Effect of Fermented Traditional Chinese Prescription With Lactobacillus Plantarum on Dysbacteriotic Diarrhea Mice Induced by Ceftriaxone Sodium


 Background: Buzhongyiqi decoction (B), Sijunzi decoction (S), and Shenlingbaizhu decoction (SH) have been extensively clinically used for the treatment of health status and diseases caused by spleen-qi deficiency for many years and microbial fermentation has been widely applied in Traditional Chinese Medicine (TCM) for thousands of years in China. This study was aimed to investigate the mitigative effect of TCM and fermented TCM (FTCM) with Lactobacillus plantarum (LP) in antibiotic-associated diarrhea (AAD), and identify the compounds of S and Fermented S (FS). Methods: The dysbacteriotic diarrhea mice induced by ceftriaxone sodium (CS) were treated with LP, B, S, SH, Fermented B, S, and SH. The diarrhea indexes, the abundance of some gut bacteria, intestinal morphometrics, and the mRNA expressions related to intestinal barrier function were assessed at multilevels. In addition, S and FS were chosen to identify and relatively quantify the compounds by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS), and different expressed compounds were analysed. Results: Results showed that CS significantly increased the fecal output weight, the total number of fecal output, and fecal water content, indicating the occurrence of diarrhea, while TCM, LP, and FTCM alleviated the diarrhea to different degrees and FTCM showed more sustained effects. Then, bacterial culture test showed above symptoms were accompanied with the disruption of some intestinal flora. Meanwhile, the diarrhea mice showed abnormal intestinal morphology and destroyed intestinal barrier manifested as reduced mRNA expression of Aquaporins (AQPs) and tight junction (TJ) protein. Notably, the above indices were alleviated in other treatments mice.Conclusions: All these findings imply that the intestinal side effects caused by antibiotics can be alleviated by TCM, LP, and fermented TCM through regulating the intestinal flora and barrier function, which provides an idea of further development and application of them in the clinical use of antibiotics.


Introduction
Antibiotics have been used for the prevention and treatment of lots of severely bacterial infections and saved countless lives. In recent years, a large number of studies have revealed that the abuse of antibiotics may cause undesirable consequences, such as antibiotic resistance, pathogen overgrowth, alteration of gut microbial composition, increased bacterial susceptibility, and the risk of repeated infections [1]. In addition, nephrotoxicity and neurotoxicity [2], emesis, antibiotic-associated diarrhea (AAD) and allergy [3] were also side-effects induced by antibiotics. Notably, antibiotics can destroy the structure of normal gut microbiota and alter the functions in children, adults and animal models, which is dysbacteriosis [4]. Because the gut microbiota shows an important role in immunity, metabolism and endocrinology, the negative impacts of antibiotics on the microbiota lead to further health complications of obesity, allergies, autoimmunity and other diseases [4,5]. The interventions of scienti c method are effective for improving antibiotic application, which raises a question of whether healthy microbiomes could regulate or restore by probiotics or Traditional Chinese Medicine (TCM).
Some studies have provided important information that probiotics alter gut microbiota and produce ora metabolites that in uence health via 1 of 3 general mechanisms: direct antimicrobial effects, enhancement of mucosal barrier integrity, and immune modulation [6]. In addition, TCM also adjusts the balance of intestinal ora [7]. TCM is one of the oldest medicine practices in human history, widely applied to the clinical diagnosis and treatments [8]. In TCM practice, tonic herbs and a few animal sourced medicines are used to strengthen the body and cure diseases caused by Qi de ciency. Some typical Qiinvigorating herbs include Astragali Radix, Ginseng Radix et Rhizoma (ginseng), yam, Codonopsis, Coix seed, Atractylodis Macrocephalae Rhizoma, Glycyrrhizae Radix et Rhizoma (licorice), and Schisandra Chinensis Fructus, and exemplary Qi-invigorating multi-ingredient decoctions (called Tang in Chinese) include "Sijunzi Tang, Lizhong Tang, Buzhongyiqi Tang, and Shenlingbaizhu Tang" [8]. TCMs are mostly relevant to intestinal ora because they inevitably interact with gut microbiota when administrated orally to treat diseases [9,10]. Many researchers have investigated metabolites of TCMs by regulating intestinal ora and realized that indeed these metabolites have important pharmacological activities and play various roles in the disease treatment [7]. Indeed, microorganisms have been employed in fermenting TCM, e.g. probiotic was used to ferment Scutellaria Radix [11], Atractylodis Macrocephalae Rhizoma [12], red ginseng [13], and some Chinese prescription, like Danggui Buxue Tang, [14], Sagunja-tang [15], Ge-Gen-Qin-Lian decoction [16].
Based on above, the three kinds of classic prescriptions (Buzhongyiqi decoction, Sijunzi decoction, Shenlingbaizhu decoction) that were fermented by Lactobacillus plantarum were applied to alleviate diarrhea and regulate the imbalance of intestinal ora induced by antibiotic. However, it has not been reported before. In this study, the diarrhea indexes, the abundance of some gut bacteria, intestinal morphometrics, and the mRNA expressions related to intestinal barrier function were assessed at multilevels. In addition, Sijunzi decoction and Fermented Sijunzi decoction due to their better effects were chosen to identify and relatively quantify the compounds by an untargeted ultra-high-performance liquid chromatography coupled with quadrupole time-of-ight mass spectrometry (UHPLC-Q-TOF/MS). in distilled water at room temperature for 1 h, were decocted twice in 2100 mL water for periods of 1 h each in a glass ask [17], and concentrated to 1 g/mL by rotary evaporation apparatus (RE3000-A, Shanghai Yarong biochemical instrument factory, China). The B and SH were decocted and concentrated as the above method. The herb compositions of B, S and SH were shown in Table 1. After one-week of acclimatization, the mice were randomly divided into nine groups (n=8). In the blank control group (BC), mice were administered with 0.3ml saline intragastrically twice per day at 9 am and 1 pm, respectively. In the ceftriaxone sodium group (CS, 4g/kg/d), mice were given 0.3ml CS and saline by gavage at 9 am and 1 pm, respectively. For the CS + LP group (CS+LP), the CS + B group (CS+B), the CS + S group (CS+S), the CS + SH group (CS+SH), the CS + Fermented B group (CS+FB), the CS + Fermented S group (CS+FS) and the Fermented SH group (CS+FSH), mice were given 0.3ml CS at 9 am and the corresponding drug at 1 pm by gavage, respectively, and treated for 7 days. On day 1, 4 and 7, diarrhea symptoms were recorded and fresh stool samples of mice in each group were taken for live bacterial culture. On day 8, the mice were euthanized. The intestinal tissues were stored at -80ºC for qRT-PCR. Three samples of each group were xed in 4% paraformaldehyde solution for intestinal histopathology.
Diarrhea assessment and live bacterial culture All mice were kept separately with one mouse one cage. During the observation period of 2h, diarrhea symptoms were assessed by three indicators: fecal output weight[18], total number of fecal output [19], and fecal water content [18]. Fecal output was determined by measuring cumulative stool weight in 2h.
Total number of fecal output in each group was obtained by counting the fecal numbers of mice. Fecal samples were weighed and dried, and then the dried solid weight and total fecal weight were measured.

Intestinal morphometrics
The duodenum, jejunum, and ileum tissues were embedded in para n and cut into 5 µm sections and three slides of each sample were stained with hematoxylin & eosin (HE) staining. Images were observed with an Olympus BX51 microscope equipped with CCD DP70 video camera (Olympus Optical, Tokyo, Japan). The villus height and crypt depth were measured using Image-Pro Plus (Version 5.1, Media Cybernetics, Silver Spring, MD, USA) and villus height to crypt depth ratio (VH/CD) was calculated.
RNA extraction and qRT-PCR After 0.5-1 cm duodenum and colon tissues were cut into pieces, the pieces were added to 1 ml Trizol for RNA extraction (Takara, Dalian, China). The quality of RNA was examined by NanoDrop ND-2000 Spectrophotometer (Nano-Drop, USA). Afterward, total RNA was reversely transcribed at 37°C for 15 min, 85°C for 5 seconds and 4°C for 10 min using the PrimeScript™ kit (Takara, Dalian, China). The sequences of reference gene and target gene, designed by Primer 3.0 plus, were as follows: GAPDH, AQP1, AQP3, AQP4, ZO-1, Occludin, and ZO-1 (  The freeze-dried samples (six S and six FS) were crushed using a mixer mill (MM400, Retsch, Laichi, Germany) for 1.5 min at 30 Hz. 100 mg powder was weighted and extracted overnight at 4℃ with 0.6 ml 70% aqueous methanol. Following centrifugation at 10, 000g for 10 min, the extracts were absorbed and ltrated before UPLC-MS/MS analysis. Same volume was taken from each sample and pooled as Quality Control (QC) samples.
The sample extracts were analyzed using a 1290 UHPLC system (Agilent Technologies, La Jolla, CA, USA) combined with quadrupole time-of-ight mass spectrometer (QTOF) 6600 (AB Sciex, Redwood City, CA, USA). The chromatographic separations were performed on an Agilent Eclipse Plus C18 column (1.8 µm, 2.1 mm*100 mm, Milford, MA, USA) with the column temperature set at 40℃. The mobile phases were pure water with 0.04% acetic acid (A) and acetonitrile with 0.04% acetic acid (B). The gradient program was performed as follows: the starting conditions of 95% A, 5% B, a linear gradient to 5% A, 95% B within 10 min, a composition of 5% A, 95% B kept for 1 min, and subsequently, a composition of 95% A, 5% B was adjusted within 0.10 min and kept for 2.9 min. The injection volume was 4µl.
According to Yang et al method [20], QTOF was operated using an Electron Spray Ionization (ESI) in positive and negative ion mode and controlled by Analyst 1.6.3 software (AB Sciex, Waltham, MA, USA) to evaluate the full scan survey MS data. The ESI source operation parameters were as follows: source temperature 550 ℃; ion spray voltage (IS) 5500 V (positive ion mode)/-4500 V (negative ion mode); ion source gas I, gas II, and curtain gas were set at 50, 60, and 30.0 psi, respectively.

Statistical analysis
All data were are represented as means±SEM. The signi cance of the difference between means was determined by analysis of variance (ANOVA) of Graph Pad Prism 6.0 (GraphPad Software Inc., San Diego, USA) followed by Tukey's test with P < 0.05 being considered signi cant.

Result
Disruption of the intestinal ora by antibiotics-triggered diarrhea in mice and alleviation of treatments In order to investigate the effects of TCM and fermented TCM (FTCM) on antibiotic-induced diarrhea, the related indexes were detected in BC, CS, CS+LP, CS+B, CS+S, CS+SH, CS+FB, CS+FS, and CS+FSH groups, respectively. Fig. 1 represents the data of stool weigh, total number of fecal output and fecal water content in 2 hours. Compared to BC group, the CS group had a signi cant increase in fecal water content on day 1 (P < 0.05) (Fig. 1A1). Notably, on day 4 and 7, the CS group was signi cantly increased in stool weigh, total number of fecal output, and fecal water content compared to the BC group (P < 0.05) ( Fig. 1B  and 1C), indicating the occurrence of diarrhea. Meanwhile, compared to CS group, the total number of fecal output was signi cantly decreased in seven groups (P < 0.05), and the fecal water content was a signi cant decrease in CS+B, CS+FB, CS+FS and CS+FSH groups (Fig. 1B2,3). As shown in Fig. 1C, the stool weight and fecal water content were signi cantly decreased in seven groups (P < 0.05), showing an alleviation by TCM, LP and FTCM. In CS+FS group, the stool weight, total number of fecal output and water content of fecal were remarkably lowed (P < 0.05) from day 4 to day 7 compared to CS group. But this did not happen in CS+S and CS+LP groups ( Fig. 1B and 1C), which suggested FS has a continuous therapeutic effect.
Then, bacterial culture test was performed to examine whether above symptoms were accompanied with the disruption of some gut bacteria. The situation of live bacteria counting was presented in Fig. 2. Compared to the BC group, the number of lactobacillus in the feces of the CS group showed a trend of signi cant decrease from day 1 to day 7 (P < 0.05). Besides the CS+LP group, there was no signi cant increase compared to CS group (P > 0.05) in other six treatment groups on day 7 ( Fig. 2A). On day 1, 4 and 7, the number of bi dobacterium in CS group was signi cantly lower than that in BC group (P < 0.05). Compared to the CS group, it was signi cantly increased in CS+LP, CS+S, CS+FS and CS+FSH groups on day 7 (P < 0.05) (Fig. 2B). In addition, the number of enterococcus in CS group was signi cantly lower than that of BC group on day 4 and 7 (P < 0.05). Slightly different from bi dobacterium, the number of enterococcus was signi cantly increased in CS+LP, CS+S, CS+FB and CS+FS groups compared to CS group on day 4 and 7 (P < 0.05) (Fig. 2C). In the CS group, an obvious decrease in the abundance of colibacillus was observed on day 1, 4 and 7 (P < 0.05), and besides CS+B and CS+S groups, the abundance had not been recovered on day 7 in other treatment groups.
Treatments improved gut barrier function in dysbacteriosis mice induced by CS The small intestinal morphology was shown in Fig. 3, 4 and 5. It was observed that CS caused intestinal mucosal injury in duodenum, jejunum and ileum, including short and broad villi, deep crypts, incomplete and fractured structures. As seen in Fig. 3A and 3C, the villus height and VH/CD of duodenum were reduced (P < 0.05) in CS group and the villus height was increased in CS+LP, CS+B, CS+S, CS+FB and CS+FS groups (P < 0.05). Interestingly, the VH/CD of duodenum was obviously increased only in CS+S and CS+FS groups (P < 0.05). Fig. 4 showed that mice with CS had lower villus height and VH/CD in jejunum than mice in BC group (P < 0.05), and the crypt depth was enhanced notably in CS+LP group compared to CS group (P < 0.05). The VH/CD was increased signi cantly in CS+S, CS+FB, CS+FS groups compared to CS group (P < 0.05). Analogously, mice with CS had shorter villus height and VH/CD in ileum than mice in BC group (P < 0.05) (Fig. 5C). Unlike jejunum, the villus height of ileum enhanced in CS+SH group, and the VH/CD greatly enhanced in CS+S, CS+SH and CS+FB groups (P < 0.05), respectively.
The mRNA relative expression of aquaporins (AQP1, 3, and 4) and TJ proteins (ZO-1 and Occludin) in duodenum and colon are presented in Fig. 6 and 7. Compared to BC group, the mRNA expression levels of AQP1 and ZO-1 were markedly reduced (P < 0.05) in CS gavaged mice. However, the AQP1 mRNA expressions was enhanced in the CS+LP, CS+B and CS+FS groups as compared to the CS group (P < 0.05). And the ZO-1 expression was obviously increased in CS+LP, CS+B and CS+S groups as compared to the CS group (P < 0.05). Furthermore, although compared to BC group, the mRNA expressions of AQP4 was not signi cantly decreased in CS group, when compared to the CS group, it was obviously enhanced in CS+FS group (P < 0.05) (Fig. 6).
As seen in Fig. 7, the mRNA expression levels of AQP1 and ZO-1 were evidently increased (P < 0.05) in other treatment groups as compared to CS group. In addition, the Occludin mRNA was obviously higher in CS+B, CS+S, CS+SH and CS+FS groups than CS group (P < 0.05). Whereas, compared with the BC group, the changes in AQP3 and AQP4 mRNA expression were not signi cant in other treatment groups.

Identi cation of the compounds in S and FS
A principal component analysis (PCA) model was constructed in Fig. 8A. The aggregation trend of two groups was absolutely separated in this model, and the spots were gathered together in their own group.
To investigate distinct signi cant compounds between S and FS, the orthogonal partial least squaresdiscriminant analysis (OPLS-DA) model was established. An obvious separation trend between the two groups was observed in the Fig. 8B, and the interpretation rate of the model to X and Y matrix parameters R2X = 0.809 (80.9%), R2Y = 1 (100%), and prediction ability Q2Y = 0.997 (99.7%) indicated that the model had an excellent reliability and prediction.
The results of heat map (Fig. 9A) showed the distribution of differential compounds in the S and FS groups. Differential compounds were screened out (FC > 1.2, P value < 0.05 and VIP > 1), and 121 signi cantly changed compounds were identi ed in volcano plot (Fig. 9B). Among them, 30 compounds in the FS group were obviously up-regulated compared to the S group, such as Pyrocatechol, Nicotinic acid and (S)-(-)-2-Hydroxyisocaproic acid, etc, and 91 compounds were signi cantly down-regulated, like Maleic acid, Nicotinamide, Poricoic acid etc. The details were shown in supplementary le 1.

Discussion
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 ora 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 ora 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 ora 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 cipro oxacin 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 bene cial bacteria, effectively restore and balance the intestinal ora, but the intestinal ora 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 de ciency type is the largest among antibiotic-associated diarrhea patients, and various tonifying Qi-invigorating TCMs are used to cure bowel diseases, treat intestinal ora 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 ora and promoting the reproduction of bene cial bacteria such as bi dobacteria 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 signi cantly reduced after continuous treatment with TCM, LP, and FTCM for 7 days, among which, FS signi cantly 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 bi dobacterium of mice in LP, FS and FSH increased signi cantly compared to the CS group level, and the number of colibacillus decreased signi cantly, which shows that LP, FS and FSH have the function of supporting the growth of bene cial ora 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 bene cial 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 modi ed [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 ora 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 signi cantly 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 bene cial 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 e cient [39]. Studies have shown that dietary supplementation of methionine is bene cial to intestinal development and antioxidant function of pigs [40]. The two AA may play a signi cant 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

Declarations
All institutional and national guidelines for the care and use of laboratory animals were followed.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Consent for publication
Not applicable. Figure 1 Stool weigh, total number of fecal output and fecal water content in 2 hours of day 1(A), 4 (B) and 7(C).         The heat maps (A) and volcano plots (B) of signi cantly different compounds in Sijunzi tang (S) and

Figures
Fermented Sijunzi tang (FS). The red spots indicate signi cantly up-regulated compounds, and the green spots represent signi cantly down-regulated compounds.