Mechanism of electroacupuncture in improving central sensitization in rats with non-erosive reflux disease of liver-stomach disharmony pattern based on NGF/TrkA signaling pathway

To investigate the effect of electroacupuncture (EA) on central sensitization in rats with non-erosive reflux disease (NERD) of liver-stomach disharmony pattern, so as to explore its underlying mechanisms in improving NERD.

Forty male SD rats were randomly divided into control, model, EA, and medication (western medicine) groups (n=10 per group). The NERD model of liver-stomach disharmony pattern was established by intraperitoneal injection of ovalbumin plus aluminum hydroxide adjuvant (basic sensitization) combined with chronic tail-pinching stress stimulation. Rats in the EA group received EA stimulation (2 Hz, a strength that could induce a slight tremor of the limbs) at “Zhiyang” (GV9), “Shendao” (GV11), “Dazhui” (GV14), and “Baihui” (GV20) for 30 min, once daily for 2 consecutive weeks. Rats of the medication group received gavage of omeprazole (1.8 mg/kg) once daily for 2 weeks. The remaining 2 groups were administered an equal volume of distilled water. Before and after the intervention, the sucrose preference test was performed, and the mechanical withdrawal threshold (MWT) was measured using von Frey filaments. After the intervention, the abdominal withdrawal reflex (AWR) test was conducted, followed by an esophageal acid perfusion test. After perfusion, the rats were euthanized for tissue collection. The number of synaptic vesicles in the spinal cord dorsal horns and the intercellular space of the esophageal mucosal epithelium were observed by transmission electron microscopy (TEM). The contents of serum gastrin (GAS) and motilin (MTL) were measured by ELISA. The protein expression levels of nerve growth factor (NGF), tropomyosin receptor kinase A (TrkA), glutamate receptor AMPA type subunit 1 (GluA1), and postsynaptic density protein 95 (PSD95) in the spinal cord tissue were detected by Western blot, and the mRNA expression levels of NGF and TrkA in the spinal cord were measured by real-time quantitative PCR. The positive expression of GluA1 and PSD95 in the spinal cord was also assessed by immunofluorescence staining.

Compared with the control group, the model group showed a significant decrease in the sucrose preference rate, MWT, body weight, visceral pain threshold, and serum GAS and MTL contents (P<0.01), and a significant increase in the AWR score of 40, 60, and 80 mmHg (P<0.01), number of spinal synaptic vesicles, expression levels of NGF, TrkA, GluA1 and PSD95 proteins, and NGF and TrkA mRNAs, as well as the immunofluorescence intensity of GluA1 and PSD95 (P<0.01). Following the intervention, the modeling induced decrease and increase of all the indexes (except for GluA1 protein expression) mentioned above in the EA group, and the modeling induced decreased levels of sucrose preference rate, MWT, body weight, visceral pain threshold, serum MTL, and the increased levels of the expression of NGF, TrkA, GluA1 and PSD95 proteins and TrkA mRNA in the medication group were reversed (P<0.01, P<0.05). The effect of EA was obviously superior to medication in up-regulating the levels of MWT and visceral pain threshold (P<0.01). Results of TEM showed that in comparison with the control group, the number of synaptic vesicles in the spinal cord was increased, and the intercellular spaces of esophageal mucosal epithelial cells were apparently widened in the model group, while after EA intervention, the number of synaptic vesicles was significantly decreased, and the intercellular spaces of esophageal mucosal epithelial cells tended to recover.

Electroacupuncture can alleviate visceral hypersensitivity and reduce central sensitization in rats with NERD of the liver-stomach disharmony type, which may be associated with its function in inhibiting NGF/TrkA signaling pathway of spinal cord.