Electroacupuncture Regulates TRPV1 through PAR2/PKC Pathway to Alleviate Visceral Hypersensitivity in FD Rats

Abstract

Visceral hypersensitivity (VH) is the predominant pathogenesis of functional dyspepsia (FD). Duodenal hypersensitivity along with nausea further reduces the comfort level in gastric balloon dilatation and inhibits gastric receptive relaxation. The potential mechanism behind electroacupuncture- (EA-) mediated alleviation of VH has not been elucidated. In an FD rat model with tail clamping stress, iodine acetamide (IA) induced VH. The rats were treated with EA with or without PAR2 antagonist FSLLRY-NH2, and the body weight, gastric sensitivity, compliance, and gastrointestinal motility were determined. Mast cells and activated degranulation were stained with toluidine blue (TB) staining and visualized under a transmission electron microscope (TEM). Immunofluorescence was used to detect the expression of PAR2, PKC, and TRPV1 in the duodenum and dorsal root ganglion (DRG) and that of CGRP, SP in DRG, and c-fos in the spinal cord. EA alone and EA + antagonist enhanced the gastrointestinal motility but diminished the expression of TRPV1, CGRP, SP, and c-fos-downstream of PAR2/PKC pathway and alleviated VH in FD rats. However, there was no obvious superposition effect between the antagonists and EA + antagonists. The effect of EA alone was better than that of antagonists and EA + antagonists 2 alone. EA-induced amelioration of VH in FD rats was mediated by TRPV1 regulation through PAR2/PKC pathway. This protective mechanism involved several pathways and included several targets.

Figure 1

Decrease in the growth rate of body weight and increase in the sensitivity in the model group. (a) The body weight in two groups at different times. (b) Stomach sensitivity and compliance. (c) HE staining of gastrointestinal tissue (× 200 magnification). No pathological alterations, including ulcer and bleeding, were observed in the intestinal mucosa of the stomach and duodenum in all groups. Scale bar: 100 um. Compared with the control group, ^※P < 0.05, ^□P < 0.0, ^#P < 0.001, and ^△P < 0.0001.

Figure 2

Electroacupuncture mediates the beneficial effects of visceral hypersensitive FD. (a) EA-mediated reduction in the residual rate in the stomach and increase in the propulsive rate of the small intestine in rats. (b) EA reduced the number and activation of mast cells in the duodenum. (c) The ultrastructural characteristics of fragmented or allergic degranulation of duodenal mast cells were observed under TEM (× 1700 magnification). Compared with the control group, ^△P < 0.0001, ^□P < 0.01; compared with the model group, ^#P < 0.001, ^☆P < 0.0001; compared with the EA group, ^※P < 0.05, ^□P < 0.01. Scale bar: 50 um.

Figure 3

EA downregulates the positive expression of TRY, PAR2, PKC, and TRPV1 in duodenal mast cells. (a) The positive expression of TRY in the duodenum was detected by immunohistochemistry (× 400 magnification). (b) The positive expressions of PAR2, PKC, and TRPV1 in the duodenum were identified by immunofluorescence (× 400 magnifications). (c) The protein expression levels of PAR2, PKC, TRPV1, and pTRPV1 in the duodenum were detected by western blot. Compared with the control group, ^△P < 0.0001, ^□P < 0.01; compared with the model group, ^☆P < 0.0001; compared with EA group, ^※P < 0.05. Scale bar: 50 um.

Figure 4

EA reduced the positive expression of PAR2, PKC, TRPV1, CGRP, and SP in DRG and c-fos in the spinal cord. (a) The positive expressions of PAR2, PKC, and TRPV1 in DRG were detected by immunofluorescence (× 400 magnification). (b) The protein expressions of PAR2, PKC, TRPV1, ptrpv1, CGRP, and SP in DRG were detected by western blot. (c) The protein expression of c-fos in spinal cord were detected by western blot. Compared with the control group, ^∗P < 0.05, ^□P < 0.01; compared with model group, ^#P < 0.05, ^☆P < 0.0001; compared with EA group, ^※P < 0.05.