Effect of traditional chinese medicine (TCM) and its fermentation using Lactobacillus plantarum on ceftriaxone sodium-induced dysbacteriotic diarrhea in mice

Abstract

Background: Buzhongyiqi decoction (BD), Sijunzi decoction (SD), and Shenlingbaizhu decoction (SHD) have been extensively used clinically for the treatment of diseases caused by spleen-Qi deficiency and microbial fermentation has historically been utilized in traditional Chinese medicine (TCM). This study aimed to investigate the mitigative effect of TCM and fermented TCM (FTCM) with Lactobacillus plantarum (LP) on antibiotic-associated diarrhea, and to select an optimal formula and then identify its compounds.

Methods: Dysbacteriosis in mice was induced by ceftriaxone sodium (CS). The mice were then treated with LP, BD, SD, SHD, fermented BD, fermented SD (FSD), and fermented SHD. Diarrhea indexes, the abundances of gut bacteria, intestinal morphometrics, and mRNA expressions of genes related to intestinal barrier function were assessed. Then, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) were employed to identify and relatively quantify the compounds in the selected decoctions.

Results: CS significantly increased the fecal output weight, the total number of fecal output, and fecal water content, indicating the occurrence of diarrhea. Bacterial culture tests showed that the above symptoms were accompanied by the disruption of specific intestinal flora. TCM, LP, and FTCM alleviated the diarrhea index and recovered the intestinal microbiota. FTCM showed more advantageous than TCM or LP alone. The mRNA expressions of aquaporins (AQPs) and tight junctions (TJs) decreased by CS were enhanced by TCM, LP, and FTCM. In addition, through UHPLC-Q-TOF/MS, (S)-(-)-2-hydroxyisocaproic acid, L-methionine, 4-guanidinobutyric acid (4GBA), and phenyllactate (PLA) in SD and FSD were identified and relatively quantified.

Conclusions: TCM, LP, and TCM fermented with LP alleviated CS-induced diarrhea symptoms, and improved the intestinal flora and barrier function. Four compounds including (S)-(-)-2-hydroxyisocaproic acid, L-methionine, 4GBA, and PLA in FSD, which were identified by UHPLC-Q-TOF/MS, might function in modulating intestinal flora and improving villi structure.

Keywords: Antibiotic; Diarrhea; Fermented TCM; Sijunzi decoction.

Fig. 1

Stool weigh, total number of fecal output and fecal water content in 2 h of 1st day (A), 4th day (B) and 7th day (C). BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 8. ^{a, b, c, d}Labeled means in the bars without a common letter were significantly different (p < 0.05)

Fig. 2

Live bacteria counting of lactobacillus (A), bifidobacterium (B), enterococcus (C) and colibacillus (D) on 1st day, 4th day and 7th day of treatments. BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 3. ^{a, b, c, d, e, f}Labeled means in the bars without a common letter were significantly different (p < 0.05)

Fig. 3

The picture of gross anatomical features of the intestines

Fig. 4

Intestinal morphology (200×) of duodenum in mice with dysbacteriosis induced by ceftriaxone sodium and the alleviation of treatments. A villus height, B crypt depth, C villus height to crypt depth ratio (VH/CD). BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. ^{a, b, c, d}Labeled means in the bars without a common letter were significantly different (p < 0.05). Red arrow: villus atrophy, dissolution, damage, and epithelial cells arranged irregularly

Fig. 5

Intestinal morphology (200×) of jejunum in mice with dysbacteriosis induced by ceftriaxone sodium and the alleviation of treatments. A villus height, B crypt depth, C villus height to crypt depth ratio (VH/CD). BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 3. ^{a, b, c}Labeled means in the bars without a common letter were significantly different (p < 0.05). Red arrow: villus dissolution, damage, irregularly arranged epithelial cells, partially detached, and intestinal gland injury

Fig. 6

Intestinal morphology (200×) ofileum in mice with dysbacteriosis induced by ceftriaxone sodium and the alleviation of treatments. A villus height, B crypt depth, C villus height to crypt depth ratio (VH/CD). BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 3. ^{a, b, c, d}Labeled means in the bars without a common letter were significantly different (p < 0.05). Red arrow: the serosal layer rupture, villus dissolution, epithelial cells arranged irregularly, intestinal gland injury, and enterocyte cytoplasmic vacuolation

Fig. 7

The mRNA relative expression levels of AQP1, AQP3, AQP4, ZO-1, and Occludin genes of duodenum in mice with dysbacteriosis induced by ceftriaxone sodium and the alleviation of treatments (A–E). AQP, Aquaporins; ZO-1, Zonula occludens-1. BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 5. ^{a, b, c}Labeled means in the bars without a common letter were significantly different (p < 0.05)

Fig. 8

The mRNA relative expression levels of AQP1, AQP3, AQP4, ZO-1, and occludin genes of colon in mice with dysbacteriosis induced by ceftriaxone sodium and the alleviation of treatments (A–E). AQP, Aquaporins; ZO-1, Zonula occludens-1. BC: Blank Control, CS: Ceftriaxone Sodium, CS + LP: Ceftriaxone Sodium + Lactobacillus Plantarum, CS + BD: Ceftriaxone Sodium + Buzhongyiqi decoction, CS + SD: Ceftriaxone Sodium + Sijunzi decoction, CS + SHD: Ceftriaxone Sodium + Shenlingbaizhu decoction, CS + FBD: Ceftriaxone Sodium + fermented Buzhongyiqi decoction, CS + FSD: Ceftriaxone Sodium + fermented Sijunzi decoction and CS + FSHD: Ceftriaxone Sodium + fermented Shenlingbaizhu decoction. n = 5. ^{a, b, c}Labeled means in the bars without a common letter were significantly different (p < 0.05)

Fig. 9

The score plots of Principal Component Analysis (PCA) (A) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) (B) of Sijunzi decoction (SD) and fermented Sijunzi decoction (FSD)

Fig. 10

The heat maps (A) and volcano plots (B) of significantly different compounds in Sijunzi decoction (SD) and fermented Sijunzi decoction (FSD). The red spots indicate significantly up-regulated compounds, and the green spots represent significantly down-regulated compounds

Fig. 11

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification maps of significantly different compounds between Sijunzi decoction (SD) and fermented Sijunzi decoction (FSD). The number behind the pathway represents the amount of significantly different compounds in this pathway; the percentage indicates the ratio of amount of significantly different compounds in the pathway to all significantly different compounds