Cistanche Deserticolapolysaccharides Alleviate Cognitive Decline in Aging Model Mice By Restoring The Gut Microbiota-brain Axis

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ABSTRACT

Recent evidence suggests alterations in the gut microbiota-brain axis may drive cognitive impairment with aging. In the present study, we observed that prolonged administration of D-galactose to mice induced cognitive decline, gut microbial dysbiosis, peripheral inflammation, and oxidative stress. In this model of age-related cognitive decline, Cistanche deserticola polysaccharides (CDPS) improved cognitive function in Dgalactose-treated mice by restoring gut microbial homeostasis, thereby reducing oxidative stress and peripheral inflammation. The beneficial effects of CDPS in these aging model mice were abolished through ablation of gut microbiota with antibiotics or immunosuppression with cyclophosphamide. Serum metabolomic profiling showed that levels of creatinine, valine, L-methionine, o-Toluidine, N-ethylaniline, uric acid, and proline were all altered in the aging model mice, but were restored by CDPS. These findings demonstrated that CDPS improves cognitive function in a D-galactose-induced aging model in mice by restoring homeostasis of the gut microbiota brain axis, which alleviated an amino acid imbalance, peripheral inflammation, and oxidative stress. CDPS thus shows therapeutic potential for patients with memory and learning disorders, especially those related to gut microbial dysbiosis.

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INTRODUCTION 

polysaccharide, Ganoderma lucidum polysaccharide, and Lycium barbarum polysaccharide [9–11]. CDA-0.05 is a galactoglucan isolated from Cistanche deserticola that promotes the growth of several beneficial intestinal bacteria, including several species of Bacteroides and Lactobacillus [12]. The underlying mechanisms of the normal aging process are also implicated in several human diseases such as neurodegenerative disorders, coronary atherosclerosis, type 2 diabetes (T2DM), and hypertension [13, 14]. Recent studies have shown that changes in the intestinal flora play a significant role in human aging [15]. Several studies have shown that prolonged administration of D-galactose in experimental mice and rats mimics the normal aging process and is a useful model to study aging-related phenotypes such as cognition decline [16]. Moreover, D-galactose-induced aging model mice show changes in the composition of gut microbiota [17]. Therefore, we hypothesized that changes in the gut microbiota composition may cause cognitive decline in the D-galactose-induced aging model mice, and investigated if Cistanche deserticola polysaccharides (CDPS) may alleviate cognitive decline by restoring gut microbiota dysbiosis. 

Cistanche can improve immunity and anti-fatigue

RESULTS 

D-galactose-induced aging model mice demonstrate cognitive decline and gut microbial dysbiosis We analyzed the behavioral performance of wild-type  (WT) mice and those treated with 150 mg D-gal per  Kg body weight for 2 months (model or Mod) using novel object recognition and Morris water maze  (MWM) tests. The preferential index values in the novel object recognition test were significantly reduced in the Mod group mice compared to the WT  group mice (Figure 1A, 1B). MWM test results showed that the escape latency time after the sixth day was significantly increased in the Mod group compared to the WT group (Figure 1C, 1D).  Moreover, target platform crossings and swimming times within the target quadrant were significantly reduced in the Mod group compared to the WT group (Figure 1E, 1F). These results demonstrated a significant decline in the learning and memory abilities of the Dgal-induced aging model mice. We then analyzed the differences in the abundance and composition of the gut microbial phyla, genera and species in the fecal samples of the Mod and WT groups of mice using 16S ribosomal RNA (rRNA) sequencing data from fecal samples. The predominant intestinal flora in the WT and Mod group mice were Firmicutes and Bacteroides. However, the abundance of  Bacteroides was significantly reduced and the abundance of Firmicutes was significantly increased in the Mod group compared to the WT group (Figure 2A).  Next, we performed linear discriminant analysis (LDA)  to determine LDA effect size (LEfSe) scores followed by Kruskal-Wallis and Wilcoxon tests to evaluate the relative abundance of different taxa in the WT and Mod group mice. The LDA results are shown in Figure 2B.  Furthermore, we constructed cladograms showing differential enrichment of various genera and species belonging to the Bacteriodes and Firmicutes phyla in the WT and model groups (Figure 2C). Overall, our results demonstrated impaired cognitive ability and gut microbial dysbiosis in the D-galactose-induced aging model mice. CDPS treatment improves cognitive ability in the Legal-induced aging model mice We analyzed if CDPS treatment alleviates cognitive decline in D-gal-induced aging model mice. During the  2 months of the administration, the body weight was measured every other day. The body weights of the model and CDPS groups of mice were similar (Figure  3A). Conduct behavioral experiments after the last dose.  Novel object recognition and Morris water maze test results showed that short-term memory was significantly higher in the CDPS groups of mice compared to the model group of mice; long-term memory in the CDPS group of mice was higher but statistically insignificant compared to the model group of mice (Figure 3B, 3C). This suggested that CDPS  treatment abrogated loss of short-term object recognition memory in D-gal treated mice. The spatial learning and memory of these mice were evaluated by the Morris water maze test, and the results showed that the escape latency times of the CDPS  group of mice were comparable to the WT group of mice and significantly shorter than the Mod group mice (Figure 3D). Furthermore, escape latency times were significantly lower on the sixth-day post-CDPS administration compared to the model group (Figure  3E). The swimming time within the target quadrant was significantly higher in the CH and CM groups compared to the model group. The CL group is higher than the model group but has no statistical significance (Figure 3F). Moreover, the number of platform crossings was significantly higher in the CM and CL  groups compared to the model group. However, the  CH group is higher than the model group and has no statistical significance (Figure 3G). These results demonstrated that CDPS treatment improved spatial learning and memory in the D-gal-induced aging model mice. 

Figure 1. Prolonged administration of D-galactose induces learning and memory impairment in mice. (A, B) Novel object recognition test results show preferential index values for WT and model group mice after (A) 24 h training and (B) 1 h testing phase.  (C–F) Morris water maze test results show (C) latency in the learning phase, (D) latency in the test phase, (E) number of plate crossings, and  (F) time in the target quadrant for the WT and model group mice. Note: *p<0.05, **p<0.01, ***p<0.001 compared with the WT group mice; by. All values are represented as means ± SEM (n=15); Data was analyzed by one-way ANOVA followed by Dunnett's post hoc test.

Monosaccharides and polysaccharides are the essential nutrients required for the growth of bacteria [18–21]. It is also reported that CDPS regulates the composition of gut microbiota [22]. Therefore, we analyzed if CDPS treatments alleviated the gut microbial dysbiosis in the model group mice by evaluating16S rRNA sequencing data of feces samples from the WT, model, and CDPS groups of mice. First, we calculated alpha diversity indices to evaluate the overall fecal microbiota richness and structural difference among these groups. We analyzed alpha diversity (α-diversity) indexes such as observed species, Shannon, Chao 1, ACE, and Simpson index values to determine changes in the composition of various bacterial species in the feces samples of different murine groups. The α-diversity (observed species, Shannon, Chao 1, ACE, and Simpson indexes) indexes were higher in the WT and CDPS groups of mice compared to the model group, but statistical significance was only observed for the Chao 1 index values between the CM group and Mod group. It indicated that administration with CDPS increases the microbiome richness (Figure 4A–4E). Next, we analyzed β-diversity indexes to identify differences in the gut microbial species between the WT, model, and CDPS groups of mice using Non-metric Multidimensional Scaling (NMDS), Principal Coordinates Analysis (PCoA), and Principal Component Analysis (PCA). PCA showed variations in the gut microbial composition of the model group mice during the aging process, including dimension reduction and maintenance of patterns and trends (Figure 4F). The differences in the fecal microbiota between the WT, model, and CDPS groups were identified based on PCoA of the unweighted UniFrac distances for the 16S rRNA genes (Figure 4G). Clustering analysis showed significant differences in NMDS between the model group and the WT and CDPS groups (Figure 4H). We evaluated the top 10 phyla of the gut microbiota and found that the abundance of the Bacteroides phyla was significantly higher in the CH, CM, and CL group compared to the model group (Figure 4I). This suggested that CDPS restored the homeostasis of the gut microbiota in D-gal-treated mice. The cladogramsshowed differential enrichment of various genera and species belonging to Bacteriodes and Firmicutes phyla in the WT, model, and the CDPS groups (Figure 4J). As shown in the heatmaps, CDPS treatments reduced the relative abundances of Thermoplasmata, Bacilli, unidentified Actinobacteria, Fusobacteriia, and unidentified Elusimicrobia and increased the relative abundances of Methanobacteria, Spirochaetia, Deltaproteobacteria, unidentified_Deferribacteres, Mollicutes, Nitrososphaeria, Anaerolineae, Erysipelotrichia, and unidentified_Cyanobacteria compared to the model group (Figure 4K). These results demonstrated that CDPS treatment significantly restored homeostasis of the gut microbiota in the D-galinduced aging model mice. 

Figure 3. CDPS treatment improves learning and memory in the D-gal aging model mice. (A) The body weights of WT, Mod, and  CDPS group mice during administration. (B, C) Novel object recognition test results show the preferential index in WT, Mod, and CDPS group mice after (B) 24 h training and (C) 1 h testing phase. Morris water maze test results show (D, E) escape latency, (F) number of plate crossings,  and (G) time in the target quadrant for the WT, Mod, and CDPS group mice. Note: *p<0.05, **p<0.01, and ***p<0.001 compared to the WT  group mice; #p<0.05, ##p<0.01, and ###p<0.001 compared to the Mod group mice. Differences between groups were analyzed by one-way  ANOVA followed by the Dunnett's post hoc test. All values are shown as means ± SEM (n=15).

We next analyzed the effects of CDPS on inflammation by analyzing the serum levels of pro-inflammatory cytokines (IL-2 and TNF-α), and anti-inflammatory cytokines (IL-4 and IL-10) in different groups of mice. The serum levels of IL-2 and TNF-α were significantly lower and the serum levels of IL-4 and IL-10 were significantly higher in the CH, CM, and CL group compared to the model group. It is shown that CDPS has anti-inflammatory effects (Figure 5A–5D). Oxidative stress is caused by increased production of reactive oxygen species (ROS) and is one of the main factors that promote aging [23]. Therefore, we analyzed the effects of CDPS on oxidative stress in the D-gal-induced aging mouse model by evaluating serum levels of the antioxidant enzyme, SOD, and the lipid peroxidation product, malondialdehyde (MDA). The serum levels of MDA were significantly higher and the serum levels of SOD were significantly reduced in the Mod group compared to the WT group, but, CDPS treatment reversed these effects (Figure 5E, 5F). These results demonstrated that oxidative stress was elevated in the D-gal-induced aging model mice, but was reduced by CDPS treatment. Furthermore, we assessed the oxidative stress levels in the brain tissues by analyzing the levels of the advanced oxidized protein product (AOPP), direct lipid peroxidation (LPO), and MDA as well as activities of antioxidant enzymes such as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) in the brain tissue homogenates. The brains of Mod group mice showed significantly reduced activities of SOD and GSH-PX and significantly increased levels of AOPP, LPO and MDA compared to the WT group, but these effects were reversed in the CH, CM and CL group (Figure 6A–6E). 

Figure 4. CDPS treatment restores gut microbial composition in the D-galactose-induced aging model mice. (A–E) The α  diversity indexes of the gut flora in the feces of WT, Mod, and CDPS (CH, CM, and CL) group mice. (F–H) The β diversity indexes show differences in the gut microbial species between WT, Mod, CH, CM, and CL groups of mice. (I) The relative abundance of top10 gut bacterial phyla in the WT, Mod, CH, CM, and CL groups of mice. (J) The top 100 gut microbial genus in the WT, Mod, CH, CM, and CL groups of mice. (K)  The heatmap shows differentially enriched gut microbiota in the WT, Mod, CH, CM, and CL groups of mice. Note: *p<0.05, **p<0.01, and  ***p<0.001 compared to the WT group mice; #p<0.05, ##p<0.01, and ###p<0.001 compared to the Mod group mice; Data were analyzed by  unpaired Student's t-tests. All values are shown as means ± SD (n=15).

DISCUSSION 

Proteobacteria [37, 38]. Furthermore, gut microbial composition regulates brain function by modulating circulating levels of several cytokines [39–43]. Our results showed that CDPS treatment decreased the relative abundance of Thermoplasmata, Bacilli, unidentified Actinobacteria, Fusobacteriia, and unidentified Elusimicrobia, and increased the relative abundance of Methanobacteria, Spirochaetia, Deltaproteobacteria, unidentified_Deferribacteres, Mollicutes, Nitrososphaeria, Anaerolineae, Erysipelotrichia, and unidentified_Cyanobacteria. 

Figure 7. CDPS alleviates learning and memory by restoring homeostasis of the gut microbiota. (A) Heat maps show long-term memory (preferential index*) and short-term memory (preferential index) in the WT, Mod, CM, ABX, and Cy groups of mice. (B) The relative abundance of top 10 gut microbial phyla in the WT, Mod, CDPS, ABX, and Cy groups of mice.(C) Venn diagram shows the number of bacterial operational taxonomic units (OTUs) in the WT, Mod, CDPS, ABX, and Cy groups of mice. (D–I) ELISA assays show levels of TNF-α, IL-2, IL-4, IL- 10, SOD, and MDA in the serum of WT, Mod, CDPS, ABX, and Cy groups of mice. (J–N) Colorimetric assay results show the levels of AOPP,  MDA, and LPO as well as activities of GSH-PX and SOD enzymes in the brains of WT, Mod, CDPS, ABX, and Cy groups of mice. Note: *p<0.05,  **p<0.01, and ***p<0.001 compared to the WT group mice; #p<0.05, ##p<0.01, and ###p<0.001 compared to the Mod group mice. The data were analyzed by one-way ANOVA followed by Dunnett's post hoc test. All values are represented as means ± SEM (n=15).

Gut microbial metabolites are released into the bloodstream and regulate the health and metabolism of the host [26, 27]. The gut microbial metabolites can be estimated by evaluating fecal metabolite composition, which changes with alterations in the composition of the gut microbes [44]. Recent studies have shown that plasma levels of citrulline, proline, arginine, asparagine, phenylalanine, and threonine are associated with neurodegenerative disorders including Alzheimer’s disease [45, 46]. Our study showed that serum levels of creatinine, valine, L-methionine, o-Toluidine, N- ethylaniline, uric acid, and proline were associated with D-gal-induced aging in mice. The innate and adaptive arms of the immune system play a significant role in maintaining host-microbial homeostasis in the intestinal luminal surface [47]. The intestinal microbiota also plays a significant role in regulating the central nervous system (CNS) and immunity by releasing cytokines and metabolites into the bloodstream [48, 49]. The pro-inflammatory cytokines play a key role in several neurodegenerative diseases [50–52]. For example, age-related macular degeneration (AMD) and glaucoma is associated with extracellular accumulation of amyloid β (Aβ) and intracellular deposition of hyper-phosphorylated tau (ptau) and iron in the retinal ganglion cells (RGC) [44]. Moreover, inflammation plays a significant role in pathogenesis associated with glaucoma [53]. Visual impairment is an early symptom of Alzheimer’s disease (AD) and is manifested before the onset of cognitive decline [54]. Our study demonstrated that CDPS protects against cognitive decline and peripheral inflammation by maintaining the homeostasis of the gut microbiota. There are several limitations to this study. Firstly, the relationship between amino acid metabolism and the composition of the gut microbiota is not well known. Secondly, the composition and molecular structure ofCDPS is not known. Therefore, future studies are required to further explore the regulatory role of CDPS in alleviating AD through the gut microbiota-brainsignaling axis. In conclusion, our study demonstrated that CDPS improved cognitive ability in D-gal-induced aging model mice by restoring the homeostasis of gut microbiota, thereby restoring amino acid imbalance, peripheral inflammation, and oxidative stress. These findings suggest that CDPS is a potential therapeutic for patients with learning and memory disorders, especially those associated with gut dysbiosis.

Gut microbial metabolites are released into the bloodstream and regulate the health and metabolism of the host [26, 27]. The gut microbial metabolites can be estimated by evaluating fecal metabolite composition, which changes with alterations in the composition of the gut microbes [44]. Recent studies have shown that plasma levels of citrulline, proline, arginine, asparagine, phenylalanine, and threonine are associated with neurodegenerative disorders including Alzheimer’s disease [45, 46]. Our study showed that serum levels of creatinine, valine, L-methionine, o-Toluidine, N- ethylaniline, uric acid, and proline were associated with D-gal-induced aging in mice. The innate and adaptive arms of the immune system play a significant role in maintaining host-microbial homeostasis in the intestinal luminal surface [47]. The intestinal microbiota also plays a significant role in regulating the central nervous system (CNS) and immunity by releasing cytokines and metabolites into the bloodstream [48, 49]. The pro-inflammatory cytokines play a key role in several neurodegenerative diseases [50–52]. For example, age-related macular degeneration (AMD) and glaucoma is associated with extracellular accumulation of amyloid β (Aβ) and intracellular deposition of hyper-phosphorylated tau (ptau) and iron in the retinal ganglion cells (RGC) [44]. Moreover, inflammation plays a significant role in pathogenesis associated with glaucoma [53]. Visual impairment is an early symptom of Alzheimer’s disease (AD) and is manifested before the onset of cognitive decline [54]. Our study demonstrated that CDPS protects against cognitive decline and peripheral inflammation by maintaining the homeostasis of the gut microbiota. There are several limitations in this study. Firstly, the relationship between amino acid metabolism and the composition of the gut microbiota is not well known. Secondly, the composition and molecular structure ofCDPS is not known. Therefore, future studies are required to further explore the regulatory role of CDPS in alleviating AD through the gut microbiota-brain signaling axis. In conclusion, our study demonstrated that CDPS improved cognitive ability in D-gal-induced aging model mice by restoring the homeostasis of gut microbiota, thereby restoring amino acid imbalance, peripheral inflammation, and oxidative stress. These findings suggest that CDPS is a potential therapeutic for patients with learning and memory disorders, especially those associated with gut dysbiosis. 

MATERIALS AND METHODS 

Preparation of CDPS About 1.0 Kg of cleaned Cistanche deserticola was air-dried in the oven at 40° C and pulverized into crude powder. The powder was extracted in hot ethanol for 3 h. The residue was filtered through gauze to remove the filtrate and then diluted with water (8X)  and refluxed sequentially for 2 h, 1.5 h, and 1 h at  90° C. At each time point, the solution was centrifuged to separate out the supernatant and combined with the brown-red filtrate. Then, the filtrate was concentrated under reduced pressure, cooled to room temperature,  added slowly to 95% ethanol (3X), and allowed to stand at 4° C for 24 h. Then, the solution was centrifuged at 6000 r/min for 20 min at 4° C. The precipitate was collected after repeating water extraction and alcohol precipitation thrice. The precipitate was reconstituted in water, deproteinized,  dialyzed, and freeze-dried to get crude Cistanche deserticola polysaccharide (CDPs). The polysaccharide content was more than 90% as evaluated by ultraviolet spectrophotometry. Animal grouping and treatments Eight-week-old male Kunming mice (SCXK License  No.2019-0010) were purchased from SPF  Biotechnology Co. Ltd (Beijing, China), housed in a  light and temperature-controlled room, and fed with food and water. All animal experiments were conducted according to protocols approved by the  Institutional Animal Care and Use Committee of Inner  Mongolia Medical University. The experiments were carried out according to the National Institutes of  Health (NIH) Guide for the Care and Use of Laboratory  Animals. After 1 week adaptation to the new surroundings, 120  mice were divided into the following 7 groups: (1) wildtype control (WT); model group (150 mg/Kg/day D-gal;  Mod); (3) CH: D-gal plus 100 mg/kg CDPS; (4) CM:  D-gal plus 50 mg/kg CDPS; (5) CL: D-gal plus 25  mg/kg CDPS; (6) ABX group: antibiotics plus D-gal  plus 50 mg/Kg CDPS; (7) Cy group: cyclophosphamide  plus D-gal plus 50mg/kg CDPS. The mice from the model, ABX, Cy, and CDPS groups received subcutaneous injections of saline-dissolved 150 mg/kg D-gal every day for 2 months. The WT group was subcutaneously injected with an equal volume of saline for 2 months. The CDPS group mice were also administered daily with an intragastric injection containing 100 mg/kg, 50 mg/Kg or 25 mg/Kg CDPS for 2 months. The ABX group mice received drinking water with 0.1 mg/mL ampicillin and 0.5 mg/mL streptomycin for 2 months in addition to D-gal and CDPS injections. Before administering D-gal, the mice received injections containing 0.1 mg/mL ampicillin, 0.5 mg/mL streptomycin, and 0.1 mg/mL colistin for 7 days in the ABX group. The Cy group mice received intraperitoneal injections of 20 mg/Kg cyclophosphamide every other day (q.o.d) for 2 months in addition to daily injections of D-gal and CDPS. 

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