The gut microbiota is a complex ecosystem that provides essential functions, including carbohydrate metabolism, interaction with immune system, and prevention against pathogen invasion[21]. The intestinal flora is susceptible to dysbacteriosis caused by external and internal factors, and antibiotic is one of the main causes[22]. To observe the alleviation of TCM, LP, and FTCM on dysbacteriosis, we established a mouse dysbacteriosis model by intragastric administration of CS. Ceftriaxone is a broad spectrum, third-generation cephalosporin that is widely used to treat gastrointestinal infections. Repeated overuse of this antibiotic can disrupt the equilibrium of intestinal flora and cause side effects such as antibiotic‐associated diarrhea[23]. In the present study, there are obvious diarrhea symptoms induced by CS, including increased stool weigh, total number of fecal output and fecal water content. Then, bacterial culture tests showed that there was a destruction of some intestinal flora in mice gavaged antibiotic, which is similar to Zeng's research[24]. Overall, the above results are consistent with our previous studies that ceftriaxone and ciprofloxacin can cause watery diarrhea and intestinal microbial disorders in animal models[25].
An increasing body of evidence showed that probiotics can not only inhibit the proliferation of harmful bacteria in the intestine, promote the proliferation of beneficial bacteria, effectively restore and balance the intestinal flora, but the intestinal flora also participates in immune regulation and enhances immunity, which help prevent and treatment of antibiotic-induced diarrhea[26]. Notably, some studies found that the proportion of spleen deficiency type is the largest among antibiotic-associated diarrhea patients, and various tonifying Qi-invigorating TCMs are used to cure bowel diseases, treat intestinal flora disorder, enhance immunity, relieve fatigue, and prolong lifespan either as clinical medications or daily diets[8]. Qiweibaizhu powder could effectively treat dysbiosis diarrhea by improving intestinal flora and promoting the reproduction of beneficial bacteria such as bifidobacteria and lactobacillius[24, 27]. Therefore, probiotics combined with TCM may regulate balance in gut microbiota to reduce dysbiosis. In this study, the water content of the above animal feces was significantly reduced after continuous treatment with TCM, LP, and FTCM for 7 days, among which, FS significantly constantly relieved diarrhea symptoms from day 4 to day 7, and on day 7, compared to S, the fecal water content of mice was obviously decreased in FS. The number of lactobacillus and bifidobacterium of mice in LP, FS and FSH increased significantly compared to the CS group level, and the number of colibacillus decreased significantly, which shows that LP, FS and FSH have the function of supporting the growth of beneficial flora and clearing intestinal pathogenic bacteria. The reason may be LP fermentation in S and SH altered the gut pH that inhibit the proliferation of colibacillus[28]. Although B and S increased the number of beneficial bacteria, they also promoted the growth of harmful bacteria (colibacillus), showing that fermentation has certain advantages. The effect of SH is unstable and may be related to its composition, which needs further study.
The intestine is one of the important visceral organs, not only for digestion and absorption of nutrients but also for its innate barrier protecting the body from pathogenic microorganisms. Small intestine tissue can be observed by H&E staining. The serious damage to the small intestine chorionic villi and the histomorphological changes suggest that the small intestine absorption function and barrier function is severely damaged[29]. Studies have shown that Chinese medicine has obvious advantages in the treatment of intestinal mucosal barrier dysfunction[30]. He et al. treated broilers with probiotic (Bacillus subtilis, Bacillus licheniformis, and Saccharomyces cerevisiae), which increased VH/CD in duodenum, as well as higher ZO-1 mRNA expression in jejunum[31]. In addition, piglets treated with LP 299v had a lower diarrhea incidence than the control, the VH/CD and ZO-1 mRNA level in the jejunum and ileum were increased, and the structure of the gut microbiota were modified[32]. After diarrhea in mice, the water in the intestine will increase, then promote the softening of feces, and stimulate the intestinal mucosa to a certain extent, thus causing mucosal damage. In the present study, the villus height of duodenum was increased after mice were fed with LP, B, S, FB and FS, and the VH/CD of duodenum was obviously increased by gavage with S and FS, which is similar to above researches. The VH/CD of jejunum was enhanced in CS+S, CS+FB, CS+FS groups. SH improved the villus height and VH/CD of ileum. Overall, the above treatments could promote the growth of the intestine, and reduce the stimulation of diarrhea on the intestinal mucosa and epithelium microvilli.
Furthermore, intestinal epithelial cells are connected through tight junctions (TJ), which regulates the intestinal barrier permeability and epithelial integrity. Therefore, the homeostasis expression of TJ protein is essential for the maintenance of human health[33]. ZO-1 and Occludin are the most critical components in the structural and functional organization of TJ[34]. The gut flora target various intracellular pathways, alter the expression and distribution of TJ proteins, then regulate intestinal barrier function[35]. Aquaporins (AQPs) are water-channel membrane proteins expressed in various tissues. Reportedly, at least 7 AQP subtypes (AQPs 1, 3, 4, 5, 7, 8, 9, and 11) are expressed in the gastrointestinal tract and play important roles in several physiological and pathological processes[36]. In particular, the distal small intestine and proximal colon are the major sites for AQP1, 3, and 4 expression[37]. Zhang et al.[18] observed that the antibiotic-associated diarrhea rats exhibited defective gastrointestinal integrity and improper epithelial organization, with decreased expression of aquaporin-encoding genes, aberrant TJ proteins, and a decrease in the number of goblet cells compared with control animals. Likewise, we found that the AQP1 and ZO-1 mRNA expression levels in the duodenum and colon were significantly attenuated in diarrhea mice. And the two gene expression levels were enhanced in mice of giving LP, B, S or FS. These results are roughly in accordance with the results observed for the intestinal morphology, suggesting that TCM, probiotic, and FTCM display beneficial properties at the molecular level.
Since FS showed better advantages in most indicators, compounds in S and FS were detected by UHPLC-Q-TOF/MS. Results showed that 30 compounds in FS were obviously up-regulated compared to S. In which, the four compounds were noteworthy of attention, including (S)-(-)-2-Hydroxyisocaproic acid, L-Methionine, 4-Guanidinobutyric acid (4GBA), and Phenyllactate (PLA). (S)-(-)-2-Hydroxyisocaproic acid also named L-Leucine, and it is a signaling amino acid in animal metabolism, which can elevate villus height of duodenum and VH/CD of duodenum and ileum[38]. That is similar to our results. L-Leucine as an up-regulated compound in FS, indicating it could improve intestinal development. In addition, L-Methionine is an essential amino acid (AA) in humans and other vertebrates. It can't be synthesized by the body and must be obtained from the diet. Methionine absorption from the gastrointestinal tract is highly efficient[39]. Studies have shown that dietary supplementation of methionine is beneficial to intestinal development and antioxidant function of pigs[40]. The two AA may play a significant role in improving intestinal health of diarrhea mice. 4GBA is an alkaloid included in guanidino compounds, and inhibits the growth of H. pylori in a dose dependent manner and might be useful in the treatment and/or protection of gastritis[41]. With the same functional group, 4-methylguanidine butyric acid inhibits bacteria and fungi, such as Staphylococcu saureus, Escherichia coli, Saccharomyces cerevisiae and Aspergillus niger[42]. In this experiment, the growth of colibacillus was inhibited in mice that gavaged FS, but further studies are needed to investigate whether it related to the increase of 4GBA. PLA is found in various foods, such as honey, pickle, sourdough and a variety of fermented foods[43]. It has versatile antimicrobial activity against food-borne pathogenic bacteria[44] and spoilage mould[45]. PLA has great potential for applications in the food, feed and pharmaceutical. Therefore, increased PLA in FS have an important antibacterial effect possibly. Perhaps the four compounds can be potential indicators of inhibiting harmful bacteria and improving intestinal development.