Lactobacillus johnsonii alleviates rhubarb-induced diarrhoea by regulating the gut microbiota and TLR4/NF-κB signalling pathway

Abstract

Background: Rhubarb (RH) is a commonly used traditional Chinese medicine (TCM) for treating digestive system diseases. However, long-term or excessive use of RH can cause gastrointestinal adverse reactions, such as diarrhoea. RH steaming with wine (PRH) can affect gut microbiota (GM) and alleviate diarrhoea caused by rhubarb. Nevertheless, the causal relationship between differential strains and wine steaming in alleviating RH-induced diarrhoea remains unclear.

Purpose: This study aimed to further elucidate the mechanism of wine steaming in alleviating the RH-induced diarrhoea through establishing a causal relationship.

Methods: The components of RH and PRH were detected using ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) technology. Differential strains were screened using metagenomic sequencing technology. To evaluate the alleviating effect of Lactobacillus johnsonii (L. john) on RH-induced diarrhoea, the faecal water rate, intestinal propulsion rate, intestinal transit time, and six-hour defecation volume were measured. Histopathological observations of the duodenum, jejunum, and ileum were conducted using the hematoxylin-eosin (HE) staining method. Meanwhile, the levels of inflammatory factors and immunoglobulins (IgG and IgA) in the ileum were detected by enzyme-linked immunosorbent assay (ELISA). The counts of CD4⁺, CD8⁺, and T regulatory cells (Treg) in peripheral blood were measured using flow cytometry. The protein expression of toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB), zonula occludens-1 (ZO-1), and occludin in the ileum tissue was detected using immunohistochemistry and Western blotting. Faecal samples were analysed using 16S rRNA sequencing technology, and the levels of short-chain fatty acids (SCFAs) were detected using gas chromatography-mass spectrometry (GC-MS). Endogenous metabolites were analysed using UPLC-MS.

Results: Thirty-seven chemical ingredients in RH and PRH were identified, mainly containing anthraquinones, phenolic acids, and flavonoids. The relative peak areas of anthraquinone components decreased in PRH. Screened from RH and PRH, the differential strain L. john alleviated diarrhoea induced by RH, decreased faecal water rate and intestinal propulsion rate, and improved the degree of pathological damage in the small intestine. L. john can downregulate the levels of inflammatory factors (IL-6, TNF-α, IL-17, and IFN-γ), inhibit the expression of TLR4/NF-κB, upregulate the expression of ZO-1 and Occludin, increase the contents of IgG and IgA, CD4⁺/CD8⁺ ratio, and Treg cell percentage. The supplementation of L. john can regulate the composition of GM, with decreased Streptococcus and increased norank_f_Muribaculaceae, and elevate the levels of SCFAs, including acetic acid, propionic acid, and butyric acid mediated by GM. Metabolic analysis showed that L. john alleviated the disorder in the glycerophospholipid metabolic pathway.

Conclusion: L. john can alleviate diarrhoea and intestinal pathological damage caused by RH by improving GM dysbiosis and SCFAs level abnormalities, restoring cellular immune function and intestinal barrier function, and regulating abnormal expression of TLR4/NF-κB and release of inflammatory factors. The findings have revealed the causal relationship between GM and the alleviation of RH-induced diarrhoea by steaming with wine and contributed to the understanding of the mechanism of wine steaming in alleviating the RH-induced diarrhoea.

Keywords: Gut microbiota; Immune function; Lactobacillus johnsonii; Rhubarb; Short-chain fatty acids.