Modified Renshen Wumei Decoction Alleviates Intestinal Barrier Destruction in Rats with Diarrhea

Abstract

Modified Renshen Wumei decoction (MRWD), a famous traditional Chinese medicine, is widely used for treating persistent diarrhea. However, as the mechanism by which MRWD regulates diarrhea remains unknown, we examined the protective effects of MRWD on intestinal barrier integrity in a diarrhea model. In total, 48 male rats were randomly distributed to four treatment groups: the blank group (CK group), model group (MC group), Medilac-Vita group (MV group) and Chinese herb group (MRWD group). After a 21-day experiment, serum and colon samples were assessed. The diarrhea index, pathological examination findings and change in D-lactate and diamine oxidase (DAO) contents illustrated that the induction of diarrhea caused intestinal injury, which was ameliorated by MV and MRWD infusion. Metabolomics analysis identified several metabolites in the serum. Some critical metabolites, such as phosphoric acid, taurine, cortisone, leukotriene B4 and calcitriol, were found to be significantly elevated by MRWD infusion. Importantly, these differences correlated with mineral absorption and metabolism and peroxisome proliferator-activated receptor (PPAR) pathways. Moreover, it significantly increased the expression levels of TLR4, MyD88 and p-NF-κB p65 proteins and the contents of IL-1 and TNF-α, while the expression levels of occludin, claudin-1 and ZO-1 proteins decreased. These deleterious effects were significantly alleviated by MV and MRWD infusion. Our findings indicate that MRWD infusion helps alleviate diarrhea, possibly by maintaining electrolyte homeostasis, improving the intestinal barrier integrity, and inhibiting the TLR4/NF-κB axis.

Keywords: Modified renshen wumei decoction; diarrhea; intestinal barrier; metabolomics; rats.

Fig. 1. Physical and physiological effects of MRWD on the colon of rats.

(A) Haematoxylin–eosin (HE) staining of colon tissue. (B) The concentration of D-lactate was detected by ELISA. (C) The concentration of DAO was detected by ELISA. *Compared with the CK group, *p < 0.05 and **p < 0.01. ^#Compared with the MC group, ^#p < 0.05 and ^##p < 0.01.

Fig. 2. Multivariate statistical analysis plot of CK, MC MV and MRWD groups.

The PCA (A) and PLS-DA (B) score plots demonstrate complete separation of the serum samples among the groups.

Fig. 3. Analysis of differential metabolites in the serum samples obtained from CK, MC, MV and MRWD groups.

(A) A volcano plot representing the significant variables for discriminating among the four groups. Significantly increased variables are presented in the red circle, while significantly decreased variables are presented in the green circle. (B) A heatmap of hierarchical clustering analysis (HCA) for differential metabolites. HCA is based on the Euclidean distance; colors from blue to red indicate elevated contents of metabolites.

Fig. 4. Pathway analysis of differential metabolites in the serum samples.

A bubble plot for the identification of the most relevant metabolic pathways. The color of the circles indicates the significance of changes in the metabolites in the corresponding pathway, while the size corresponds to the pathway impact score. The pathway impact score represents the cumulative percentage from the matched metabolite to the total pathway importance.

Fig. 5. Effect of MRWD on the serum parameters.

(A) Validation of the representative metabolites by the metabolomics approach using ELISA. (B) The analysis of serum electrolyte contents. *Compared with the CK group, *p < 0.05, **p < 0.01 and ***p < 0.001. ^#Compared with the MC group, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001.

Fig. 6. MV and MRWD infusion alleviated the destruction of tight junctions (TJs).

(A) The colon samples were analyzed by western blotting using antibodies against claudin-1, ZO-1, occludin and β-actin (used as the protein loading control). (B) The occludin/β-actin ratio in the colon. (C) The claudin-1/β-actin ratio in the colon. (D) The ZO-1/β-actin ratio in the colon. *Compared with the CK group, *p < 0.05, **p < 0.01 and ***p < 0.001. ^#Compared with the MC group, ^#p < 0.05, ^##P < 0.01 and ^###p < 0.001.

Fig. 7. MV and MRWD infusion decreased the inflammatory response in rats with diarrhea.

(A) The colon samples were analyzed by western blotting using antibodies against TLR4, MyD88, p-NF-κB p65, NF-κB p65 and β-actin (used as the protein loading control). (B) The TLR4/β-actin ratio in the colon. (C) The MyD88/β-actin ratio in the colon. (D) The p-NF-κB p65/NF-κB p65 ratio in the colon. (E) The IL-1, IL-6, TNF-α and IFN-γ contents were assessed by ELISA. *Compared with the CK group, *p < 0.05, **p < 0.01 and ***p < 0.001. ^#Compared with the MC group, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001.