Part 1：Effects Of The Cistanche Tubulosa Aqueous Extract On The Gut Microbiota Of Mice With Intestinal Disorders

Please contact oscar.xiao@wecistanche.com for more information

Disorders of the gut microbiota are associated with many diseases. *e aqueous extract from Cistanche tubulosa (CT), a traditional Chinese herbal formula, has been reported to play a role in protecting the human intestine. However, little is known about its effects on the gut microbiota. *e present study was carried out to determine whether the CT aqueous extract can modulate the gut microbiome in mice with intestinal disorders. We found that the damaged intestinal morphology resulting from treatment with cefixime could be rescued using the CT aqueous extract. *e comparison of microbial diversity between mice treated with the CT extract and control mice also indicated that the disorder in the microbiome community of model groups could be restored by treatment with high and medium concentrations of the aqueous extract. Treatment with cefixime led to a significant decrease in lactic acid bacteria; however, the supplementation of the CT aqueous extract recovered the growth of these lactic acid bacteria. Furthermore, the CT aqueous extract was able to moderate the dramatic changes in the metabolic pathways of the gut microbiome induced by cefixime. *ese findings provided an insight into the beneficial effects of the CT aqueous extract on gut microbiota, and they also provided an important reference for the development of related drugs in the future.

Cistanche for relaxing Bowels, please click here to know more

Introduction

have demonstrated that C. deserticola polysaccharides induce melanogenesis in melanocytes, reduce oxidative stress[15], alleviate cognitive dysfunction by regulating antioxidant and anti-inflammatory processes in rats [16], protectPC12 cells against OGD/RP-induced injury [17], enhance echinacoside absorption in vivo, and affect the gut microbiota [18]. Probiotics are live nonpathogenic microorganisms that have health benefits and confer microbial balance in the gastrointestinal tract when administered in adequate amounts [19]. *ey can enhance nonspecific cellular immune responses characterized by the activation of macrophages, natural killer (NK) cells, and antigen-specific cytotoxic T lymphocytes and the release of various cytokines in a strain-specific and dose-dependent manner [20]. Probiotic strains improve the properties of the intestinal epithelium via TJ modulation, and specific probiotic strains have been demonstrated to regulate mucin expression, thereby influencing the properties of the mucus layer and indirectly regulating the gut immune system [21]. Strains of lactic acid bacteria (LAB) and Bifidobacterium are major probiotics that have been used in many fields [22–26]. *eir health benefits are numerous, with their antioxidant capacity being an important factor in their health-related functions[27]. Probiotics can chelate metal ions to prevent them fromcatalyzing oxidation [28, 29]; they can also increase theexpression of antioxidant enzymes [30, 31], produce variousmetabolites with antioxidant activity [32, 33], mediate antioxidant signaling pathways [34–36], and regulate the enzymes producing reactive oxygen species (ROS) and theresponse of intestinal microorganisms to oxidative stress[37].A recent study demonstrated that the polysaccharidesof CD could stimulate the growth of some lactic acidbacteria, which could benefit human health [38]. However, the content of polysaccharides in CD is different from that in CT [7, 39], and this difference may lead to difffferenteffffects in intestinal microorganisms. Furthermore, although CD polysaccharides can reduce oxidative stress byactivating the NRF2/HO-1 pathway [15], the effects ofsingle polysaccharides may differ from the overall effect ofmultiple compositions in CT. *us, it is necessary to precisely define the effects of CT aqueous extracts on intestinal microorganisms. In addition, fans can alsoresist oxidative stress [40] and suppress lipolysaccha ride-mediated inflammatory responses by activating theKeap1/Nrf2/HO-1 pathway [41]. *before, determining the effect of the CT aqueous extract is of great value. Inaddition, the effects of certain constituents of the aqueousCD extract on oxidative stress and intestinal flora suggestthat the resistance to oxidative stress might be correlatedwith intestinal flora changes. In order to fill the gaps in the knowledge on the topicsmentioned above, we investigated the effects of the CTaqueous extract on the gut microbiota of mice with intestinalflflora disorders. *ese results will provide valuable information about the possible mechanisms through which changes the intestinal flora and confers gut resistance to oxidative stress.

2. Materials and Methods

administered daily at 12:00 h, and other substances were administered daily at 15:00 h. During the experiments, the C, D, E, and F groups were kept in the model state of intestinal disorders. *e feces were collected every seven days on a sterile operable table and stored at −20°C.2.4. Histopathological Observation of the Mice Colon. At the end of the experiment, the mice were killed by cervical dislocation, and their colon contents were collected on a sterile operable table and stored at −80°C; at the same time, colonic tissue samples were fixed in 10% neutral formalin.*en, the samples were dehydrated using gradient concentration of ethanol, hyalinized using xylene, embedded in paraffin, sectioned, and stained with hematoxylin-eosin. Morphological changes in the colonic mucosa were observed and compared using an optical microscope. Villus length and crypt depth in the colon was measured, and the ratio of villus length to crypt depth (V/C value) was calculated (51).2.5. DNA Extraction and Library Construction. DNA was extracted from the feces using the E.Z.N.A. ®Soil DNA Kit(Omega Bio-Tek, Norcross, GA, USA) according to the manufacturer's protocol. DNA quality was determined using a fluorometer (QuantiFluor™–ST, Promega Corporation, USA). Paired primers in the V3-V4 region of 16s rDNA were designed to amplify the region and produce 466 bp DNA fragments. *e forward primer was 341F (-5-CCTACGGGNGGCWGCAG-3-), and the reverse primerwas 806R (-5-GGACTACHVGGGTATCTAAT-3-). EachPCR volume was 25 μL, containing 2.5 μL of 10 × PCR buffffer,2 μL of dNTPs, 1 μL of each primer, and 20–30 ng of template DNA. *en, the indexed adapters were attached to the end of the amplicons to generate sequencing libraries. *libraries were validated using a QuantiFluor™ fluorometer and quantifified to 10 nmol.2.6. 16s rRNA Gene Sequencing and Microbial CommunityAnalysis. *e Illumina platform (Illumina MiSeq) was used to obtain 2 × 250 bp paired-end data. Operational taxonomic units (OTUs) were obtained using Uparse software through standard clustering with 97% similarity. *e naive Bayesianassignment algorithm of the RDP classififier was used to align the OTUs with the Greengene database Release 13.5 and perform species annotation. *e alpha diversity of gut microbiota was calculated using the Shannon and Simpson indices, and the difffferences between groups were analyzed by linear discriminant analysis Effffect Size (LEfSe). *e beta diversity was analyzed by principal coordinate analysis (PCoA) of Brady–Curtis dissimilarities. PICRUSt2 was used to estimate the microbial metabolic capacity of the gut microbiome [42].2.7. Statistical Data Analysis. SPSS 20 was used for one-way ANOVA, and the experimental data were expressed as X ± S; X indicates the average value, and S indicates the standard deviation.

Cistanche for improving immunity

3. Results

3.1. 8 Effect of the CT Aqueous Extract on Colon Morphology.*e representative compounds (echinacoside and acetonide)and their concentrations of the CT extract were validated by HPLC (Figure S1). To determine the effect of the aqueous extract on the gut, we investigated the length of colon villi and depth of recesses following the treatment with the aqueous extract. *e colon villi in the normal and high-dose groups (A, B, and D) were longer and fingerlike, whereas the colon villi in the model and low-dose groups (C and F) were short, and the tips of the colon villi were broken (Figure 1). Accordingly, high-dose CT aqueous extract significantly increased the length of colon villi and reduced recess depth in mice with intestinal disorders compared with the mice in the model group (P < 0.01). In contrast, recess depth was not significantly different between the high-dose group and the normal group (P > 0.05) (Table S1). *ese results indicated that the high dose of the CT aqueous extract can improve the morphology inside the colon of mice with intestinal disorders.3.2. 8e Effffect of the CT Aqueous Extract on the Diversity ofGut Microbiota. We performed 16s rRNA gene sequencing to investigate the potential cause of the morphological changes inside the colon and investigate the changes in gut microbiota following treatment with the CT aqueous extract. An average of 100,553 effective tags, ranging from 77,734 to125,144, was obtained from the raw data (Table S2). *stags were clustered into 4932 OTUs (Table S3). We then analyzed the diversity of the gut microbiota based on Theseus. *e Shannon and Simpson indexes showed no difference between the A group (normal with the CT aqueous extract) and the B group (normal without the CT aqueous extract) (Figure 2(a)). *is indicated that in the mice without the cefixime treatment, the CT aqueous extract might have had no additional beneficial or harmful effects on the theα-diversity of the gut microbiota. However, the α-diversity in the model group (C) showed a decreasing trend compared to that in the normal groups. *e mice treated with highand middle-dose CT aqueous extracts showed signs ofα-diversity recovery, whereas such a phenomenon was not observed in mice treated with the low-dose CT aqueous extract (Figure 2(a)). Meanwhile, the PCoA revealed that the normal groups (A and B) and intestinal disorder groups administered high-dose (D) and middle-dose (E) CTaqueous extracts tended to have shorter intersample distances than those in the model group and in the low-dose CTaqueous extract supplement group (F) (Figure 2(b)). *results indicated that the CT aqueous extract could help improve the diversity of the gut microbiota in mice with intestinal disorders.

3.3. Changes in the Composition of Gut Microbiota Treatedwith the CT Aqueous Extract. *e microbiota composition profiles were compared among different groups. At the phylum level, the relative abundance of Proteobacteria in the model group was higher than that in the other groups.

Figure 1: *e effect of the aqueous Cistanche tubulosa (CT) extract on colon morphology: A, a normal group with middle-dose aqueousCistanche tubulosa (CT) extract added; B, normal group; C, model group; D, the model group with high-dose CT aqueous extract added;E, a model group with middle-dose CT aqueous extract added; F, a model group with low-dose CT aqueous extract added. (a) GroupA. (b) Group B. (c) Group C. (d) Group D. (e) Group E. (f ) Group F.

(Figure 3(a)). *e increase in Proteobacteria suggested that the microbiome of model mice was changed by cefixime and that the CT aqueous extract might benefit the intestinal microbiota as the increased prevalence of Proteobacteria is a hub marker of disordered intestinal flora [43–45]. In addition, at the genus level, the relative abundance of Lactobacillus in the model group decreased compared with that in the normal and high-dose groups; however, it increased compared with that in the middle- and low-dose group(Figure 3(b)). *these results indicated that the high-dose CTaqueous extract might promote the growth of some bacteria from the genus Lactobacillus. Differential microbiota between the studied groups was further determined according to the LEfSe analysis. *analysis showed that, after the treatment with cefixime, the relative abundances of Turicibacter, Alphaproteobacteria, Acidobacteria, Betaproteobacteriales, and Chloroflflexi significantly increased, whereas the relative abundances of lactobacillus, Eubacterium_nodatum_group, Pseudonocardiales, and Christensenellaceae_R-7_group significantly decreased compared with those in the normal group(Figure 4(a)). Strikingly, when the model group was supplemented with the high-dose CT aqueous extract, the relative abundances of Muribaculaceae, Lactobacillus, Kineosporiaceae, Eubacterium no datum group, and Pedobacterwere significantly increased compared to those in the model group. Meanwhile, the relative abundances of Rhodobacter, Ruminococcaceae UCG_013, Roseburia, Ruminiclostridium_9, and Candidatus Stoquefifichus decreased significantly compared to those in the model group (Figure 4(b))

Figure 2: Gut microbiota diversity analysis of mice feces. (a) α-Diversity of bacteria communities measured by the Shannon index (A)and the Simpson index (B); (b) the principal coordinate analysis (PCoA) plot visualizing the data based on Bray–Curtis dissimilarities.A, normal group with middle-dose aqueous Cistanche tubulosa (CT) extract added; B, normal group; C, model group; D, model group with high-dose CT aqueous extract added; E, model group with middle-dose CT aqueous extract added; F, model group with low-dose CT aqueous extract added.