Electroacupuncture at ST36 ameliorates gastric dysmotility in rats with diabetic gastroparesis via the nucleus tractus solitarius-vagal axis

Abstract

Background: Diabetic gastroparesis (DGP), characterized by delayed gastric emptying and impaired motility, poses significant therapeutic challenges due to its complex neural and molecular pathophysiology. Emerging evidence suggests that electroacupuncture (EA) at ST36 modulates gastrointestinal function; however, the precise neuromolecular pathways underlying its efficacy in DGP remain incompletely defined.

Aim: To elucidate the neural mechanisms underlying EA at ST36 improving DGP gastric motility through the nucleus tractus solitarius (NTS)-vagal axis.

Methods: The DGP model was established via a single high-dose intraperitoneal injection of 2% streptozotocin combined with an 8-week high-sugar/high-fat diet. Interventions included EA at ST36, pharmacological modulation [choline acetyltransferase (ChAT) agonist polygalacic acid (PA) and inhibitor antagonist alpha-NETA], and subdiaphragmatic vagotomy. Post-intervention observations included body weight and blood glucose levels. Gastric emptying was evaluated using phenol red assays, gastric slow-wave recordings, and dynamic positron emission tomography-computed tomography imaging. Histopathological analysis (hematoxylin-eosin staining) and molecular assessments (Western blot, immunofluorescence) were performed to quantify gastric smooth muscle-associated factors [neuronal nitric oxide synthase (nNOS), cluster of differentiation 117 (C-kit), stem cell factor (SCF)] and vagal targets [ChAT, α7 nicotinic acetylcholine receptor (α7nAChR)] in the ST36 acupoint region, L4-L6 spinal segments, and NTS. Gastrointestinal peptides [gastrin (Gas), motilin (MLT) and vasoactive intestinal peptide (VIP)] were measured via enzyme-linked immunosorbent assay.

Results: The study found that EA significantly increased the rate of gastric emptying, restored the slow-wave rhythms of the stomach, and improved the architecture of the smooth muscles in the stomach. This was evidenced by a reduction in inflammatory infiltration and an increase in the expression of nNOS, C-kit, and SCF. Mechanistically, EA activated vagal targets (ChAT and α7nAChR) at ST36, transmitting signals via spinal segments L4-L6 to the NTS, subsequently regulating gastrointestinal peptides (Gas, MLT, VIP) and restoring interstitial cells of Cajal (ICCs) function via subdiaphragmatic vagal efferent pathways. It is crucial to note that subdiaphragmatic vagotomy led to the abrogation of EA-induced enhancements in gastric motility and ICC recovery, thereby confirming the indispensable role of vagal efferent signalling.

Conclusion: EA provides a novel molecular mechanism for improving gastrointestinal motility in DGP via a peripheral stimulation (ST36), spinal afferent (L4-L6), brainstem integration (NTS), vagal efferent (gastric) circuit.

Keywords: Diabetic gastroparesis; Electroacupuncture; Gastric motility; Interstitial cells of Cajal; Positron emission tomography-computed tomography imaging; Vagus nerve.

Figure 1

Electroacupuncture has been demonstrated to promote gastric emptying in diabetic gastroparesis rats. A: Body weight (n = 10); B: Non-fasting blood glucose level (n = 10); C: Electrographic recording of gastric motor activity; D: Number of slow wave discharges in the gastric antrum within 5 minutes (n = 5); E: Gastric emptying rate (n = 5); F: Small intestine propulsion rate (n = 5); G: Representative positron emission tomography images; H: Region of interest in the stomach; I: Radioactive concentration in the region of interest in the stomach (n = 5); J: Gastric emptying rate at 3600 seconds (n = 5). Data are expressed as the mean ± SD. ^aP < 0.05. STZ: Streptozotocin; DGP: Diabetic gastroparesis; EA: Electroacupuncture.

Figure 2

Electroacupuncture improves the dysfunction of gastric smooth muscle in diabetic gastroparesis rats. A: Representative images of hematoxylin-eosin in the stomach. Scale bar: 100 μm; B-E: Western blotting analysis and quantification of neuronal nitric oxide synthase, cluster of differentiation 117 (C-kit) and stem cell factor protein levels in stomach tissue (n = 5); F: Representative immunofluorescence images of C-kit. Scale bar: 50 μm. Data are expressed as the mean ± SD. ^aP < 0.05. DGP: Diabetic gastroparesis; EA: Electroacupuncture; nNOS: Neuronal nitric oxide synthase; C-kit: Cluster of differentiation 117; SCF: Stem cell factor; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 3

Electroacupuncture improves gastric motility and is associated with vagus nerve targets. A-C: Western blotting analysis and quantification of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor protein levels in stomach tissue (n = 5); D: Expression of cluster of differentiation 117 +/ChAT + in the stomach in each group. Scale bar: 50 μm; E-G: Enzyme-linked immunosorbent assay to detect the concentration of gastrin, motilin and vasoactive intestinal peptide in supernatant (n = 5). Data are expressed as the mean ± SD. ^aP < 0.05. C-kit: Cluster of differentiation 117; DGP: Diabetic gastroparesis; EA: Electroacupuncture; ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; Gas: Gastrin; MLT: Motilin; VIP: Vasoactive intestinal peptide.

Figure 4

Electroacupuncture activates the vagus nerve target in the ST36 acupoint area. A-C: Western blotting analysis and quantification of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor protein levels in acupoint skin (n = 5); D: Representative immunofluorescence images of ChAT. Scale bar: 50 μm. Data are expressed as the mean ± SD. ^aP < 0.05. DGP: Diabetic gastroparesis; EA: Electroacupuncture; ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; PA: Agonist polygalacic acid; AP: Antagonist alpha-NETA; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 5

Electroacupuncture activates the vagus nerve target at the L4-L6 segment. A-C: Western blotting analysis and quantification of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor protein levels in L4-L6 (n = 5); D: Representative immunofluorescence images of c-FOS. Scale bar: 50 μm; E: Expression of c-FOS +/ChAT + in the stomach in each group. Scale bar: 50 μm. Data are expressed as the mean ± SD. ^aP < 0.05. DGP: Diabetic gastroparesis; EA: Electroacupuncture; ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; PA: Agonist polygalacic acid; AP: Antagonist alpha-NETA; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 6

Electroacupuncture activates the vagus nerve target of nucleus tractus solitarius. A-C: Western blotting analysis and quantification of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor protein levels in nucleus tractus solitarius (n = 5); D: Representative immunofluorescence images of c-FOS. Scale bar: 50 μm; E: Expression of c-FOS +/ChAT + in the stomach in each group. Scale bar: 50 μm. Data are expressed as the mean ± SD. ^aP < 0.05. DGP: Diabetic gastroparesis; EA: Electroacupuncture; ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; PA: Agonist polygalacic acid; AP: Antagonist alpha-NETA; DAPI: 4’,6-diamidino-2-phenylindole.

Figure 7

The regulatory effect of electroacupuncture stimulation on gastric emptying after subdiaphragmatic vagotomy. A-C: Western blotting analysis and quantification of choline acetyltransferase (ChAT) and α7 nicotinic acetylcholine receptor protein levels in stomach tissue (n = 5); D: Electromyogram of the rat stomach; E: Number of slow wave discharges in the antrum of the stomach within 5 minutes (n = 5); F: Gastric emptying rate (n = 5); G: Small intestine propulsion rate (n = 5); H: Representative positron emission tomography images; I: Radioactive concentration in the gastric region of interest (n = 5); J: Gastric emptying rate at 3600 seconds (n = 5). Data are expressed as the mean ± SD. ^aP < 0.05. ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; DGP: Diabetic gastroparesis; EA: Electroacupuncture; GER: Gastric emptying rate; SDV: Subdiaphragmatic vagotomy.

Figure 8

The effect of electroacupuncture on the function of smooth muscle after subdiaphragmatic vagotomy. A: Representative images of hematoxylin-eosin in the stomach. Scale bar: 100 μm; B-E: Western blotting analysis and quantification of neuronal nitric oxide synthase, cluster of differentiation 117 (C-kit) and stem cell factor protein levels in stomach tissue (n = 5); F: Representative immunofluorescence images of C-kit. Scale bar: 50 μm; G: Expression of C-kit +/choline acetyltransferase + in the stomach in each group. Scale bar: 50 μm; H-J: Enzyme-linked immunosorbent assay to detect the concentration of gastrin, motilin and vasoactive intestinal peptide in supernatant (n = 5). Data are expressed as the mean ± SD. ^aP < 0.05. DGP: Diabetic gastroparesis; EA: Electroacupuncture; SDV: Subdiaphragmatic vagotomy; nNOS: Neuronal nitric oxide synthase; C-kit: Cluster of differentiation 117; SCF: Stem cell factor; ChAT: Choline acetyltransferase; DAPI: 4’,6-diamidino-2-phenylindole; Gas: Gastrin; MLT: Motilin; VIP: Vasoactive intestinal peptide.

Figure 9

This schematic diagram illustrates the vagus nerve pathway through which electroacupuncture at ST36 enhances diabetic gastroparesis. Electroacupuncture activates the choline acetyltransferase target of the vagus nerve by intervening in the ST36 acupoint area, which is transmitted up the spinal cord from L4-L6 to the intracranial nucleus tractus solitarius. This, in turn, regulates the gastric smooth muscle-related factors neuronal nitric oxide synthase, cluster of differentiation 117 and stem cell factor, as well as gastrointestinal peptides, through the subdiaphragmatic vagus nerve. This, in turn, improves the gastric motility disorder of diabetic gastroparesis. ChAT: Choline acetyltransferase; α7nAchR: α7 nicotinic acetylcholine receptor; nNOS: Neuronal nitric oxide synthase; C-kit: Cluster of differentiation 117; SCF: Stem cell factor; Gas: Gastrin; MLT: Motilin; VIP: Vasoactive intestinal peptide; NTS: Nucleus tractus solitarius; DMV: Dorsal motor nucleus of the vagus.