NMR-based metabonomics reveals the dynamic effect of electro-acupuncture on central nervous system in gastric mucosal lesions (GML) rats

Abstract

Background: Gastric mucosal lesions (GML) are common in gastric diseases and seriously affect the quality of life. There are inevitable side effects in drug therapy. Acupuncture is an important part of traditional Chinese medicine. Electro-acupuncture (EA) has unique curative effect in treatment of GML. However, there are still few reports on the central mechanism of electro-acupuncture in treatment of GML. In this study, NMR metabonomics was used to explore the central metabolic change mechanism of electro-acupuncture in treatment of GML.

Methods: SD rats were randomly divided into Control, GML and EA groups. According to different intervention time, each group was further divided into 3 subgroups. This study mainly established GML model rats by 75% ethanol. Dynamic expressions of metabolites in cerebral cortex and medulla were observed by 1D ¹H Nuclear Magnetic Resonance (NMR) metabolomics, combined with gastric mucosal histopathological examination to evaluate the time-effect relationship of electro-acupuncture at Zusanli (ST36) and Liangmen (ST21) points for 1 day, 4 days and 7 days treatment of GML.

Results: The results showed that the repair effect of electro-acupuncture on gastric mucosal injury was the most obvious in 4 days and stable in 7 days, and 4 days electro-acupuncture can effectively inhibit GML gastric mucosal inflammation and the expression of inflammatory cells. Meanwhile, the NMR spectrum results of medulla and cerebral cortex showed that, 21 potential metabolites were identified to participate in the mechanism of pathogenesis of GML and the regulation of electro-acupuncture, including 15 in medulla and 10 in cerebral cortex. Metabolic pathway analysis showed that the differential metabolites involved 19 metabolic pathways, which could be divided into energy, neurotransmitters, cells and cell membrane and antioxidation according to their functions. The correlation analysis of stomach, medulla and cerebral cortex shows that the stimulation signal of GML may reach the cerebral cortex from the stomach through medulla, and electro-acupuncture can treat GML by regulating the central nervous system (CNS).

Conclusions: 4 days electro-acupuncture treatment can significantly improve gastric mucosal injury, and the curative effect tends to be stable in 7 days treatment. Meanwhile, the pathogenesis of GML and the efficacy of electro-acupuncture involve metabolic pathways such as energy, neurotransmitters, cells and antioxidation, and electro-acupuncture can treat GML by regulating CNS.

Keywords: Dynamic expression; Electro-acupuncture; Gastric mucosal lesions; NMR metabonomics.

Fig. 1

Histopathological observation of gastric mucosa in each group. a1 gastric mucosa of rats in Control-T1 subgroup; b1 gastric mucosa of rats in GML-T1 subgroup; c1 gastric mucosa of rats in EA-T1 subgroup; a2 gastric mucosa of rats in Control-T4 subgroup; b2 gastric mucosa of rats in GML-T4 subgroup; c2 gastric mucosa of rats in EA-T4 subgroup; a3 gastric mucosa of rats in Control-T7 subgroup; b3 gastric mucosa of rats in GML-T7 subgroup; c3 gastric mucosa of rats in EA-T7 subgroup (magnification × 400; Scale bar, 50 µm)

Fig. 2

Observation of inflammation and immune cell expression in gastric mucosa in each group. a Representative photomicrographs of gastric NF-κB p65 immunohistochemical staining in each group. b Representative photomicrographs of gastric CD3 immunohistochemical staining in each group. c Representative photomicrographs of gastric Ly6G/Ly6C immunohistochemical staining in each group (magnification × 200; Scale bar, 100 µm). d AOD analysis of IHC results of NF-κB p65. e AOD analysis of IHC results of CD3.f AOD analysis of IHC results of Ly6G/Ly6C. ^***P < 0.01 compared with the control group; ^###P < 0.001, ^##P < 0.01compared with the GML group

Fig. 3

The typical ¹H NMR spectra extracted from the tissues of stomach, medulla and cerebral cortex. (1, Low density lipoprotein (LDL); 2, Very low density lipoprotein (VLDL); 3, Isoleucine; 4, Leucine; 5, Valine; 6, 2-isoprene; 7, Ethanol; 8, Methylmalonic acid; 9, Lactate; 10, Alanine; 11, Lysine; 12, Gamma aminobutyric acid (GABA); 13, Acetate; 14, Acetyl aspartic acid; 15, Glutamate; 16, Glutamine; 17, Glutathione; 18, Pyruvic acid; 19, Oxaloacetate; 20, Succinate; 21, α-Ketoglutarate; 22, Citric acid; 23, Sarcosine; 24, Aspartate; 25, DMG; 26, Creatine; 27, Creatinine; 28, Phenylalanine; 29, Ethanolamine; 30, Choline; 31, Phosphocholine; 32, Glycerophosphocholine; 33, β-Glucose; 34, Taurine; 35, Myo-inositol; 36, Methanol; 37, α-Glucose; 38, Glycine; 39, Glycerol; 40, Glycogen; 41, Guanosine acetate; 42, Serine; 43, Hippurate; 44, Phosphocreatine; 45, Inosine; 46, Adenosine; 47, Allantoin; 48, Uridine 5'-diphosphoglucose; 49, Uracil; 50, Cytidine; 51, Uridine; 52, Fumarate; 53, Tyrosine; 54, 3-Methylhistidine; 55, Histidine; 56, Xanthine; 57, Nicotinamide; 58, Formate)

Fig. 4

OPLS-DA scores plots and corresponding S-plots from medulla of rats. a and d scores plots and S-plots in Control-T1 and GML-T1 (R2X = 0.66cum, R2Y = 0.896cum, Q2 = 0.798cum); b and e scores plots and S-plots in Control-T4 and GML-T4 (R2X = 0.49cum, R2Y = 0.918cum, Q2 = 0.719cum); c and f scores plots and S-plots in Control-T7 and GML-T7 (R2X = 0.538cum, R2Y = 0.797cum, Q2 = 0.527cum); g and j scores plots and S-plots in GML-T1 and EA-T1 (R2X = 0.651cum, R2Y = 0.989cum, Q2 = 0.925cum); h and k scores plots and S-plots in GML-T4 and EA-T4 (R2X = 0.413cum, R2Y = 0.954cum, Q2 = 0.596cum); i and l scores plots and S-plots in GML-T7 and EA-T7 (R2X = 0.584cum, R2Y = 0.928cum, Q2 = 0.8cum)

Fig. 5

OPLS-DA scores plots and corresponding S-plots from cerebral cortex of rats. a and d scores plots and S-plots in Control-T1 and GML-T1 (R2X = 0.637cum, R2Y = 0.892cum, Q2 = 0.793cum); b and e scores plots and S-plots in Control-T4 and GML-T4 (R2X = 0.318cum, R2Y = 0.977cum, Q2 = 0.795cum); c and f scores plots and S-plots in Control-T7 and GML-T7 (R2X = 0.538cum, R2Y = 0.797cum, Q2 = 0.527cum); g and j scores plots and S-plots in GML-T1 and EA-T1 (R2X = 0.842cum, R2Y = 0.999cum, Q2 = 0.991cum); h and k scores plots and S-plots in GML-T4 and EA-T4 (R2X = 0.738cum, R2Y = 0.979cum, Q2 = 0.966cum); i and l scores plots and S-plots in GML-T7 and EA-T7 (R2X = 0.812cum, R2Y = 0.958cum, Q2 = 0.807cum)

Fig. 6

Metabolic pathways related to ¹H NMR based differential metabolites in rats. (1, Alanine, aspartate and glutamate metabolism; 2, Glycine, serine and threonine metabolism; 3, D-Glutamine and D-glutamate metabolism; 4, Taurine and hypotaurine metabolism; 5, Starch and sucrose metabolism; 6, Glutathione metabolism; 7, Pyruvate metabolism; 8, Aminoacyl biosynthesis; 9, Glyoxylate and dicarboxylate metabolism; 10, Arginine biosynthesis; 11, Arginine and proline metabolism; 12, Glycolysis/Gluconeogenesis; 13, Methyl butyrate metabolism; 14, Citrate acid cycle (TCA cycle); 15, Primary bile acid biosynthesis; 16, Galactose metabolism; 17, Glycerophospholipid metabolism; 18, Cysteine and methionine metabolism; 19, Purine metabolism). The purple square represents different metabolites in cerebral cortex; the blue circle represents different metabolites in medulla. Black font means intermediate products between differential metabolites; red means difference metabolites between control and GML group

Fig. 7

Correlation analysis of potential metabolites in stomach, medulla and cerebral cortex of rat. a correlation analysis of differential metabolites in GML-T1 subgroup; b correlation analysis of differential metabolites in GML-T4 subgroup; c correlation analysis of differential metabolites in EA-T1 subgroup; d correlation analysis of differential metabolites in EA-T4 subgroup. The red font represents different metabolites in gastric tissue; the green font represents different metabolites in medulla; the blue font represents different metabolites in cerebral cortex. The red area represents a positive correlation between the two metabolites, and the blue area represents a negative correlation