How Tongxie-Yaofang Regulates Intestinal Synaptic Plasticity by Activating Enteric Glial Cells and NGF/TrkA Pathway in Diarrhea-Predominant Irritable Bowel Syndrome Rats

Abstract

Purpose: Diarrhea-predominant irritable bowel syndrome (D-IBS) is a frequent functional gastrointestinal disease that affects health and quality of life owing to its high incidence and recurrence rate. Tongxie-Yaofang (TXYF) is a traditional Chinese medicine prescribed for D-IBS. However, the therapeutic mechanism of TXYF has not been fully elucidated. This study aimed to investigate the effects of TXYF on visceral hypersensitivity in stress-induced D-IBS rats and the underlying mechanisms.

Methods: Electromyographic (EMG) activity of the external oblique muscles and the abdominal withdrawal reflex (AWR) score captured by Barostat were used to quantify the effect of TXYF on visceral sensitivity. Transmission electron microscopy (TEM) was used to observe the ultrastructure of the enteric nervous system (ENS). For molecular detection, the colonic expression of enteric glial cell's (EGC's) activation markers, glial fibrillary acidic protein (GFAP) and calcium-binding protein S100β, NGF, TrkA, synaptic plasticity-related factors, synaptophysin (SYN) and postsynaptic density-95 (PSD-95), glutamate, glutamate receptors α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR), and N-methyl-D-aspartate receptor (NMDAR) were detected by immunohistochemistry, enzyme-linked immunosorbent assay, and real-time PCR. An ex vivo experiment was conducted to measure the EGC-induced NGF release.

Results: TXYF decreased the EMG activity and AWR scores in rats with D-IBS. Under TEM, TXYF improved the dense and irregular nerve arrangement, narrowed the synaptic cleft, and decreased the number of synaptic vesicles in D-IBS rats. In addition, TXYF decreased the expression of GFAP, S100β, SYN, and PSD-95; down-regulated the levels of NGF, TrkA, and glutamate; and reduced the mRNA expression of AMPAR1, NMDAR1, and NMDAR2B. In an ex vivo experiment, TXYF decreased NGF release in D-IBS rats, and this trend disappeared under EGC inhibition.

Conclusion: TXYF alleviated visceral hypersensitivity in D-IBS rats possibly by improving synaptic plasticity through inhibiting the activity of EGCs and the NGF/TrkA signaling pathway in the colon.

Keywords: Tongxie-Yaofang; diarrhea-predominant irritable bowel syndrome; enteric glial cell; synaptic plasticity; visceral hypersensitivity.

Figure 1

Experimental design. The neonatal maternal separation and restraint stress were conducted respectively during postnatal days 2 to 21 and 50 to 59. From postnatal days 60 to 74, the rats in TXYF group were given TXYF formula. After drug intervention, the rats were arranged for the detection of visceral sensitivity and ex vivo experiment. And the colonic tissues were obtained from the post-intervention rats for real-time PCR, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC) and transmission electron microscope (TEM) detection.

Figure 2

Procedure of ex vivo experiment. The NGF release detection was conducted by the ex vivo experiment. The colonic segments from the three groups were incubated in Krebs-Hensleit’s solution (K-HS) for 30 min, next the tissues were incubated with vehicle (K-HS) or DL-fluorocitrate for 30 min. Then the supernatants were collected for NGF level measurement. The experimental environment was maintained at 37°C and gassed with 95%O₂ and 5%CO₂.

Figure 3

Major components of TXYF formula detected by UPLC-MS/MS. (A) Positive ion chromatogram, (B) negative ion chromatogram.

Figure 4

Effect of TXYF on colonic sensitivity in D-IBS rats. (A) Abdominal withdrawal reflex (AWR) score and (B) area under the curve (AUC) of electromyography (EMG) in response to graded distension pressure were measured to evaluate the colonic sensitivity. (C) Typical images of EMG activity in the external oblique muscle in response to graded colorectal distension. *P < 0.05, **P < 0.01 vs Normal group, ^#P < 0.05, ^##P < 0.01 vs D-IBS group. Data are presented as mean ± SEM (n=6).

Figure 5

Effect of TXYF on the expression of glial fibrillary acidic protein (GFAP) and calcium- binding protein S100β in the colon. (A) Immunohistochemical staining of GFAP. (B) Immunohistochemical staining of S100β (Scale bar 100μm). (C) Integrated optical density (IOD) of GFAP in the colon. (D) Integrated optical density (IOD) of S100β in the colon. *P < 0.01 vs Normal group, ^#P < 0.05, ^##P < 0.01 vs D-IBS group. Data are presented as mean ± SEM (n=5).

Figure 6

Effect of TXYF on NGF/TrkA level and NGF release in the colon. (A) Level of NGF. (B) Level of TrkA. (C) Basal NGF release. (D) NGF release pretreated with EGC inhibitor DL-fluorocitrate. *P < 0.05, **P < 0.01 vs Normal group, ^#P < 0.05, ^##P < 0.01 vs D-IBS group. Data are presented as mean ± SEM (n=6).

Figure 7

Effect of TXYF on the ultrastructure of ENS in the colon (Scale bar 1.0μm). Black arrows indicated synaptic vesicles; red arrows indicated the spaces between each nerve.

Figure 8

Effect of TXYF on the expression of synaptophysin (SYN) and postsynaptic density-95 (PSD-95) in the colon. (A) Immunohistochemical staining of SYN. (B) Immunohistochemical staining of PSD-95. (Scale bar 100μm). (C) Integrated optical density (IOD) of SYN in the colon. (D) Integrated optical density (IOD) of PSD-95 in the colon. *P < 0.05, **P < 0.01 vs Normal group, ^#P < 0.05, ^##P < 0.01 vs D-IBS group. Data are presented as mean ± SEM (n=5).

Figure 9

Effect of TXYF on the glutamate pathway in colon. (A) Level of glutamate; (B) Relative mRNA expression of AMPAR1; (C) Relative mRNA expression of NMDAR1; (D) Relative mRNA expression of NMDAR2B. *P < 0.05 vs Normal group, ^#P < 0.05, ^##P < 0.01 vs D-IBS group. Data are presented as mean ± SEM (n=6).

Figure 10

The model diagram of TXYF mechanism pathway on D-IBS. TXYF could inhibit EGC activation and NGF/TrkA signaling pathway, then affect neural synaptic plasticity to alleviate intestinal hypersensitivity in D-IBS.