Part 2：Effects Of The Cistanche Tubulosa Aqueous Extract On The Gut Microbiota Of Mice With Intestinal Disorders

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Figure 3: *e composition and relative abundance of gut microbiota in mice feces at the phylum and genera levels. (a) Phylum level classification composition and relative abundance. (b) Genera level classification composition and relative abundance. A, normal group with middle-dose aqueous Cistanche tubulosa (CT) extract added; B, normal group; C, model group; D, a model group with high-dose CTaqueous extract added; E, a model group with middle-dose CT aqueous extract added; F, a model group with low-dose CT aqueous extract added.

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3.4. Functions of the Gut Microbiota Related to the Treatment with the CT Aqueous Extract. We used PICRUSt2 software to predict the metabolic pathways of the gut microbiota, and the normal group was used as a reference to analyze the changes in other groups. Under the cefixime treatment, the relative abundance of ethylbenzene degradation, biosynthesis of siderophore group nonribosomal peptides, and metabolism of xenobiotics by cytochrome P450 pathways increased; after the treatment with high- and middle-dose aqueous extracts, their relative abundance returned to normal levels. Meanwhile, the relative abundance of thecyanoamino acid metabolism pathway decreased under thecefifixime treatment; however, it increased after the treatment with the high-dose CT aqueous extract. Furthermore, in general, the changes in different metabolite pathways after the treatment with cefixime were significant compared with those in the normal group; however, the addition of the aqueous extract was able to prevent excessive changes(Figure 5).

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4. DiscussionColon morphology can be altered by growth, digestion and absorption, immune regulation, and intestinal injury repair[46–50]. *e V/C ratio can comprehensively reflect the digestive status of the intestinal tract and is directly proportional to the digestive and absorption capacity of the gut tract [51, 52]. In the present study, the villi and recess biopsy and statistical data showed that the high-dose aqueous extract could partly improve the defective morphology inside the colon. To investigate how the aqueous extract changes gut morphology and affects the intestinal microbiota, we worked backward from changes in the intestinal flora. We found that the relative abundance of Proteobacteria, a hub marker of disordered intestinal flflora, increased under the treatment with cefixime compared to that without cefixime treatment.*e relative abundances of other hub markers, Bacteroidetesand Firmicutes, had no significant changes, although these,

Figure 4: Results of the LEfSe analysis between different groups. (a) Relative abundance of bacteria between the normal group (B) and model group (C); (b) relative abundance of bacteria between the model group (C) and model group with high-dose aqueous Cistanchetubulosa (CT) extract added (D).

groups are predominant in the human gut; the ratio ofBacteroidetes/Firmicutes was found to be decreased in obese people compared with that in lean people, and this ratio was found to increase with weight loss in people on two types of low-calorie diet [38, 41, 43–45, 48, 53, 54]. Meanwhile, Turicibacter, which is associated with obesity [55], was significantly elevated in the model group compared to that in the other groups. Notably, the diversity of gut microbiota in the model mice was improved by the addition of the aqueous extract. We noted some specific intestinal bacteria in mice under different treatments; for example, Lactobacillus and Muribaculaceae were the two main bacterial genera that increased in the group treated with the high-dose aqueous extract compared to those in the model group(Figure 4). Recent studies have indicated that the polysaccharides of CT aqueous extracts possess significant

Figure 5: Changes in the relative abundance of metabolic pathways in the normal group with middle-dose aqueous Cistanche tubulosa(CT) extract added (A), model group (C), a model group with high-dose CT aqueous extract added (D), a model group with middle-dose aqueous extract added (E), and model group with low-dose CTaqueous extract added (F). *e abundances are normalized according to the values in the normal group (B). Color depth indicates the magnitude of the ratio.

antioxidant activities in vitro [56] and can promote the growth of some lactic acid bacteria, which could benefit host health [43]. In parallel, Muribaculaceae are probiotics organisms linked to longevity [57]. *ese suggested that the mechanism by which the CT aqueous extract improves the gut microbiota may be the promotion or protection of the growth of probiotic organisms. Another bacterium worthy of note was the bacterium YE57. Although the high-dose CTaqueous extract promoted the relative abundance of bacterium YE57 in the present study (Figure 4), previous studies have found that its abundance was higher in the normal gut than that in the gut treated with high-concentration herbal tea residue [58] and that its abundance was reduced after the intervention with Bacillus licheniformis combined with XOS(xylooligosaccharides) [59]. *us, the role of this bacterium in the gut microbiota deserves further study. Besides, the relatively small sample number in this study might cause a measure of false positive and false negative, and future study on larger samples is suggested to validate the identified bacterial markers.CT aqueous extract composition might be important for its effects on the composition and functional changes in the gut microbiota of mice with intestinal disorders. PSGs are common active components found in CD andCT, and echinacoside was identified as the major PNG in [60]. In the past decades, echinacoside has been shown to possess many pharmacological activities, such as antiaging and neuroprotective effects, improvement of cardiac function, reduction of hyperlipidemia and hyperglycemia, and prevention of obesity-induced diabetes and metabolic syndrome [53, 61–65]. In fact, we detected changes in the metabolic pathways of the gut microbiota.*e treatment of cefixime led to the enrichment of bacteria related to ethylbenzene degradation and biosynthesis of siderophore group nonribosomal peptides, while the treatments with the high- and middle-dose CT aqueous extract could alleviate these changes, indicating that this extract moderated the bacterial community-related to these functions. In addition, the increased bacterial enrichment related to the cyanoamino acid metabolism pathway under the treatment with the high-dose aqueous extract and its decreased enrichment in model mice indicated that the CT aqueous extract can promote the metabolism of cyanoamino acid. *e changes in relevant metabolites might provide this aqueous extract with pharmacological activities.

Although the mechanism by which the CT aqueous extract changes the composition and function of intestinal microbiota is complex, there are some clues to speculate about the potential mechanism. It has been reported that both lactic acid bacteria and CT aqueous extracts can antagonize oxidative stress. Oxidative stress occurring during inflammation is a common factor that exacerbates intestinal disorders by strongly reducing gut microbial diversity and promoting the surge of specific bacteria (4).On the contrary, reactive oxygen species also promote the selective growth of bacterial groups through nitrate and tetrathionate respiration [66–68]; for example, bacteria from the family Enterobacteriaceae can grow rapidly as a consequence of changes in the composition of the intestinal flora under oxidative conditions during inflammation [69, 70]. Most living organisms evolve enzymatic defenses, nonenzymatic antioxidant defenses, and repair mechanisms to scavenge oxygen radicals [71]. However, these native antioxidant systems are generally not sufficient to prevent oxidative damage in living organisms. Several additional synthetic antioxidants, including butylated hydroxyanisole and butylated hydroxytoluene, have been widely used to decrease oxidation, but their safety has been questioned [72, 73]. *before, researchers have turned to find safer and more natural antioxidants obtained from naturally occurring substances. Due to the ability of both polysaccharides and lactic acid bacteria to eliminate oxidative stress, determining the precise antioxidative mechanism of CT aqueous extracts on the intestinal microbiota requires further investigation in the future. In conclusion, we found that the CT aqueous extract was able to improve intestinal gut microbiota in mice with intestinal disorders by promoting diversity, moderating the metabolic changes, and remodeling the structure of gut microbiota, and these results may provide a reference for the development of related drugs in the future.

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