Gastrointestinal neuroregulation in diabetic gastroenteropathy: Integrating molecular mechanisms and diagnostic approaches

Abstract

Diabetic gastroenteropathy (DGP) is a chronic complication of diabetes caused by gastrointestinal (GI) neuropathy. More than 50% of people with diabetes experience GI discomfort, which not only leads to increased glycemic variability but also negatively affects daily quality of life. The sympathetic, vagal and enteric nervous systems and their coordination are necessary to support the normal functioning of GI motility. In addition, neuroimmune processes and the brain-gut axis also play important roles in the complex GI regulatory network. Specifically, diabetes can mediate GI dysfunction through multiple pathways such as chronic low-grade inflammation, neuroimmune disorders, oxidative stress, nerve injury and regulation imbalance, and neuromuscular coupling disorders. Many cytokines including 5-hydroxytryptamine, nitric oxide, vasoactive intestinal peptide, transient receptor potential ankyrin 1, advanced glycation end products, nitric oxide synthase, γ-aminobutyric acid, and short-chain fatty acids. These key neuroregulatory mediators and cytokines participate in multiple pathways to regulate GI peristalsis, thereby promoting the occurrence and progression of DGP. This article aims to systematically analyze the independent and synergistic effects of sympathetic nerve, vagus nerve, and enteric nervous system in DGP, integrate the role of neuroimmunity, gut microbiota-GI-brain axis and other neuropathy-related pathological mechanisms in the pathogenesis of DGP, and integrate the mechanism of key regulatory mediators according to functional categories to comprehensively reveal the multi-level neural regulatory network of DGP. The advantages and limitations of various GI motility detection methods recorded in the existing literature are integrated, aiming to provide valuable insights into the pathophysiology and clinical diagnosis of DGP.

Key Words: Diabetes; Gastrointestinal; Nerve; Cytokine; Clinical diagnosis

Core Tip: Diabetic gastroenteropathy (DGP) arises from multi-level neural regulatory dysfunction; this study systematically clarifies the synergic dysregulation of the sympathetic-vagus-enteric nervous system, constructs a DGP neural regulatory network, and integrates gastrointestinal motility detection technologies to provide novel insights for DGP’s pathophysiological research and clinical diagnosis.

INTRODUCTION

Current epidemiology

Diabetes mellitus, as the most common endocrine disease, has caused a major global health crisis, contributing to chronic hyperglycemia, affecting the metabolism of carbohydrates, fats, and proteins[1]. Among its numerous complications, diabetic gastrointestinal (GI) dysmotility are a severe yet often overlooked condition. According to the International Diabetes Federation, the global prevalence of diabetes mellitus among adults aged 20-79 years reached 537 million in 2021, with projections indicating an increase to 783 million by 2045[2]. Long-term hyperglycemia can induce vascular and nervous system complications in multiple systems of the body. Diabetic gastroenteropathy (DGP) is a chronic pan-GI complication with a high incidence but is easily overlooked. The latest clinical guidelines have clarified that it includes diabetic gastroparesis, diabetic diarrhea, diabetic constipation and other phenotypes[3,4]. About 50% of patients with diabetes will have varying degrees of GI symptoms[5].

Clinical manifestations and burden of disease

Typical clinical symptoms of DGP include persistent nausea, vomiting, early satiety, abdominal distension, abdominal pain, abnormal defecation, etc. These symptoms can significantly reduce the quality of life of patients, and cause mental health problems such as anxiety and depression. Psychological disorders can further aggravate GI symptoms and form a vicious circle[6,7]. As a result, it is difficult to control blood glucose and aggravate other complications, which brings additional clinical diagnosis and treatment challenges[8].

Pathophysiological mechanisms

The pathogenesis of DGP is attributed to glucose-induced histomorphological and biomechanical remodeling of the GI tract, and the movement of the GI tract is precisely regulated by various nerves. GI nerve dysfunction caused by sympathetic nerve, vagus nerve, and enteric nervous system (ENS) disorders is considered to be a key factor in the occurrence and development of DGP[9]. Impaired excitatory pathway function affects gastric contraction, leading to delayed gastric emptying and food retention. Changes in inhibitory pathways lead to impaired postprandial relaxation, and these two mechanisms together lead to GI symptoms in patients with diabetes. Low-level immune inflammation caused by long-term hyperglycemia will produce neurotoxic effects, and then impair neurological function[10,11]. As a part of the microenvironment surrounding the nervous system, the interaction between immune cells and nerve cells is also involved in the regulation of GI motility[12]. In addition, the gut microbiota (GM) mediates bidirectional communication between the gut and the brain by modulating neural function, synthesizing and releasing neuroactive compounds, and maintaining intestinal barrier integrity, representing a higher-level regulatory mechanism of the central nervous system over intestinal function[13]. Many key mediators and molecules involved in oxidative stress, neuroimmunity, low-grade chronic inflammation, and neuromuscular coupling are involved in a complex regulatory network that regulates the nervous system[14].

Research gaps

To date, the neural regulatory mechanisms underlying DGP have not been systematically integrated. This review aims to provide a comprehensive summary of the pathophysiology related to GI disorders and neural dysfunction induced by diabetes, to identify potential therapeutic approaches based on these mechanisms. Describe the alterations in the autonomic nervous system at the molecular level by biomarkers that regulate and maintain GI neurologic function, thereby providing insight into the pathophysiologic basis of delayed gastric emptying and slowed intestinal motility. Additionally, clinical observations of symptoms such as delayed gastric emptying in DGP are often vague, making it challenging to differentiate and assess the severity of these symptoms based solely on clinical presentation. We have synthesized the existing methods for GI motility testing documented in the literature, with the goal of providing valuable references for further research on the diagnosis and treatment of DGP.

GI NERVES REGULATE GI MOTILITY

The GI tract contains millions of neurons that receive signal inputs from the brain and spinal cord[15,16]. Normal GI functions, including gastric emptying and intestinal peristalsis, are precisely regulated by the GI nervous system composed of the sympathetic, vagus, and enteric nerves. Sympathetic and vagus nerve pathways constitute central regulatory circuits, transmitting signals to the GI tract through nuclei located in the caudal brainstem via thoracolumbar and sacral nerves[17]. The sympathetic nervous system primarily exerts inhibitory effects on GI smooth muscle, suppresses mucosal secretion, and regulates GI blood flow via neuromediated vasoconstriction. In contrast, the vagus nerve exerts both excitatory and inhibitory control over gastric and intestinal tone and motility. Additionally, the ENS, termed the “second brain”, functions as intrinsic local nerves to establish independent local neural circuits while coordinating with central pathways[18,19].

Multiple intrinsic and extrinsic factors, including intrinsic neural plexuses, autonomic neurotransmitters, neurohormonal mechanisms, and neuroimmune regulation, are involved in this process[17]. Neuroendocrine cells densely distributed in the intestine connect the nervous system with the gut microbiome and brain to constitute the GM-GI-brain axis (GMGBA)[20]. This multilevel coordinated mechanism ensures the stability and precision of GI motor functions[21,22]. The cytokine and neuroimmune interactions among sympathetic, vagus, and enteric nerves and the mechanisms of the gut-brain axis in DGP are described in Figure 1, and the microscopic cytokine mechanisms are described in Figure 2.

Figure 1 Mechanisms of cytokine and neuroimmune interactions and gastrointestinal brain axis among sympathetic, vagus, and enteric nerves in diabetic gastrointestinal lesions. VIP: Vasoactive intestinal peptide; GLP-1: Glucagon-like peptide-1; 5-HT: 5-hydroxytryptamine; Ach: Acetylcholine; NO: Nitric oxide; SCFA: Short chain fatty acids; GABA: Gamma-aminobutyric acid; IL-6: Interleukin-6; Trpa 1: Transient receptor potential anchor protein 1; nNOS: Neuronal nitric oxide synthase; NE: Norepinephrine.

Figure 2 Microscopic mechanistic diagram of cytokines in diabetic gastroenteropathy. 5-HT: 5-hydroxytryptamine; NO: Nitric oxide; VIP: Vasoactive intestinal peptide; TRPA1: Transient receptor potential anchor protein 1; NOX: NADPH oxidase; AGEs: Advanced glycation end products; GABA: Gamma-aminobutyric acid; SCFA: Short-chain fatty acids; IECs: Intestinal epithelial cells; FFAR: Free fatty acid receptor; MCT: Monocarboxylate transporter; GMGBA: Gut microbiota-gut-brain axis; Ach: Acetylcholine; nNOS: Neuronal nitric oxide synthase; POMC: Pro-opiomelanocortin; sGC: Soluble guanylate cyclase; ICC: Interstitial cells of Cajal; GLP-1: Glucagon-like peptide-1; GPCR: G protein-coupled receptor; VPAC1: Vasoactive intestinal peptide receptor 1; IL-22: Interleukin-22; ASIC3: Acid-sensing ion channel 3; GFAP: Glial fibrillary acidic protein; NPY/AgRP: Neuropeptide Y/agouti-related protein; CCK: Cholecystokinin; GAS: Gastrin; Gsα-AC-cAMP: G protein stimulatory α subunit-adenylyl cyclase-cyclic adenosine monophosphate; HDAC: Histone deacetylase.

Sympathetic nervous system

Sympathetic neurons are distributed throughout all anatomical layers of the GI tract, exhibiting primary activation under stress conditions. They respond to various hormonal signals originating from abdominal and visceral organs via central autonomic nuclei, transmitting bidirectional excitatory and inhibitory outputs to the stomach and pancreas[22]. This system regulates GI muscle motility, local blood flow modulation, and hormone secretion while suppressing ENS activity. Sympathetic neurons innervating the GI tract are localized in paired celiac ganglia, superior mesenteric ganglia, inferior mesenteric ganglia, and prevertebral ganglion regions. The celiac-superior mesenteric ganglion complex provides neural innervation to the proximal portions of the stomach, small intestine, and colon, while the remaining colonic segments receive innervation from the inferior mesenteric ganglia[23,24].

The sympathetic nervous system influences glucose regulation. Studies demonstrate that one subset of sympathetic neurons selectively modulates GI food transit, while another population controls digestion and glucagon secretion independently of intestinal motility[25], both playing critical roles in glycemic control. Furthermore, sympathetic nervous activity has been shown to alter insulin resistance responses, potentially contributing to the development of type 2 diabetes and its complications[26]. The sympathetic nervous system also interacts extensively with GI hormones affecting motility.

Leptin released from white adipose tissue acts on the hypothalamus to modulate intracellular AMP-activated protein kinase activity, thereby regulating efferent sympathetic nerve activity projecting to the stomach, spleen, and kidneys[26,27]. Additionally, sympathetic nerves closely associate with L cells. Adrenergic receptor α2 transmits sympathetic signals and inhibits postprandial glucagon-like peptide 1 (GLP-1) release from L cells, impacting glucose homeostasis[28]. Sympathetic nerves can also act on submucosal ganglion neurons containing secretomotor-type vasoactive intestinal peptide (VIP) and contractile intestinal arterioles to inhibit water and electrolyte secretion, thereby modulating GI motility[27].

Vagus nerve

The vagus nerve, recognized as the longest cranial nerve[29], comprises 80% afferent fibers transmitting visceral and somatic signals and 20% efferent fibers regulating gastric emptying and secretion[30]. Neuroanatomical tracing demonstrates its central projections in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), and nucleus ambiguus. Notably, during its intraperitoneal course, the DMV serves as a critical regulator of gastric function by modulating smooth muscle cells and glandular activity. Anselmi et al[31] first demonstrated that abundant dopamine receptors on DMV neurons play essential roles in gastric tone regulation and segmental contractions. Vagus nerve afferent terminals in the gastric antrum function as mechanoreceptors, collaborating with sympathetic nerve fibers through ATP to modulate receptor sensitivity. This innervation architecture, surrounding sensitive structures within the myenteric plexus, enhances mechanosensory detection in the gastric antrum[32,33]. Vagal efferent fibers release the excitatory neurotransmitter acetylcholine (ACh), which promotes gastric motility by interacting with two major postsynaptic receptor systems: Nicotinic receptors (N) and muscarinic receptors (M)[34].

Clinical studies indicate that diabetes mellitus is associated with structural and functional abnormalities of the vagus nerve. Chronic hyperglycemia induces oxidative stress and mitochondrial dysfunction, leading to reduced vagal fiber density. Pathologically, this manifests as decreased DMV neuron count and axonal demyelination[35]. A specific protein, neuroregulin 1 (NRG1), is expressed in the DMV. NRG1 stimulates ACh release and activates α7 nicotinic ACh receptors (α7nAChRs), thereby reducing inflammation and restoring gastric motility via the vagus nerve. Experiments demonstrate that NRG1 and choline acetyltransferase (ChAT) expression levels are significantly reduced in diabetic gastropathy models, directly correlating with impaired gastric motility[33].

In addition, the mechanism of hyperglycemia on vagal afferents includes that peptide YY affects the hypothalamic feeding center by activating Y2 receptors, which are expressed in the vagus nerve endings of sensory afferents that innervate the gut, leading to feeding behavior disorder[36,37]. Diabetes also acts on the incretin receptors such as GLP-1 in the vagus nerve afferents. This creates a vicious cycle that exacerbates metabolic disorders and GI motility disorders[38].

ENS

As the third division of the autonomic nervous system, the ENS constitutes a ganglionated network embedded within the GI wall. Comprising over 100 million neurons and 400 million enteric glial cells (EGCs)[39], it receives local inputs, integrates information, and executes precise reflexes while functioning autonomously from extrinsic innervation[35]. The ENS exhibits structural diversity, with submucosal plexus reflex circuits embedded in connective tissue exhibiting monosynaptic organization for secretory regulation, whereas myenteric plexus circuits typically involve polysynaptic pathways governing GI motility[40-42]. Functionally, ENS neurons are categorized into excitatory cholinergic neurons co-expressing ChAT and releasing ACh and substance P for muscle contraction, and inhibitory nitrergic [neuronal nitric oxide synthase (nNOS)+] neurons secreting nitric oxide, ATP, and VIP[43,44].

Structures closely associated with the ENS include the syncytium SIP (smooth muscle-interstitial cell-PDGFβ+), comprising smooth muscle cells, Cajal interstitial cells (ICC), and PDGFRβ+ cells, which collectively regulate GI homeostasis[45]. Experimental observations reveal that ICC and PDGFRα+ cells encircle synaptic regions of enteric motor neurons, receiving nitric oxide released by enteric motoneurons. This induces conductance changes across SIP syncytium cells, modulating voltage-dependent processes in neighboring cells to trigger neurotransmitter responses that propagate throughout the syncytium[46].

Degenerative changes in enteric nerves, including axonal swelling, have been observed in diabetic animal models. Studies demonstrate that glucose is sensed by SGLT1 expressed in enteric neurons, triggering phosphorylation of the Ca²⁺/calmodulin-dependent kinase II, which reflects activated neuronal states[47]. Diabetic mice exhibit reduced calmodulin-dependent kinase II phosphorylation in response to glucose stimulation, a key biomarker for enteric neuronal activation. GI dysmotility disorders such as DGP correlate with reduced enteric nerve density. Patients with severe diabetic motor dysfunction show a 2.2-fold decrease in neuron numbers per myenteric ganglion[48]. Hyperglycemia induces reduced phosphoinositide 3-kinases activation, decreased numbers of S-100-positive EGCs, and increased neuronal apoptosis in both cultured embryonic enteric neurons and streptozotocin-induced diabetic rat models[14].

Coordination and discoordination among nervous systems

Under physiological conditions, the sympathetic nervous system exerts inhibitory control of GI motility mainly through noradrenergic signaling, preferentially acting on cholinergic excitatory neurons in the myenteric plexus to inhibit ACh release and regulate colonic motility[49]. In contrast, the vagus nerve enhances intestinal motility and secretion through cholinergic and peptidoergic pathways, and serves as the main afferent branch of the gut-brain axis, transmitting nutritional and inflammatory signals to the brain stem[50]. The ENS operates autonomously through a complex network of nitriergic, cholinergic, and serotonergic neurons, coordinating local reflex activity.

In DGP, this delicate balance is disrupted. Chronic hyperglycemia causes selective loss of nNOS+ neurons in the myenteric plexus of the ENS, which triggers delayed gastric emptying and constipation[51]. At the same time, 5-hydroxytryptamine (5-HT) signaling is dysregulated and 5-HT positive neurons increase in the submucosal plexus, resulting in diarrhea and visceral hypersensitivity. The sympathetic nervous system shows paradoxical effects: Although sympathetic hyperexcitation in diabetes exacerbates insulin resistance and lipolysis, its inhibitory effects on GI motility may be impaired by a reduced density of noradrenergic nerve endings adjacent to cholinergic neurons[51,52]. Vagal dysfunction further complicates the picture, since diminished afferent signaling inhibits postprandial satiety and the glucose-regulatory reflex, whereas efferent vagal stimulation improves glucose uptake independent of insulin[53]. Together, these feedback disturbances constitute the core closed loop of DGP neurological dysfunction, amplifying local movement disorders into systemic metabolic and emotional comorbidities through neuroimmune interactions, such as impaired macrophage α7nAChR signaling and abnormal response of 5-HT/transient receptor potential ankyrin 1 (TRPA1) signaling in the gut-brain axis.

GMGBA: UPPER REGULATION OF THE NERVOUS SYSTEM

The GM, recognized as a mature endocrine organ in humans, is predominantly composed of Bacillota and Bacteroidetes phyla. Through complex interactions with the nervous system, it forms a bidirectional regulatory axis with the brain termed the GMGBA. Dysregulation of the GMGBA contributes to disease pathogenesis. Studies on neurodegenerative diseases have confirmed that the imbalance of GM promotes the abnormal aggregation of α-synuclein, induces intestinal glial inflammation, and destroys the integrity of the intestinal barrier, which accelerate the neuronal loss and functional decline of ENS. It has become one of the key factors for the early initiation and progression of diseases, proving the important role of GM in neurological diseases. In the context of diabetes, dysbiosis of flora amplizes oxidative stress and inflammation to further damage intestinal neurons and glial cells[54-56]. Decades of research have established associations between GMGBA dysfunction and chronic conditions such as type 2 diabetes mellitus and functional GI disorders[57,58]. Bidirectional biochemical communication within the GMGBA involves key metabolites including short-chain fatty acids (SCFAs), neurotransmitters [e.g., 5-HT, dopamine, γ-aminobutyric acid (GABA)], and lipopolysaccharides[59]. A canonical pathway involves SCFAs, primary GM-derived metabolites, acting through two mechanisms: (1) Local interaction with G protein-coupled receptors (GPCRs) on enteroendocrine cells to regulate GI hormones and motility; and (2) Blood-brain barrier penetration followed by microglial engagement, exerting systemic effects on central nervous system functions while relaying feedback signals to the gut, thereby establishing a bidirectional GMGBA circuit[60].

Recent studies have provided additional evidence for the gut-brain axis. Nishimura et al[61] employed real-time calcium imaging of the ENS while simultaneously recording evoked cortical field potentials, offering empirical validation for neuro-neuronal connectivity within the gut-brain axis. Mechanistic investigations reveal that lipopolysaccharides primarily activate Toll-like receptors (TLR2/TLR9) in the ENS to mediate anti-inflammatory responses, initiating nuclear factor-κB signaling and triggering pro-inflammatory cytokine release [e.g., interleukin (IL)-6, tumor necrosis factor-α]. This gut-derived inflammatory storm damages synaptic connections within enteric plexuses and exacerbates central neuroinflammation via vagal afferent transmission to the brain[62]. Key brain regions integrating gut-brain information have been identified in the hypothalamus, amygdala, and prefrontal cortex[63]. Clinical studies in diabetic patients with GI symptoms further demonstrate significant neuroplastic changes in these regions using retrograde tracing, connectome mapping, and chemogenetic manipulation. These alterations include neuronal remodeling and gliosis, providing neuroanatomical evidence for diabetes-induced gut-brain destruction[64].

GI NEUROIMMUNE SYSTEM: THE NERVOUS SYSTEM MICROENVIRONMENT

The immune system and GI nervous system jointly surveil the intestinal and extraintestinal microenvironments[65]. Anatomical studies demonstrate that high-density immune cell clusters surround distinct populations of GI neurons[66]. Notably, gastric biopsies from DGP patients reveal significant myenteric plexus pathologies: Increased immune cell infiltration coupled with progressive reduction of circular muscle layer nerve fibers, suggesting that neuroimmune imbalance may serve as a critical mechanism underlying GI dysmotility.

Macrophages represent the most abundant immune cell population in the GI tract[67], exhibiting close interactions with neural networks and GM. Notably, sympathetic nerve fibers directly innervate Peyer’s patches and adjacent macrophage clusters. Intestinal muscularis macrophages specifically express α7nAChRs, which have been identified as critical mediators in vagal anti-inflammatory signaling pathways[33]. Neurotransmitters and neuropeptides released by enteric neurons bind to receptors on immune cells, thereby modulating their effector functions. This neuroimmune symbiosis sustains enteric neuron survival and functionality. EGCs and stromal cells collaborate with macrophages to form integral components of enteric neural circuits. Activated by hyperglycemia, EGCs undergo M1/M2 phenotypic switching, regulating mast cell degranulation and cross-talking with T/B lymphocytes to activate autophagy[68]. Their depletion induces apoptosis in submucosal and myenteric neurons, leading to diminished GI secretion and motility[69]. GM modulate neuroimmune processes by mediating crosstalk between ENS signaling and IL-6-driven regulatory T cell differentiation, providing mechanistic insights into the gut-brain axis[70].

KEY REGULATORY MEDIATORS OF DPG

Emerging studies have identified novel biomarkers of DGP, while increasing evidence has revealed the critical role of various regulatory mediators in its pathogenesis[71,72]. The molecules of each family regulate each other and form a network to jointly mediate GI nerve injury, dynamic imbalance and microenvironmental disorder. Some molecules have cross-family attribution characteristics due to their multiple functions. The following is a systematic review of their mechanism of action according to functional family.

Inhibitory signaling mediators

Nitric oxide-inhibitory neurotransmitters: Nitric oxide, a multifunctional free radical signaling molecule produced by NOS, is predominantly expressed as the Nos1/nNOS isoform in rat intestines, accounting for > 95% of total NOS activity[73]. Nitric oxide is widely distributed in the GI tract, serving as a primary inhibitory neurotransmitter and inflammatory mediator that induces muscle relaxation. Neurons mediating these effects are termed nitrergic nerves. Animal models reveal progressive nNOS depletion and dysfunction during diabetic pathology: Early-stage reduction in nNOS levels followed by late-stage nitrosative stress, culminating in complete loss of nitrergic innervation[74].

ICC-submucosal layer, primary effectors of nitrergic signaling, exhibit reduced density in DGP. Mechanistic studies demonstrate that nitric oxide activates guanylate cyclase to enhance pacemaker activity in ICC-deep muscular plexus, establishing GI rhythmicity[75]. WWv mutant mice with ICC-submucosal layer deficiency show approximately 60% reduction in NOS1 expression and significantly diminished nitric oxide release, suggesting nitric oxide’s regulatory role in ICC via direct synthesis or signal amplification. NOS knockout mice exhibit altered colonic migrating motor complexes, underscoring this pathway’s critical role in GI motility[76,77].

Iino et al[77] further discovered that TLR2 receptors, highly expressed on murine colonic enteric neural progenitor cells, promote nNOS-rich neuron formation upon activation, crucial for maintaining GI neural function. Crosstalk between NOS-containing myenteric neurons and GLP-1 receptors enhances nitric oxide production through G protein stimulatory α subunit-adenylyl cyclase-cyclic adenosine monophosphate signaling, synergistically amplifying GI relaxation[78].

VIP-inhibitory regulatory peptide: VIP an endogenous neuropeptide, exerts its biological activity primarily through GPCR-mediated signaling via VPAC1 receptor binding. In mice, VPAC1 mRNA expression demonstrates a colonic > ileal > jejunal gradient, with the highest human expression observed in the sigmoid colon, mirroring murine distribution patterns[79].

VIP plays critical roles in enteric neuroprotection: (1) By modulating EGCs and stimulating neurotrophic factor synthesis; and (2) By activating IL-22 production in innate lymphoid cells type 3 localized near intestinal neurons to preserve epithelial barrier integrity[80,81]. VIP exhibits intricate interactions with GM. Experimental findings demonstrate that VIP treatment increases the Firmicutes-to-Bacteroidetes ratio, the two predominant bacterial phyla in the intestine. Animal studies reveal that VIPergic fibers of the ENS are closely associated with CX3CR1+ cells, promoting their expression. Furthermore, ENS-CX3CR1+ cell interactions mediated by VIP signaling have been demonstrated to modulate neuroimmune responses, including macrophage activation and pro-inflammatory cytokine reduction[82,83].

GABA: GABA functions as both a neurotransmitter and endocrine mediator in the GI tract[84], primarily modulating vagal input via its action on the DMV[85]. Microinjection of the GABAb receptor agonist baclofen into the rat DMV demonstrated enhanced gastric motility through excitatory cholinergic vagal pathways. GABA’s effects on the ENS are mediated by activation of ionotropic GABAa/GABAc receptors and metabotropic GABAb receptors. Specifically, GABAa/GABAc receptor activation stimulates cholinergic and non-adrenergic non-cholinergic enteric neurons to release neurotransmitters, thereby dynamically modulating excitatory/inhibitory signaling in the ENS.

Furthermore, GABA secretion is regulated by estrogen, which enhances GABA currents and gut-brain axis communication by modulating GABA synthesis, transport, release, and receptor expression in brain regions such as the preoptic area and bed nucleus of the stria terminalis[86]. GABA also exerts immunomodulatory effects by negatively regulating pro-inflammatory cytokine production and immune cell proliferation in neuroimmune processes[87].

Excitatory signaling mediators

5-HT: Serotonin (5-HT), functioning as a paracrine factor and enteric neurotransmitter, participates in enteric neural regulation and gut-brain axis signaling[88]. It serves as a critical regulator of GI neuroactivity, with 90% of systemic 5-HT originating from enterochromaffin cells in the GI tract, while the remaining 10% is secreted by enteric neurons to act as a neurotransmitter modulating GI motility[89]. Co-expression studies reveal that 5-HT is colocalized with GI hormones in enterochromaffin cells, such as cholecystokinin and gastrin in the small intestine[90]. Hao et al[91] demonstrated that platycodon polysaccharide intervention in constipated patients significantly enhances 5-HT secretion, along with upregulated expression of tryptophan hydroxylase 1, 5-HT4 receptors, and TRPA1, thereby improving intestinal peristalsis.

Under diabetic conditions, enterochromaffin cells exposed to high-glucose diets exhibit augmented 5-HT release, resulting in an increased number of 5-HT positive neurons[92] modulating 5-HT3 receptor-mediated activation of the ENS and vagal afferent signaling to the brain, while impacting cerebral regions governing emotion, behavior, and GI functions[93]. Beyond GI regulation, 5-HT participates in the gut-brain axis by acting centrally: Hypothalamic 5-HT inhibits pro-opiomelanocortin neurons via 5-HT2C receptors and suppresses neuropeptide Y/agouti-related protein neurons through 5-HT1B receptors, thereby modulating appetite control[94]. In neuroimmune interactions, 5-HT activates T cells via 5-HT3R signaling, inducing intracellular sodium influx and enhancing T cell proliferation[95]. Pharmacological agents like thalidomide have been shown to inhibit ascending release of neurotransmitters (e.g., 5-HT) and activation of their receptors (e.g., 5-HT3R) in the brain and colon. This restores microbial homeostasis by increasing abundances of Clostridium, Lactobacillus, Bifidobacterium, and Ruminococcus species, ultimately modulating GI motility via the gut-brain axis[96].

TRPA1: TRPA1 is a Ca²⁺-permeable dynamically expressed channel expressed in enteroendocrine cells, participating in sensory transduction and GI motility regulation[97]. Someya et al[98] investigated TRPA1 distribution in the GI tract, highlighting its presence in enteric neurons and immune cells. TRPV1 and acid-sensing ion channels 3 channels colocalize with ghrelin receptors in gastric afferent nerves of nodose ganglia[99]. Activation of TRPA1 β enteroendocrine cells has been demonstrated to directly stimulate vagal sensory ganglia and trigger neurotransmitter release via microbial, pharmacological, or optogenetic modulation[62].

TRPA1 activation elevates intracellular Ca²⁺ levels, inducing 5-HT release from enterochromaffin cells to modulate gut reflexes and motility. TRPA1 also stimulates GLP-1 secretion, while glucose-dependent insulinotropic polypeptide is co-released during feeding to regulate blood glucose. Notably, TRPV1 or TPA1 neuron-dependent Ca²⁺ increases in dorsal root ganglia form a feedback loop with GLP-1 signaling[99,100].

Notably, anxiety and stress-related emotional disorders coexist with GI pathologies in clinical practice, with approximately 50% of DGP patients exhibiting comorbid affective disturbances. Emerging evidence suggests that TRPA1 exacerbates symptom severity through bidirectional gut-brain crosstalk in GI diseases[98]. Preclinical studies confirm that TRPA1 antagonists (e.g., A-967,079) significantly reduce neuroinflammatory markers (AIF1, GFAP) in the hippocampus while suppressing reactive oxygen species (ROS) generation, thereby attenuating oxidative stress-induced damage to GI neurons[101,102].

Pro-oxidative damage media

NADPH oxidase family: NADPH oxidase (NOX) was first identified as an enzyme responsible for producing ROS in macrophages[103]. Among the seven different isoforms of the NOX family that have been identified, NOX4 plays a key role in ROS production and the regulation of oxidative stress[104]. Recent studies have identified post-diabetic ecological dysbiosis characterized by reduced butyrate-producing bacterial abundance, leading to overexpression of inflammatory cytokines and NOX4 in the colons of diabetic mice, ultimately resulting in GI complications[105]. In vitro experiments further demonstrate that NOX4 inhibition under diabetic hyperglycemia increases mitochondrial density and insulin secretion in EndoC-βH1 cells, confirming that NOX4 exacerbates pancreatic β-cell dysfunction through mitochondrial pathways during diabetes progression[106].

Advanced glycation end products: Advanced glycation end products (AGEs) are formed through non-enzymatic glycation reactions between glucose, fructose, and proteins within the body[107]. The accumulation of AGEs in neural tissues can damage axoplasmic transport, impair intracellular signaling, and lead to axonal degeneration, directly harming nerve tissues.

Animal studies have demonstrated that AGEs and endogenous nitric oxide synergistically induce neuronal apoptosis in vitro[108]. Although no differences in AGEs accumulation were observed between early-decedent neurons (nNOS-positive cholinergic/nitrogenic neurons) and late-decedent neurons (TH-positive, nNOS-negative sympathetic neurons) during diabetes progression, their AGEs sensitivity differs significantly. However, AGEs exhibit uniform accumulation across neural ganglionic compartments. Nitrogenic neurons (nNOS-positive) demonstrate heightened susceptibility to AGE-induced damage compared to sympathetic neurons lacking nNOS. Consequently, chronic hyperglycemia-induced AGE accumulation predominantly causes nitrogenic neuron injury, ultimately leading to detrimental GI symptoms[109].

Microbiota-associated neuroactive mediators

SCFA: SCFA are GI bacterial-derived metabolites produced by anaerobic fermentation of indigestible polysaccharides such as resistant starch and dietary fibers, including acetate, propionate, and butyrate, which attach to free fatty acid receptors on the surface of the intestinal epithelial cells[110]. Chen et al[111] has observed that diabetic patients develop alterations in SCFAs that are most pronounced with butyrate. Cellular experiments demonstrated that butyrate treatment exacerbates histone H3 lysine 9 acetylation in primary cultured enteric neurons and EGCs, thereby impairing ENS functionality[101], and animal studies reveal that SCFAs restore neuronal numbers following antibiotic-induced neuron loss, thereby demonstrating their critical role in maintaining ENS integrity and functionality in vivo[112].

SCFA also crosses the blood-brain barrier via monocarboxylate transporter in endothelial cells and is involved in the intracerebral portion of neurotransmission in the brain-gut axis. In neuroimmunity, SCFA also activates free fatty acid receptor or inhibits histone deacetylase interacting locally with intestinal epithelial cells and immune cells such as macrophages and neutrophils, thereby disrupting neuroimmune homeostasis[113].

TECHNOLOGICAL ADVANCES IN GI NEUROFUNCTIONAL TESTING

Currently available detection methods for DGP differ significantly in principle, scope of application, sensitivity and specificity. In clinical practice, individualized selection should be made according to the specific clinical phenotype of patients. Traditional GI neurophysiological methods mainly rely on mechanical testing of the GI tract. Scintigraphy, as the gold standard for gastric emptying, has high specificity for the observation of food distribution in the stomach, but has the limitations of standardized diet intolerance and radioactive exposure. The 13C gastric emptying breath test has the advantages of non-invasiveness, non-radiation and high patient acceptance, which is suitable for children, pregnant women and radiation-sensitive people. The sensitivity can meet the basic clinical screening. However, it is an indirect detection method, and its specificity is limited when combined with other digestive diseases. Antral and duodenal manometry (barometric measurement) can accurately capture the changes of GI pressure through multiple sensors, and has strong specificity for the pathophysiological characterization of motility disorders. It can combine manual and automated quantitative analysis to improve the reliability of the results. However, it is a case of abnormal GI peristalsis with abdominal pain and bloating in invasive operation. Gastric electromyography combined with surface gastric markers can be used for initial screening in patients with gastric electromyography disorder, and antral and duodenal manometry can be used for further verification in suspected severe cases[114].

Emerging detection technologies provide a new direction for the accurate diagnosis of DGP. Functional magnetic resonance imaging can realize the synchronous evaluation of multiple gastric parameters, accurately reveal the peristalsis pattern by personalized gastric peristalsis and emptying curve, and improve the detection accuracy by combining the correction of respiratory motion error algorithm. Intelligent high-frequency intracavitary ultrasound integrates ultrasonic transducer and pressure catheter, which can simultaneously obtain the morphological and mechanical parameters of the GI tract, clearly observe the peristaltic contraction sequence and the correlation between anatomical structure and mechanical properties. It makes up for the defects of the loss of acoustic coupling of traditional ultrasound, and its sensitivity and specificity are significantly improved compared with those of traditional ultrasound. Artificial intelligence-based detection technology can deeply analyze the original data of traditional tests such as scintigraphy and breath test, optimize the registration and segmentation process of gastric emptying test through machine learning, improve the quantitative accuracy and diagnostic efficiency of detection results, and integrate multimodal detection data to realize early screening and disease grading of DGP. In future clinical diagnosis, the combined application of emerging technologies and traditional detection can be promoted, and the integrated analysis of multimodal detection data can be realized with the help of artificial intelligence to further improve the sensitivity, specificity and clinical practicability of DGP diagnosis.

Quantifying GI dysfunction is the foundation for diagnosing GI neuropathy. Currently, methods for assessing GI motility have been widely documented. However, there is still a lack of comprehensive summaries of these testing techniques. The advantages and limitations of the existing diagnostic methods are summarized in the Table 1[115-129].

Table 1 The advantages and limitations of the existing diagnostic methods for assessing gastrointestinal motility.

Technology	Introduction to the methodology	Vantage	Disadvantage	Ref.
Wireless capsule endoscopy	A battery-powered capsule instrument, wearing a monitor, records data transmitted from the capsule until the capsule is excreted, for 3-5 days	Longer recording time, more comprehensive image recording	The capsule part is hard to swallow	[115]
Flexible piezoelectric sensors	It consists of 12 modules connected in series, each consisting of 10 polyimide polymers connected in parallel to detect GI pressure	Reduced risk of tissue perforation, device retention and GI tract obstruction	Invasive methods may cause trauma	[116]
Scintigraphy	Oral or gastric tube instillation of fluids containing trace amounts of radioisotopic 99mTc and capturing changes in the distribution of these tracers in the stomach by imaging equipment	Use of common foods; the ability to view intragastric meal distribution	Intolerance to standardized diets (most commonly eggs or gluten) in some patients	[117]
Mechanical magnetic gastrography	Magnetic imaging of the tracer was measured after administration of 5 μCi of a 99mTc -colloidal sulfur-labeled diet, punching the magnetic field (30mT over a 5 milliseconds period) for initial stimulation	Combining scintigraphy with magnetic resonance technology, biomagnetic imaging is safe and effective	Larger application costs	[118]
Gastric emptying breath test	Labeling of meals with carbon 13 into the stomach, conversion to coeliac radiolabeled substrate for digestion and absorption in the duodenum prior to entry into the duodenum, and then metabolism were measured in 7 breath samples taken over a 4-hour period to measure the percentage of 13 C	Safe, radiation-free and child-friendly; Non-invasive, high patient acceptance	Indirect measurements that may mask symptoms if combined with other conditions	[119]
Barium radiopaque marker	After fasting overnight, a capsule containing the barium marker was given in the middle of the test meal at 8:00 a.m. The first abdominal radiographs (posteroanterior views in supine and standing positions) were taken 2 hours later and then hourly until 6 hours after ingestion	Low equipment requirements, suitable for mass screening, visualization of stomach contents	Radiation exposure, long testing times are hard for patients to accept	[120]
Gastric power wave transform record	Long-term multi-channel recording signal processing integrates detection, segmentation, and summarization to monitor GI motility through spatiotemporal pattern recognition	Non-invasive, flexible, easy to operate, comfortable for long-term use	Can be affected by outside noise	[121]
High frequency intracavitary ultrasound	Integration of ultrasonic transducers with pressure catheters in conventional high-frequency ultrasound systems enables simultaneous acquisition of morphological and mechanical parameters for enhanced GI motility analysis	Observation of peristaltic contraction sequences; simultaneous observation of peristaltic anatomy and mechanics	Loss of acoustic coupling in air-filled organs during swallowing	[122]
Gastric sinus duodenal manometry	Perfusion of a gastroduodenal power catheter with high-resolution water, including 13 sensors 1 cm apart and 2 sensors 10 cm and 20 cm from the last of the 13 closely placed sensors, and confirmation of sensor placement by fluoroscopy across the gastric sinus duodenal (antropyloroduodenal) junction	Combined manual and automated quantitative methods for assessment are scientifically valid	A combination of other techniques is often needed to more accurately characterize gastric emptying	[123]
Body surface gastric labeling	High-resolution water-perfused power catheter with a 64-electrode array connected to a wearable reader to measure gastric electromyographic activity and associated contractions while performing validated symptom analysis	Complex bioamplifier and signal processing system for gastric electrophysiology with proven artifact detection and suppression	The reference range is not yet fully defined	[124]
Autonomic function testing	Includes changes in finger capillary pulsation position, cold vasoconstriction, and changes in electrocardiogram R-R interval during deep breathing	Non-invasive neurological function tests with high patient acceptance; does not involve dietary tests, less burden on patients with GI dysfunction	Indirect response to gastric emptying through autonomic function, influenced by neurologic factors	[125]
Time-series magnetic resonance imaging	Gastric emptying and motility were continuously assessed in a rapid image acquisition sequence. And 4D magnetic resonance imaging images were processed using a dedicated pipeline that included respiratory motion correction, gastric segmentation and partitioning, volumetric analysis of gastric emptying, and surface-reflection-based analysis of the frequency, amplitude, and phase relationships of gastric motility	Personalized gastric peristalsis and emptying curves to reveal peristaltic patterns; simultaneous assessment of multiple gastric parameters; algorithmic correction of respiratory motion errors	Gas in the stomach can have an effect on the results	[126]
Endoscopic combined constant voltage	Using an ultra-thin endoscope and a pressure-regulated endoscopic blow-in device, air is blown into the GI tract until a preset pressure is reached. Measurement of actual intragastric pressure using a fiber optic manometer placed in the stomach	GI manometry with observation of internal gastric changes	Complex and technically demanding, and may take a long time to complete the examination, increasing the patient's waiting time and discomfort	[127]
Satiation drinking test	Individuals consume fluids of known composition and/or calorie content at a rate for a predetermined period of time. At maximal satiety, the volume of liquid consumed is recorded, and gastric regulation and sensation are estimated	Simple operation and high patient acceptance	Possible patient discomfort due to overfeeding	[128]
Gastric electrostatic labeling	Skin electrodes were placed on the abdominal skin above the stomach to record gastric electromyographic activity	Non-invasive, radiation-free assay that directly reflects gastric electromyography activity	Motion artifacts and electrical disturbances in other internal organs can affect the results	[129]

GI: Gastrointestinal.

CONCLUSION

Although the neural regulation mechanism of DGP has received much attention, most of the existing studies have focused on a single neural pathway or isolated molecular target. This study breaks through the fragmented interpretation of the neural regulation mechanism of DGP, and systematically integrates the synergic dysregulation mechanism of sympathetic-vagus-ENS for the first time. The close-loop of multi-dimensional interaction of neuro-immune-GM in DGP was clarified. The upstream and downstream pathways of key regulatory molecules were sorted out according to functional categories, and a multi-level neural regulatory network of DGP was constructed.

We sort out the upstream and downstream pathways of key regulatory molecules by functional categories and construct a multi-level neural regulatory network of DGP: Chronic hyperglycemia induces selective loss of ENS myenteric plexus nNOS+ inhibitory neurons and abnormal upregulation of submucosal plexus 5-HT-positive neurons, leading to GI excitatory-inhibitory signal imbalance; AGEs synergize with nitric oxide to induce nitrergic neuron apoptosis, and NOX4 overproduces ROS to cause oxidative damage to enteric neurons and pancreatic β-cells, forming a mutual amplification loop between GI nerve dysfunction and metabolic disorders. Vagal dysfunction is characterized by reduced fiber density and downregulated NRG1/ChAT expression, impairing the α7nAChR-mediated cholinergic anti-inflammatory pathway; the sympathetic nervous system shows hyperexcitation-induced insulin resistance and weakened GI motility inhibition due to reduced noradrenergic nerve endings. GM dysbiosis reduces SCFA synthesis, destroying intestinal barrier integrity and mediating GMGBA bidirectional regulation disorder via GPCR and histone deacetylase-dependent pathways. Inhibitory mediators (nitric oxide, VIP, GABA) lose regulatory function while excitatory mediators (5-HT, TRPA1) are abnormally activated, disrupting GI peristalsis and mediating comorbidity of GI symptoms and emotional disorders via gut-brain axis crosstalk.

At the same time, this review comprehensively integrates the principles, advantages and limitations of existing GI motility detection technologies, and establishes the correspondence between technology selection and clinical phenotype of DGP, which makes up for the lack of systematic guidance for the application of detection technologies in existing studies. However, there are still some key gaps in the existing research, such as the pathological differences of neuronal heterogeneity, the causal direction of neuro-immune-microbial interaction, the threshold of neuropathy reversibility, and the lack of specific biomarkers. In the future, multi-omics prospective cohort studies should be carried out to identify early molecular markers, and machine learning should be used to integrate multimodal data to construct early risk diagnosis models, so as to promote the paradigm shift of DGP from symptom management to neural repair and achieve a breakthrough from symptomatic treatment to etiological treatment.