impairment, in a rat model of HMA-AD. We first clarified the effects of rhubarb on the intestinal microbiome in the rat model of HMA-AD. Much of the work regarding the microbiome has been conducted in animal models in which the composition of the microbiome can be manipulated.33 The intestinal microbiome of rodents is similar to that of humans at the phylum level, but there are substantial differences at the species level.34 Thus, the mechanisms of disease pathogenesis and drug action in rodents are likely to be quite different from those of humans. To circumvent this limitation, HMA models can be established by transplanting fecal microbes from human donors into gnotobiotic animals or animals treated with broad-spectrum antibiotics.35 In this way, some of the phenotypic characteristics of the donor can be reproduced in the animals, including the composition of the intestinal microbiome,36 and the metabolic phenotype of the HMA animal can also be transmitted to its offspring.37 Behavioral and pathologic characterization of the HMAAD rat model showed that features of AD developed, and the reproducibility of the model and its similarity to human AD have been verified systematically by our research team (these findings have recently been corroborated (XYG, PhD, unpublished data, July 2019)). The intestinal microecological environment of the model is similar to that of humans, which makes it suitable for studying the role of the intestinal microbiome in the mechanism, whereby rhubarb ameliorates the symptoms of AD.
Discussion AD is a primary degenerative disease of the central nervous system, which clinically manifests as deteriorations in cognitive function and memory.24 b-amyloid precursor protein is cleaved by b- and g-secretase to form Ab, which comprises peptides with a range of chain lengths, but Ab25e35 in particular is known to form toxic amyloid fibers. It has been reported that Ab25e35 can induce neuronal apoptosis in the hippocampus and cortex of rats in vivo,25 which causes cognitive impairment,26,27 and the intracranial administration of Ab25e35 has been used to induce AD in rodents. In the present study, we injected Ab25e35 into the hippocampus of rats and this induced brain neuronal injury detectable by light microscopic examination. This manifested as larger gaps between hippocampal neurons and a
Figure 5 Analysis of differences in the intestinal microbial composition of rats in the control, model, and rhubarb-treated groups. Note: one -way analysis of variance test was used. Red box: control group; blue box: rhubarb-treated group; green box: model group. CG: control group; Rhu_G: rhubarb-treated group; MG: model group.
242 We have shown in the present study that the intestinal microbiome of HMA-AD rats is disturbed. However, rhubarb administration returns the sizes of the Lactobacillus and Lachnospiraceae_NK4A136_group populations to normal. The Lachnospiraceae_NK4A136_group and Lactobacillus are common inhabitants of the human intestine. It has been reported that Lactobacillus is associated with central nervous system disease and specifically with AD.38 Early studies showed that mice lacking a microbiome often demonstrate cognitive impairment and anxiety, to varying degrees. However, when a Lactobacillus population is introduced, behavioral and cognitive functions are markedly improved. The underlying mechanism is that Lactobacillus can metabolize 5-HT, which improves the cognitive ability of the host. Further analysis of hippocampal tissue showed that this effect was mainly related to the expression of 5HT receptor 1A in the brain.39 Now, Lactobacillus is used as a probiotic for the prevention and treatment of AD in the clinic. In the present study, we have shown that the concentration of 5-HT in the hippocampus of HMA-AD model rats is significantly lower, suggesting that the injection of Ab25e35 into the hippocampus causes changes in the concentrations of neurotransmitters involved in cognition. However, the concentration of 5-HT in the hippocampus was significantly higher in rats administered rhubarb, suggesting that rhubarb may affect the level of 5-HT in the hippocampus by regulating the abundance of Lactobacillus. Lactobacillus can also metabolize glutamic acid (Glu) to produce g-aminobutyric acid (GABA), which has also been implicated in cognitive dysfunction.40 GABA and Glu maintain the balance of excitation and inhibition in the brain, which is important for consciousness, learning, and memory. Changes in GABA content and receptor expression can lead to neuronal damage in the hippocampus and vertebral body of AD patients. Glu is one of the most important excitatory neurotransmitters in the central nervous system, having strong excitatory effects on the cerebral cortex, hippocampus and thalamus. It also plays an important role in learning, memory, neuronal plasticity, and brain development. Thus, a low Glu level in the brain can cause longterm learning and memory impairment. However, a high Glu concentration can have neurotoxic effects, which are important components of the pathogenesis of AD.41 The present study has shown that rhubarb may be able to alleviate the cognitive impairment associated with AD and regulate the intestinal microbial structure in AD patients, but the potential causative mechanism linking the two requires further investigation.
Conclusion Rhubarb improves the cognitive dysfunction and increases the concentration of 5-HT in the hippocampus of HMA-AD rats, potentially by modulating the intestinal abundance of Lachnospiraceae_NK4A136_group and Lactobacillus.
Funding This work was financially supported by the National Natural Science Foundation of China (81673562).
H. Zhao et al.
Conflicts of interest The authors declare that no competing financial interests exist.
CRediT authorship contribution statement Huizhen Zhao: Conceptualization, data curation, and formal analysis, visualization, and writing e original draft. Demin Gao: Formal analysis, and writing e review & editing. Xiaoyan Gao: Conceptualization, methodology, funding acquisition, supervision and writing e review & writing.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtcms.2019.08.004.
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29. Carabotti M, Scirocco A, Maselli MA, Severi C. The gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems. Ann Gastroenterol. 2015;28(2): 203e209. 30. Foster JA, McVey Neufeld KA. Gutebrain axis: how the microbiome influences anxiety and depression. Trends Neurosci. 2013;36(5):305e312. 31. Yang L, Akao T, Kobashi K, Hattori M. Purification and characterization of a novel sennoside-hydrolyzing beta-glucosidase from Bifidobacterium sp. strain SEN, a human intestinal anaerobe. Biol Pharm Bull. 1996;19(5):705e709. 32. Belkaid Y, Hand TW. Role of the microbiota in immunity and inflammation. Cell. 2014;157(1):121e141. 33. Goodman AL, Kallstrom G, Faith JJ, et al. Extensive personal human gut microbiota culture collections characterized and manipulated in gnotobiotic mice. Proc Natl Acad Sci USA. 2011; 108(15):6252e6257. 34. Dethlefsen L, McFall-Ngai M, Relman DA. An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature. 2007;449(7164):811e818. 35. Arrieta MC, Walter J, Finlay BB. Human microbiota-associated mice: a model with challenges. Cell Host Microbe. 2016;19(5): 575e578. 36. Marcobal A, Kashyap PC, Nelson TA, et al. A metabolomic view of how the human gut microbiota impacts the host metabolome using humanized and gnotobiotic mice. ISME J. 2013; 7(10):1933e1943. 37. Edwards CA, Rumney C, Davies M, et al. A human floraassociated rat model of the breast-fed infant gut. J Pediatr Gastroenterol Nutr. 2003;37(2):168e177. 38. Wang HY, Lee IS, Braun C, Enck P. Effect of probiotics on central nervous system functions in animals and humans: a systematic review. J Neurogastroenterol Motil. 2016;22(4):589e605. 39. Caracciolo B, Xu WL, Collins S, Fratiglioni L. Cognitive decline, dietary factors and gut-brain interactions. Mech Ageing Dev; 2014. https://doi.org/10.1016/j.mad.2013.11.011. 40. Bravo JA, Forsythe P, Chew MV, et al. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA. 2011;108(38):16050e16055. 41. Riese F, Gietl A, Zo ¨lch N, et al. Posterior cingulate g-aminobutyric acid and glutamate/glutamine are reduced in amnestic mild cognitive impairment and are unrelated to amyloid deposition and apolipoprotein E genotype. Neurobiol Aging. 2015;36(1):53e59.
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Rhubarb ameliorates cognitive dysfunction in a rat model of Alzheimer’s disease through regulation of the intestinal microbiome Huizhen Zhao, Demin Gao, Xiaoyan Gao* School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, 102488, China Received 26 June 2019; received in revised form 10 August 2019; accepted 10 August 2019
Available online 16 August 2019
KEYWORDS Rhubarb; Intestinal microbiome; Alzheimer’s disease; Cognitive function; 5-hydroxytryptamine
Abstract Objective: To determine the mechanism whereby rhubarb (Rheum tanguticum MAXIM. Ex BALF.) may ameliorate cognitive dysfunction through regulation of the intestinal microbiome. Methods: We used a rat model of human microbiome-associated (HMA)-AD to characterize the therapeutic effect of rhubarb on cognitive dysfunction by assessing learning and spatial memory, tissue pathology, and neurotransmitter expression in brain tissue. Then, 16S rRNA gene sequencing was used to analyze the intestinal microbial composition of the rats before and after rhubarb intervention, to determine whether changes in the intestinal microbiome might underpin the beneficial effect of rhubarb on cognitive dysfunction. Results: Morris water maze experiments showed that the learning and spatial memory of HMAAD rats were improved after rhubarb administration. Examination of brain sections showed that rhubarb had a protective effect on neurons in the brain tissue of HMA-AD rats. Brain tissue neurotransmitter analysis showed that rhubarb significantly reduces the 5-hydorxytryptamine concentration in the hippocampus of HMA-AD rats (P Z .0013). Furthermore, rhubarb affected the abundance of the Lachnospiraceae_NK4A136_group and Lactobacillus in the large intestine. Conclusion: This study suggests that rhubarb ameliorates cognitive dysfunction in rats with HMA-AD by regulating the abundance of beneficial bacteria, which likely affects the concentration of 5-hydorxytryptamine in the hippocampus. ª 2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
* Corresponding author. E-mail address: [email protected] (X. Gao). Peer review under responsibility of Beijing University of Chinese Medicine. https://doi.org/10.1016/j.jtcms.2019.08.004 2095-7548/ª 2019 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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Introduction
Materials
Alzheimer’s disease (AD), the most common form of dementia, is a degenerative disease of the central nervous system characterized by progressive memory loss, cognitive impairment, and personality changes. According to the ‘World 2018 AD Report’ issued by the International Alzheimer’s Association, there were about 50 million patients with AD worldwide in 2018, and the incidence of AD was rapidly increasing,1 such that it represents a serious threat to human health and social development. Microbial dysbiosis may mediate or affect the pathogenesis of AD, because the microbiome and its metabolites might affect signaling pathways2e4 and the production of proinflammatory cytokines associated with the pathogenesis of AD.5e8 Therefore, targeting of the intestinal microbiome may provide a means of preventing and/or treating AD. Rhubarb (Rheum tanguticum MAXIM. Ex BALF.) is obtained from the roots and rhizomes of Rheum officinale Baills., R Palmatum L., and R. tanguticum Maxim. ex Baill., which is a laxative with actions of unblocking the bowels and directing the turbid downward, clearing heat and purging fire, detoxifying, activating blood circulation and dissolving blood stasis, as recorded in the Pharmacopoeia of the People’s Republic of China.9 It is also indicated for the treatment of dementia as a traditional Chinese medicine (TCM), targeting phlegm and blood stasis obstructing collaterals and poisoning brain collaterals.10 It is one of the most common TCMs used for the treatment of AD. However, although a therapeutic effect is reported to have been achieved in the clinic,11 the mechanism involved in the amelioration of AD remains to be established. A limited number of studies in vitro have shown that anthraquinones, which are found in rhubarb, can reduce the deposition of Ab and Tau proteins,12,13 can reduce the inflammatory response of nerve cells,14 and are neuroprotective.15 There has been recent interest in the interactions between TCMs and the intestinal microbiome, and accumulating evidence suggests that rhubarb, and especially anthraquinones, affect the composition and function of the intestinal microbiome.16,17 Therefore, we hypothesized that rhubarb may ameliorate the symptoms of AD by correcting the associated intestinal dysbiosis. To address this hypothesis, we established a human microbiome-associated rat model of AD (HMA-AD), in which the intestinal environment of the rats would mirror the human intestinal environment as closely as possible. The effects of rhubarb were investigated in this model with respect to learning and spatial memory, tissue pathology, and neurotransmitter expression in brain tissue. The structure of the intestinal microbiome before and after rhubarb administration, and the effect of rhubarb on the intestinal microbiome of the rat model of AD were analyzed.
We used a high performance liquid chromatograph (Alliance 2695, Waters, Milford, MA) with an electrochemical detector (ECD2465, Waters), a Morris water maze (Institute of Chinese Materia Medica, Chinese Academy of Chinese Medical Sciences, Beijing, China), centrifuges (N13462C, 5430R, and 5424R, Eppendorf, Hamburg, Germany), an ultramicrospectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA), an electrophoresis instrument (DYY-6C, Beijing Liuyi Biotechnology, Beijing, China), a PCR cycler (ABI GeneAmp 9700, Thermo Fisher Scientific), MISEQ and HISEQ sequencers (Illumina hiseq, Illumina, San Diego, CA), a microplate reader (BioTek ELx800, Biotek, Winooski, Vermont), a microfluorometer (TBS380, TurnerBioSystems, Sunnyvale, CA), a focused-ultrasonicator (Covaris M220, Gene, Hong Kong, China), vortex mixers (QL-901, Kylin-Bell Lab Instruments, Haimen, China), a grinding instrument (TL-48R, Wonbio, Shanghai, China), and DNA extraction kits (OMEGA-soil DNA Kit, Omega Bio-Tek, Norcross, GA). Donepezil hydrochloride was supplied by Eisai (Suzhou, China). Rhubarb herbal medicine was provided by the Modern Research Center for TCMs (Peking University, Beijing, China) and had been authenticated by Professor Pengfei Tu (Peking University) and tested using high-performance liquid chromatography (HPLC; Supplementary Fig. 1). Imipenem and cilastatin sodium were obtained from Merck Sharp & Dohme (Kenilworth, NJ), Ab25e35 (907532V) and 5hydorxytryptamine (5-HT; 091M5163V) from Sigma (Burlington, MA), dopamine (DA; 100070e201507) from the National Institutes for Food and Drug Control (Beijing, China), 2% agarose (50164) from Biowest (Madrid, Spain), Fast Pfu (120428) from TransGen (Beijing, China), and AxyPrep DNA Gel Extraction Kits (AP-GX-250) from Axygen (Tewksbury, MA).
Materials and methods
Experimental animals Three-week-old male specific pathogen-free Sprague-Dawley rats, weighing 70 5 g, were kept in the barrier facility of Beijing University of Chinese Medicine. The rats were maintained at 20 2 C, with a relative humidity of 60% 5%, and under a 12-hour light/dark cycle, and were acclimated for one week prior to the commencement of the study. Standard chow and Milli-Q water were provided ad libitum. All rats were fasted overnight, with free access to water, before drug administration. All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals. Rats were allocated randomly to a conventional group, a control group, a model group, a rhubarb-treated group, and a positive control group (n Z 10 per group).
Establishment of a human microbiome-associated rat model of AD
Ethical approval The study protocol was approved by the Ethics Committee for Animal Care and Treatment of Beijing University of Chinese Medicine (BUCM-4-2018122801-4066).
Conventional group rats were maintained without any treatment. Microbe-free rats were obtained by gavaging the control, model, rhubarb-treated, and positive control groups with imipenem and cilastatin sodium (5 mg$mL1).
236 Humanization of the gut microbiome was then performed using human fecal samples collected from two healthy male donors. Fresh feces were diluted in nine volumes of anaerobic medium and 1.5 mL of fecal suspension was administered to the four groups of rats by gavage, once per day for 3 weeks. These rats were then fed a standard diet for 1 week, by which time a stable HMA-AD rat model had been established. After this, rats in the model, rhubarbtreated, and positive control groups were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1), prior to the attachment of a stereotaxic apparatus and exposure of the anterior fontanelle. Each rat was injected with 2.5 mL of an Ab25e35 (2 mg$mL1) into the right lateral ventricle, according to the rat brain stereotaxic map. The coordinates used were 1.2 mm caudal to the anterior fontanelle and 1.2 mm to the right side of the sagittal suture. The needle was inserted 3.2 mm deep to the surface of skull, remained in place for 8 min, and was extracted at 1 mm$min1. The rats were kept in their cage to recover. The rats in the control group were similarly injected with the same volume of saline.
Preparation of drug solutions Small pieces of rhubarb herbal medicine (w30 g) were weighed and soaked in 300 mL distilled water for 1 hour and decocted twice in boiling water for 30 minutes on each occasion. The combined products of this water decoction were filtered and dried in a 60 C water bath, then made up to 50 mL with ultrapure water, to yield a 0.6 g$mL1 solution. Donepezil hydrochloride solution was prepared by crushing one tablet, containing 5 mg donepezil hydrochloride, and dissolving it in 100 mL of normal saline by ultrasonication for 30 minutes, to obtain a 0.05 mg$mL1 solution.
Administration of treatments After the intracranial administration of Ab25e35, rhubarb (2.1 g$kg1) or donepezil hydrochloride solution (0.6 mg$kg1) were orally administered to the rats in the rhubarb-treated and positive control groups, respectively, for 12 days. Normal saline (1 mL) was similarly administered to rats in the control and model groups.
Morris water maze test Morris water maze testing was conducted as previously described,18 for rats in the conventional, control, model, rhubarb-treated, and positive control groups from 8 days after intracranial injection of Ab25e35. Navigation training was conducted on days 8e11 of the interventions. Each trial started 30 minutes after drug administration, with the rat being placed against the edge of the pool, which was divided into four virtual quadrants. Each rat underwent a trial every half-day for eight successive half-days, making a total of eight trials over 4 successive days. The starting positions for the eight trials were evenly distributed among the four quadrants. Each rat was given 60 seconds to locate the platform during each trial. The platform locating latency (PLL) was
H. Zhao et al. defined as the time taken for the experimental animal to locate and ascend the platform. If the rat had failed to ascend the platform within 60 seconds, it was guided to the platform and allowed to stay here for 30 seconds, which should have assisted with knowledge acquisition and memory formation, and in these instances the PLL was recorded as 60 seconds. After navigation training, spatial training was performed on day 12 of the intervention, in which the platform was removed and the rats were placed against the edge of the pool, as before. Then, the swimming time and the number of times the rat crossed the quadrant where the original platform was located, over 60 seconds were recorded for each rat.
Hematoxylin and eosin staining of brain tissue After behavioral testing, three rats were selected randomly from the control, model, rhubarb-treated, and positive control groups for hematoxylin and eosin (H&E) staining of brain sections. Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1), then their thoracic cavities were opened to expose the heart. A needle was inserted from the apex of the heart, into the left ventricle, and from there into the ascending aorta, and the right atrium was incised to collect blood. Simultaneously, normal saline was quickly perfused. When the liquid emerging from the right atrium became clear and the liver turned white, pre-cooled 10% neutral formalin was infused until the neck and upper limbs became stiff. Then, the brain was quickly removed and fixed in 10% neutral formalin for subsequent sectioning and H&E staining. The histology of the nerve cells in each rat was then characterized by microscopic examination.
Quantification of neurotransmitters in brain tissue Preparation of brain tissue samples After 12 days of treatment, rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg$kg1) and then decapitated. After craniotomy on ice, the whole brain was removed and the hippocampus quickly separated, weighed, and frozen at 80 C for subsequent analysis.2.9.2 Preparation of brain homogenates. Brain tissue was placed in test tubes and 0.1 mol$L1 perchloric acid containing 100 mmol$L1 ethylenediamine tetraacetic acid disodium salt was added at a volume ratio of 1:3 to deproteinize each sample. Whole brain and hippocampal homogenates were prepared in homogenizers, and then centrifuged twice at 13 523.2g at 4 C for 10 minutes, and the supernatant was collected. Twenty microliters of each supernatant were separated by HPLC. The neurotransmitter concentrations in each sample were calculated using a standard curve. Neurotransmitter standard curve construction Precise quantities of 5-HT and DA were weighed out and placed into a 5-mL volumetric bottle, in which they were dissolved in citric acid-sodium acetate, to create a 1 mg$mL1 standard solution for each neurotransmitter. This was diluted to yield 150, 300, 600, 1200, 2400, and
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4800 ng$mL1 solutions of the two neurotransmitters, which were refrigerated at 4 C.
indices. Rank-abundance curves directly reflected species abundance and evenness. The composition of the intestinal microbiome in the fecal samples of rats in the control, model, and rhubarb-treated groups were analyzed on the basis of genus taxonomy. The R tool was used to construct community pie charts to visually characterize the species of intestinal bacteria in the feces of rats in each group, and the proportion of each bacterial taxon in each group was calculated according to the relative abundance of each.
HPLC conditions for brain tissue samples An Acquity HPLC C18 column (4.6 mm 150 mm, 5 mm, Waters) was used to separate the brain samples. Mobile phase A comprised 50 mM citric acid-sodium acetate (pH 3.5), 1 mM B8 ion-pair reagent, 1.8 mM dibutylamine, and 0.3 mM ethylenediamine tetraacetic acid disodium salt, and mobile phase B was methanol. The volumes of mobile phases A and B were always kept at 97% and 3%, respectively in each elution. The flow rate was 0.8 mL$min1 and the detection voltage was þ0.75 V.
454 pyrosequencing of the V3eV4 regions of the 16S rRNA genes of the gut microbiome Fecal samples from three groups of rats were collected in 5mL sterilized tubes and snap-frozen in liquid nitrogen. Samples were stored at 80 C until further processed. The total genomic DNA of the gut microbiome was extracted from the fecal samples using an Omega-stool DNA Kit in accordance with the manufacturer’s protocol. The concentrated and purified DNA samples were quantified using a NanoDrop 2000 UV-vis spectrophotometer (Thermo Fisher Scientific), and the DNA quality was verified by 2% agarose gel electrophoresis. The V3eV4 regions of the 16S rRNA genes in the fecal samples were amplified using barcoded primers (ID-P2 [50 -sampleIDtag-ATTACCGCGGCTGCT3’] and ID-P3 [50 -sampleIDtag-CCTACGGGAGG CAGCAG-3’]) and a GeneAmp 9700 thermocycler (ABI). Purified amplicons were pooled in equimolar quantities and paired-end sequenced (2 300) on an Illumina MiSeq platform (Illumina), using the standard protocol of Majorbio Bio-Pharm Technology (Shanghai, China).
Analysis of the microbial population of fecal samples The sequences of DNA fragments obtained by paired-end sequencing on the Illumina platform were spliced into long reads within the highly variable region (V3eV4) on the basis of the overlaps between reads using PANDAseq. Low-quality data were filtered to remove reads with a mean mass value < 20 and those with more than three N bases, leaving only clean reads of 220e500 nucleotides in length. Alpha analysis was used to analyze the richness and diversity of microbial communities in fecal samples, and quantitative insights into microbial ecology software was used to evaluate the diversity of microbial communities with a series of Table 1
Statistical analysis The results were expressed as mean (standard deviation (SD)). One-way analysis of variance (ANOVA) was used to analyze the Morris water maze data, the levels of neurotransmitters, and the abundance of intestinal microbiota from 16S rRNA gene sequencing among the control, model, and rhubarb-treated groups. P < .05 was considered statistically significant. Analyses were carried out using SPSS18.0 software (IBM SPSS, Armonk, NY).
Results The learning and memory of HMA-AD rats was improved by rhubarb treatment Navigation training in a Morris water maze was used to evaluate the learning and memory of the rats with regard to spatial location and direction. The PLLs in each group gradually shortened with training time. To determine the effect of colonization with a human intestinal microbial population on cognitive function, the PLLs were compared between the conventional and control group rats. This showed that there was no significant difference in cognitive function between conventional rats and HMA-AD rats, indicating that colonization with human intestinal microbes had no effect on the learning or memory of the rats. The PLLs of rats in the model group were higher than those of the control group, and there were significant reductions in the PLLs from the third day of the experiment (P Z .018). The injection of Ab25e35 into the hippocampus causes cognitive impairment in rats.19 After drug intervention, the PLLs of the rhubarb-treated and positive control groups were also shorter than that of the model group, with significant differences appearing on the fourth day (P Z .0211 and .0003, respectively) (Table 1). Here, there was no significant difference in the PLL after 4 days of training between the positive control and rhubarb-treated groups, implying that rhubarb and donepezil were similarly effective.
Changes in the mean escape latency in each group during the 4 days of training.
Group
1st day (s)
2nd day (s)
3rd day (s)
4th day (s)
Conventional Group Control Group Model Group Rhubarb-treated Group Positive control Group
50.54 53.38 57.32 56.73 44.87
39.45 41.71 45.42 37.71 43.16
21.34 20.02 38.22 29.31 31.38
22.15 22.56 34.92 16.07 10.36
(0.89) (10.68) (0.68) (2.92) (20.05)
(11.54) (15.83) (17.38) (18.63) (15.62)
Note: Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; #P < .05 and
###
(7.65) (14.27) (20.91)* (18.71) (20.06)
(12.65) (15.01) (15.02)** (15.55)# (3.86)###
P < .001 vs the model group.
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In the spatial training in the Morris water maze, the frequency of crossing and the mean swimming speed were used to assess the formation and application of spatial memory of the rats. There was no significant difference in the frequency of the rats crossing the target quadrant between the conventional and control groups (P > .05), implying that colonization with healthy human intestinal microbes does not affect the cognitive function of normal rats. Compared with the control group, the frequency with which the rats crossed the target quadrant was significantly lower in the model group (P Z .0265), showing that the spatial memory of model rats was relatively poor. Compared with the model group, the frequencies with which the rats crossed the platform in the rhubarb-treated and positive control groups were significantly higher (P Z .005 and .0097, respectively), implying that administration of rhubarb ameliorates the Ab25e35-induced defect in spatial memory. However, the mean swimming speeds of rats in the rhubarb-treated and positive control groups were not significantly different from those of rats in the model group. Moreover, there were no significant differences in the frequency of rats crossing the platform or the mean swimming speeds between the rhubarb-treated and positive control groups (P > .05), implying that rhubarb and donepezil have similar effects to ameliorate the defect in spatial learning in rats with AD (Table 2).
Effects of rhubarb administration on the brain histology of HMA-AD rats The ‘Ab cascade hypothesis’ holds that the loss of neurons induced by high levels of Ab in brain tissue is one of the main factors leading to cognitive impairment. Therefore, microscopic inspection of H&E-stained sections was used to characterize the histology of brain neurons and to determine the effect of rhubarb on the apoptosis of neurons induced by Ab in the brains of rats with AD. In the control group, the brain tissue structure was clear and intact, the nerve cells were densely packed and orderly, and the glial cells were intact and well organized. There was no edema in the pericellular space and the neurons contained abundant lightly-stained cytoplasm. The neuronal nuclei were centrally located and obvious, with a uniform light blue and purple color. In comparison, rats in the model group demonstrated many fewer neurons in all the regions of the hippocampus. Moreover, the neuronal cytoplasm was
Table 2 Crossing frequency and mean swimming speed of each group of rats during the spatial training. Group
Frequency of Average swimming rats crossing speed (cm$sL1) the platform
Conventional Group Control Group Model Group Rhubarb-treated Group Positive control Group
7.34 7.11 5.50 7.44 7.67
(0.95) (1.37) (1.36)* (1.17)## (1.70)##
22.64 24.02 27.92 26.41 22.67
(1.87) (4.37) (5.61) (2.11) (2.03)
Note: Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; ##P < .01 vs the model group.
concentrated, the nucleus was pyknotic, and the cells were stained deep red. The neurons were also more disordered than those of rats in the control group, such that there were wider gaps between neurons, and some of them demonstrated cytoplasmic vacuolation. In rats that had been rhubarb-treated, the structure of parts of the brain was more normal, with the nerve cells showing greater organization. There were no observable differences between the positive control and model groups with regard to the neuronal histology of the brain (Fig. 1).
Effects of rhubarb on 5-HT and DA concentrations in the brains of HMA-AD rats 5-HT and DA are the major neurotransmitters involved in cognitive function. Clinical studies have shown that the levels of 5-HT and DA are low in the brain of AD patients.20 We measured the 5-HT and DA concentrations in the whole brain and hippocampus of rats in each group to delineate the mechanism whereby rhubarb might ameliorate ADassociated cognitive impairment. The concentrations of 5-HT and DA in the brain tissue of the model and control groups were similar (P > .05). After treatment with rhubarb or donepezil, the whole-brain concentrations of 5-HT tended to be higher (P > .05), but there were no effects of these substances on the wholebrain concentrations of DA (P > .05). The hippocampus plays a key role in learning and memory formation; specifically, it is responsible for the consolidation of short-term memory and its transformation into long-term memory. We measured the concentrations of 5-HT and DA as key mediators of learning and memory formation in the hippocampus. The results showed that the concentration of 5-HT in the hippocampus of the model group was significantly lower than that of the control group (P Z .0212). In rhubarb-treated rats, the concentration of 5-HT in the hippocampus was significantly higher compared with that in the model group (P Z .0013), as was that of the positive control group, but this difference did not reach significance (P > .05). Moreover, there was no significant difference in hippocampal 5-HT concentration between the positive control and rhubarb-treated groups (P > .05). The concentrations of DA in the hippocampi of each group were similar (P > .05) (Fig. 2).
Rhubarb administration influences the intestinal microbial structure in HMA-AD rats After high-throughput screening of the V3eV4 variable region sequence of the 16S rRNA genes in the fecal samples of rats in each group, 666 285 valid 16S rRNA gene sequences were obtained, with a mean of 39 193 (4589) per sample. The mean length of each sequence was 439 bp (range 423e486 bp). These sequences were classified as 270 operational taxonomic units (OTUs) at the 97% similarity level. Alpha diversity analysis of the sequencing data from the fecal samples showed that the Shannon diversity curves of bacterial species evenness and richness for each group were flattened (Fig. 3), indicating that the sequencing data adequately reflected the diversity of the major components of the bacterial population in each group.
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Figure 1 Histopathology of the rat brains (light microscope 40). (a) control group; (b) model group; (c) rhubarb-treated group; (d) positive control group.
Figure 2 Whole-brain and hippocampal concentrations of 5-HT and DA in each group. (a) The concentrations of 5-HT in the whole brain; (b) The concentrations of DA in the whole brain; (c) The concentrations of 5-HT in the hippocampus; (d) The concentrations of DA in the hippocampus. Note: 5-HT: 5-hydorxytryptamine; DA: dopamine. Data are expressed as mean (SD). N Z 10. *P < .05 vs the control group; ##P < .01 vs the model group.
Previous studies have shown that the intestinal microbial structure is disordered in AD.21 In the present study, norank_f_Bacteroidales_S24-7_group, Alloprevotella, Prevotella_1, Lactobacillus, and Prevotellaceae_UCG-001 were the dominant intestinal bacterial taxa in fecal
samples from rats in the control group, with respective percentages of 28.80%, 7.80%, 7.78%, 6.63%, and 6.15% (Fig. 4A). Norank_f_Bacteroidales_S24-7_group, Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, Prevotella_1, and Alloprevotella were the dominant intestinal
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Figure 3 Rarefaction curves for fecal samples from rats in each group. Abbreviations: CG: control group; Rhu_G: rhubarb-treated group; MG: model group.
H. Zhao et al. bacterial taxa in fecal samples from rats in the model group, with respective percentages of 24.91%, 9.00%, 6.86%, 6.05%, and 5.39% (Fig. 4B). Thus, the intestinal microbiome differed in the HMA-AD and control rats. However, Norank_f_Bacteroidales_S24-7_group, Lactobacillus, Prevotella_9, and unclassified_f_Lachnospiraceae were the dominant intestinal bacterial taxa in fecal samples from rats in the rhubarb-treated group, with percentages of 33.00%, 7.86%, 7.08%, and 5.40%, respectively (Fig. 4C), implying that rhubarb may be able to modify the intestinal microbial structure in AD. We next determined the intestinal microbes that influenced AD pathology and the bacterial genera that were modified by rhubarb administration. First, the differences in abundance of specific microbial taxa among the control, model, and rhubarb-treated groups were analyzed using the ANOVA test. We found that the Lachnospiraceae_NK4A136_group was more abundant, while Lactobacillus was less abundant in the model than in the control group (Fig. 5), which was consistent with previously reported findings.22 Thus, the pathologic changes associated with AD may be the result of changes in the abundance of
Figure 4 Intestinal microbial structure of three groups of rats, at the genus level. (a) control group; (b) model group; (c) rhubarbtreated group.
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the Lachnospiraceae_NK4A136_group and Lactobacillus in the intestinal tract of the host. Lactobacillus is known to be able to metabolize and synthesize neurotransmitters, such as 5-HT and g-aminobutyric acid, which affect the function of the central nervous system via neuroendocrine or vagal pathways that comprise the brain-intestinal axis.23 Rhubarb treatment was associated with lower abundance of the Lachnospiraceae_NK4A136_group and greater abundance of Lactobacillus in fecal samples from HMA-AD rats (Fig. 5). Thus, rhubarb may affect the metabolic behavior of intestinal bacteria and the cognitive function of the host by regulating the abundance of the Lachnospiraceae_NK4A136_group and Lactobacillus.
smaller number of hippocampal C3 neurons in the model group. Furthermore, the rats showed impaired learning and spatial memory, all of which are consistent with the successful establishment of a model of AD that can be used for mechanistic research. Rhubarb administration ameliorated the neuron loss in the AD rats, suggesting that it could significantly improve their learning and spatial memory, and it also had a protective effect on hippocampal neurons, suggesting that it might alleviate cognitive dysfunction. Rhubarb is used as a TCM with the action of unblocking the bowels and directing the turbid downward and can ameliorate cognitive impairment in AD, as shown in the present study, but the mechanism involved remains to be established. Because of accumulating evidence of a relationship between AD and the microbiome, many researchers are now assessing the influence of the intestinal microbiome on brain function. This interaction between intestinal microorganisms and the central nervous system is referred to as the ‘microbial-intestinal-brain’ axis. Researchers are now exploring the relationships between intestinal microbial diversity and the pathology of AD,28e30 and the intestinal microbiome has become a potential new target for the treatment of AD. It has been shown that rhubarb ingestion affects the composition and function of the intestinal microbiome. It can directly promote intestinal motility, facilitate the excretion of various toxins, prevent intestinal microbial translocation, reduce endotoxin absorption,31 and protect intestinal barrier function. It has also been shown to preserve the intestinal microbial balance,32 and thus the local microecological environment. Therefore, in the present study we determined whether rhubarb influences the intestinal microbial structure and whether this might be linked to amelioration in cognitive impairment, in a rat model of HMA-AD. We first clarified the effects of rhubarb on the intestinal microbiome in the rat model of HMA-AD. Much of the work regarding the microbiome has been conducted in animal models in which the composition of the microbiome can be manipulated.33 The intestinal microbiome of rodents is similar to that of humans at the phylum level, but there are substantial differences at the species level.34 Thus, the mechanisms of disease pathogenesis and drug action in rodents are likely to be quite different from those of humans. To circumvent this limitation, HMA models can be established by transplanting fecal microbes from human donors into gnotobiotic animals or animals treated with broad-spectrum antibiotics.35 In this way, some of the phenotypic characteristics of the donor can be reproduced in the animals, including the composition of the intestinal microbiome,36 and the metabolic phenotype of the HMA animal can also be transmitted to its offspring.37 Behavioral and pathologic characterization of the HMAAD rat model showed that features of AD developed, and the reproducibility of the model and its similarity to human AD have been verified systematically by our research team (these findings have recently been corroborated (XYG, PhD, unpublished data, July 2019)). The intestinal microecological environment of the model is similar to that of humans, which makes it suitable for studying the role of the intestinal microbiome in the mechanism, whereby rhubarb ameliorates the symptoms of AD.
Discussion AD is a primary degenerative disease of the central nervous system, which clinically manifests as deteriorations in cognitive function and memory.24 b-amyloid precursor protein is cleaved by b- and g-secretase to form Ab, which comprises peptides with a range of chain lengths, but Ab25e35 in particular is known to form toxic amyloid fibers. It has been reported that Ab25e35 can induce neuronal apoptosis in the hippocampus and cortex of rats in vivo,25 which causes cognitive impairment,26,27 and the intracranial administration of Ab25e35 has been used to induce AD in rodents. In the present study, we injected Ab25e35 into the hippocampus of rats and this induced brain neuronal injury detectable by light microscopic examination. This manifested as larger gaps between hippocampal neurons and a
Figure 5 Analysis of differences in the intestinal microbial composition of rats in the control, model, and rhubarb-treated groups. Note: one -way analysis of variance test was used. Red box: control group; blue box: rhubarb-treated group; green box: model group. CG: control group; Rhu_G: rhubarb-treated group; MG: model group.
242 We have shown in the present study that the intestinal microbiome of HMA-AD rats is disturbed. However, rhubarb administration returns the sizes of the Lactobacillus and Lachnospiraceae_NK4A136_group populations to normal. The Lachnospiraceae_NK4A136_group and Lactobacillus are common inhabitants of the human intestine. It has been reported that Lactobacillus is associated with central nervous system disease and specifically with AD.38 Early studies showed that mice lacking a microbiome often demonstrate cognitive impairment and anxiety, to varying degrees. However, when a Lactobacillus population is introduced, behavioral and cognitive functions are markedly improved. The underlying mechanism is that Lactobacillus can metabolize 5-HT, which improves the cognitive ability of the host. Further analysis of hippocampal tissue showed that this effect was mainly related to the expression of 5HT receptor 1A in the brain.39 Now, Lactobacillus is used as a probiotic for the prevention and treatment of AD in the clinic. In the present study, we have shown that the concentration of 5-HT in the hippocampus of HMA-AD model rats is significantly lower, suggesting that the injection of Ab25e35 into the hippocampus causes changes in the concentrations of neurotransmitters involved in cognition. However, the concentration of 5-HT in the hippocampus was significantly higher in rats administered rhubarb, suggesting that rhubarb may affect the level of 5-HT in the hippocampus by regulating the abundance of Lactobacillus. Lactobacillus can also metabolize glutamic acid (Glu) to produce g-aminobutyric acid (GABA), which has also been implicated in cognitive dysfunction.40 GABA and Glu maintain the balance of excitation and inhibition in the brain, which is important for consciousness, learning, and memory. Changes in GABA content and receptor expression can lead to neuronal damage in the hippocampus and vertebral body of AD patients. Glu is one of the most important excitatory neurotransmitters in the central nervous system, having strong excitatory effects on the cerebral cortex, hippocampus and thalamus. It also plays an important role in learning, memory, neuronal plasticity, and brain development. Thus, a low Glu level in the brain can cause longterm learning and memory impairment. However, a high Glu concentration can have neurotoxic effects, which are important components of the pathogenesis of AD.41 The present study has shown that rhubarb may be able to alleviate the cognitive impairment associated with AD and regulate the intestinal microbial structure in AD patients, but the potential causative mechanism linking the two requires further investigation.
Conclusion Rhubarb improves the cognitive dysfunction and increases the concentration of 5-HT in the hippocampus of HMA-AD rats, potentially by modulating the intestinal abundance of Lachnospiraceae_NK4A136_group and Lactobacillus.
Funding This work was financially supported by the National Natural Science Foundation of China (81673562).
H. Zhao et al.
Conflicts of interest The authors declare that no competing financial interests exist.
CRediT authorship contribution statement Huizhen Zhao: Conceptualization, data curation, and formal analysis, visualization, and writing e original draft. Demin Gao: Formal analysis, and writing e review & editing. Xiaoyan Gao: Conceptualization, methodology, funding acquisition, supervision and writing e review & writing.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.jtcms.2019.08.004.
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