Sini San ameliorates duodenal mucosal barrier injury and low‑grade inflammation via the CRF pathway in a rat model of functional dyspepsia

Abstract

The gut‑brain interaction is associated with impaired duodenal mucosal integrity and low‑grade inflammation, which have been proven to be important pathological mechanisms of functional dyspepsia (FD). Sini San (SNS) is a classical Chinese medicine used to treat FD, but its underlying mechanisms are poorly understood. The aim of the present study was to evaluate the effects of SNS on duodenal mucosal barrier injury and low‑grade inflammation with FD, and to assess its potential molecular mechanisms on the brain‑gut axis. FD rats were established using the iodoacetamide and tail‑squeezed methods. The expression of corticotropin‑releasing factor (CRF), CRF receptor 1 (CRF‑R1) and CRF‑R2, were determined by western blot analysis and/or immunohistochemistry (IHC). In addition, mast cell (MC) migration was assessed by IHC with an anti‑tryptase antibody, and histamine concentration was quantified using ELISA. The mRNA expression levels of tryptase and protease‑activated receptor 2 (PAR‑2) were quantified using reverse transcription‑quantitative PCR, and the protein expression levels of zona occludens protein 1 (ZO‑1), junctional adhesion molecule 1 (JAM‑1), β‑catenin and E‑cadherin were determined via western blot analysis. It was demonstrated that the expression level of CRF was downregulated in the central nervous system and duodenum following SNS treatment, and that SNS modulated the expression of both CRF‑R1 and CRF‑R2. In addition, SNS suppressed MC infiltration and the activity of the tryptase/PAR‑2 pathway in the duodenum. Furthermore, treatment with SNS restored the normal expression levels of ZO‑1, JAM‑1 and β‑catenin in FD rats. These findings suggested that the therapeutic effects of SNS on FD were achieved by restoring mucosal barrier integrity and suppressing low‑grade inflammation in the duodenum, which was at least partially mediated via the CRF signaling pathway.

Keywords: Sini san; functional dyspepsia; corticotropin-releasing factor; mucosal barrier integrity; low-grade inflammation; mast cell.

Figure 1

(A) Western blot analysis of CRF in the hypothalamus and duodenum. The relative grey value of CRF in tissues of the (B) hypothalamus and (C) duodenum. Expression levels of CRF were elevated in the hypothalamus and duodenum. SNS reduced the expression level of CRF in these two tissues. N=6; ^**P<0.01 compared with the control group; ^##P<0.01 compared with the model group. CRF, corticotropin-releasing factor; SNS, Sini San.

Figure 2

Immunohistochemical staining of CRF in the spinal cord and duodenum. Expression levels of CRF were increased in the (A) spinal cord and (B) the duodenum. SNS reduced the expression level of CRF in these tissues. Magnification, ×200; N=4. CRF, corticotropin-releasing factor; SNS, Sini San.

Figure 3

(A) Western blot analysis of CRF-R1 and CRF-R2 in the duodenum. Relative grey value of (B) CRF-R1 and (C) CRF-R2 expression levels in duodenal tissues. Expression levels of CRF-R1 were elevated in FD rats and reduced in the SNS group. Expression patterns of CRF-R2 were the reverse of those for CRF-R1. N=6. ^**P<0.01 compared with the control group; ^#P<0.05 and ^##P<0.01 compared with the model group. CRF, corticotropin-releasing factor; CRF-R1, corticotropin-releasing factor receptor 1; CRF-R2, corticotropin-releasing factor receptor 2; SNS, Sini San; FD, functional dyspepsia.

Figure 4

(A) Immunohistochemical staining of mast cells in the duodenum with an anti-tryptase antibody. Magnification, ×100. (B) Concentration of histamine, as determined by ELISA. The expression of mast cell tryptase was elevated in the model group, and reduced following SNS treatment. The concentration of histamine was markedly higher in FD rats, and was decreased in the SNS group. N=4 (A); N=6 (B); ^**P<0.01 compared with the control group; ^##P<0.01 compared with the model group. SNS, Sini San; FD, functional dyspepsia.

Figure 5

Relative mRNA expression levels of (A) tryptase and (B) PAR-2. mRNA expression levels of tryptase and PAR-2 were increased in the FD group, and SNS treatment reduced the expression of tryptase and PAR-2. N=6 for each group. ^**P<0.01 compared with the control group; ^##P<0.01 compared with the model group. PAR-2, protease-activated receptor 2; FD, functional dyspepsia; SNS, Sini-san.

Figure 6

(A and D) Western blot analysis of ZO-1, JAM-1, β-catenin and E-cadherin expression in duodenal tissues. Relative grey values of ZO-1, JAM-1, β-catenin and E-cadherin expression levels. (B, C, E and F) Compared with the control group, the protein expression level of ZO-1, JAM-1 and β-catenin was markedly reduced in the model rats, and SNS treatment increased the expression levels of these three proteins. There was no significant difference in the protein expression level of E-cadherin between groups. N=6 for each group. ^**P<0.01 compared with the control group; ^#P<0.05 and ^##P<0.01 compared with the model group. ZO-1, zona occludens protein-1; JAM-1, junctional adhesion molecule-1; SNS, Sini San; FD, functional dyspepsia.