Effects of Acetylcholine on Endothelial Function and Nitric Oxide Levels in Patients with Type 2 Diabetes Mellitus: A Randomized Sham-Controlled Study.

Introduction

Diabetic neuropathy is a significant and frequently overlooked health issue impacting hundreds of millions globally, particularly among individuals with diabetes. Therefore, it is imperative to promptly develop effective treatments for this condition. Diabetes mellitus (DM) is a chronic metabolic disorder associated with numerous long-term complications, such as neuropathy and endothelial dysfunction resulting from angiopathy. 1 Chronic hyperglycemia is a significant contributor to these complications. Research has shown that abnormalities in the NO pathway are present in diabetic individuals even before clinically evident macrovascular disease develops. 2

Chronic hyperglycemia in diabetes leads to peripheral neuropathy, which is characterized by nerve damage. This damage frequently leads to symptoms including numbness, loss of sensation, and, in certain instances, pain in the hands, legs, or feet. Nerve conduction studies serve as reliable and sensitive diagnostic tools for validating the presence of neuropathy. 3 Common symptoms include paresthesia, dysesthesia, and lancinating pain, primarily affecting the lower limbs. Physical examination reveals symmetrical deficits in light touch, vibratory sensation, and nociception. Reduced protective sensation increases the risk of unnoticed trauma and pressure-induced injuries during walking, leading to a higher likelihood of foot ulcers. These ulcers can lead to significant complications, including permanent immobility of the lower leg or amputation. Diabetic neuropathy is characterized histologically by segmental demyelination and the degeneration of myelinated and unmyelinated nerve fibers. These changes contribute to the progression of the condition and its associated symptoms. 3

Under normal physiological conditions, Plasminogen Activator Inhibitor-1 (PAI) is present in plasma at relatively low levels, typically ranging from 5 to 20ng/mL. 4 This protein serves as the primary inhibitor of tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA), enzymes that play a role in the degradation of blood clots. PAI-1 neutralizes the activity of t-PA through binding, thereby regulating fibrinolysis and preventing excessive clot breakdown, which may result in detrimental outcomes. 5 The production of PAI-1 is affected by various factors, including inflammatory cytokines, angiotensin II, and metabolic conditions such as high blood sugar (hyperglycemia), elevated lipid levels (hyperlipidemia), and oxidative stress. 6

Hyperglycemia, a significant risk factor for diabetic neuropathy, impacts peripheral sensory nerves through various mechanisms. Additionally, other physiological changes associated with diabetes may contribute to the onset of peripheral neuropathy. Reduced blood flow to peripheral nerves is one of the earliest functional abnormalities observed in diabetes. Local hypoxia in peripheral nerves is regarded as a key pathogenic factor in neuropathy development. 7

The vascular endothelium plays a critical role in the regulation of microvascular tone through the release of substances that influence vasodilation and vasoconstriction. Endothelial cells produce vasodilators, including NO, prostacyclin, and endothelium-derived hyperpolarizing factor. Additionally, they secrete vasoconstrictors such as endothelin and specific prostaglandins. Nitric oxide serves as the primary vasodilator in endothelium-dependent relaxation. Upon release from the endothelial layer, NO diffuses into adjacent smooth muscle cells, subsequently activating the enzyme guanylate cyclase. The activation induces the synthesis of cyclic guanosine monophosphate (cGMP), facilitating smooth muscle relaxation and resulting in vasodilation. NO is synthesized in the endothelium from the amino acid L-arginine through the enzymatic activity of NOS. This process plays a critical role in promoting the relaxation of vascular smooth muscle cells, thereby modulating vascular tone. 8

Acetylcholine (ACh) induces microcirculatory vasodilation through two endothelium-dependent mechanisms. The first mechanism involves ACh binding to muscarinic receptors located on endothelial cells. This interaction stimulates the production of NO via the activation of NOS and the utilization of L-arginine. NO subsequently diffuses into vascular smooth muscle cells, inducing relaxation and vasodilation. 9 , 10 The second mechanism involves ACh-mediated activation of C-nociceptive fibers, which triggers the Lewis triple-flare response, a nerve axon reflex-mediated vasodilation that contributes to vascular dilation. 11 Acetylcholine sensitizes these fibers, augmenting substance P and calcitonin gene-related peptide (CGRP) release. Substance P stimulates the release of histamine from mast cells, whereas CGRP directly causes vasodilation, resulting in an indirect vasodilatory effect. 12

Prevailing evidence suggests that cholinergic microvascular modulation is mediated by two mechanisms: vasodilation resulting from the nerve-axon reflex and direct stimulation of the vascular endothelium by acetylcholine. However, research in this area remains limited, and there are conflicting findings regarding the relative contributions of these mechanisms to overall vasodilation. In the foot, studies have shown that neuropathy reduces the vasodilator response, even in the absence of peripheral vascular disease. A recent study revealed that the nerve-axon-mediated response to acetylcholine-induced C fiber stimulation in diabetic patients was significantly reduced, similar to the response observed with sodium nitroprusside. This finding suggests that neuropathy contributes to functional ischemia in diabetic foot ulcers by impairing the ability to increase blood flow during periods of stress. 13

Iontophoresis is a non-invasive technique that enables the transdermal delivery of ionized chemical agents through the skin by using a low-intensity electrical current applied via two adhesive electrodes. 14 This method has been examined as a potential treatment for conditions such as plantar fasciitis, palmoplantar and axillary hyperhidrosis, migraines, enhanced wound healing, and digital ischemia associated with systemic sclerosis. 15 This method is extensively utilized in dermatology and physiotherapy as a non-invasive therapeutic alternative. 16 In vascular medicine, previous studies have examined the vasodilator effects of acetylcholine (ACh) iontophoresis to assess endothelial dysfunction in individuals with diabetes mellitus and its associated microvascular complications. However, its therapeutic potential remains underexplored. This study aims to assess the efficacy of ACh iontophoresis in enhancing endothelial function in diabetic patients, both with and without peripheral neuropathy. 17 , 18 Hyposis of study, this study aims to evaluate the effects of Acetylcholine (Ach) iontophoresis as a vasodilator on endothelial NOS activity to develop a non-invasive transdermal treatment that is effective and devoid of systemic adverse side effects.

Previous studies have predominantly utilized acetylcholine iontophoresis as a diagnostic modality to assess endothelial-dependent microvascular reactivity. However, its potential application as a repeated therapeutic intervention aimed at modulating microcirculatory function has not been systematically investigated, particularly in patients with diabetic neuropathy. Therefore, a clear gap exists regarding whether repeated (as a treatment) acetylcholine iontophoresis can induce measurable biochemical, microvascular, and functional neurophysiological changes. We hypothesized that repeated acetylcholine iontophoresis would significantly increase nitric oxide bioavailability and enhance microcirculatory parameters in patients with type 2 diabetes. Furthermore, we investigated whether this improvement in microvascular function would lead to a secondary enhancement in peripheral nerve conduction and electrophysiological outcomes over a four-week period.

Materials and Methods

This active-controlled randomized study involved 80 patients diagnosed clinically by the clincian with diabetic polyneuropathy, referred from the internal medicine department one month prior to the study. Patient enrollment occurred between April 2024 and December 2024. The project is registered in clinical trials under the unique identifier NCT06219590 . The original ethics approval No was P.T-BAS-06/2023-515 South Valley University. All participants signed a consent form for agreement to participate in the study. The consent form explained the study objectives and assured participants of their confidintality and right to withdraw at any time. It also auured that no harm will occurs to participants. No names or personal informations will be disclosed and no one got access to the data. The study complies with the Declaration of Helsinki. This clinical trial was reported in adherence to the CONSORT guidelines.

Participant and Sample Size

A group of 80 patients, comprising both males and females, was diagnosed with type 2 diabetes mellitus and referred from the internal medicine outpatient clinic at South Valley University Hospital. The participants’ ages varied between 40 and 60 years, and they had been diagnosed with type 2 diabetes for more than 10 years. Participants were randomly assigned to either the experimental or control group using a computer-generated random sequence (simple randomization). To ensure allocation concealment, the assignments were placed in sequentially numbered, opaque, sealed envelopes, which were opened by an independent researcher not involved in the intervention or data collection. The study employed a double-blind design: patients in the control group received a sham iontophoresis procedure (using the same equipment and duration but without active current or acetylcholine) to maintain participant blinding. Furthermore, the assessors performing the nerve conduction studies and nitric oxide analysis were blinded to the group assignments to eliminate observer bias. Patients were randomly divided into two groups as follows.

Group A (n=40): Received iontophoresis using an acetylcholine concentration of 1 mg of sterilized saline per 100 cm 2 , resulting in a 1% concentration. This treatment was administered for 15 minutes during three sessions per week over a duration of four weeks. The patient must exhibit comfort, with no reported or visible signs of pain or burning.

Group B (n=40): Received sham iontophoresis using a placebo electrode, with the machine powered on to deliver galvanic current for 15 minutes during three sessions per week for four weeks.

To prevent cutaneous irritation, current intensity for both cohorts was maintained at 3–5 mA, with dermal inspection every 3–5 minutes for signs of erythema or irritation. 9

Both groups received conventional medical treatment and a healthy diet.

Author 1 who enrolled participants, and who assigned participants to interventions.

Therapeutic Equipment - Neuromuscular Electrical Stimulation

The ASTAR Swit 33, 43–382 dual-channel stimulators (Bielsko-Biała, Poland) were utilized for the procedure. This device operates at 230V, 50/60Hz, with a maximum power of 70W and a 100VA rating. Iontophoresis employs low-voltage galvanic current to deliver ionized compounds, specifically acetylcholine (ACh) at 1% concentration, into soft tissue structures. As acetylcholine carries a positive ionic charge, it was administered via the anode (the active electrode), which maintains an equivalent polarity. The active electrode incorporates a chamber with a semi-permeable membrane housing the acetylcholine solution. This electrode was positioned laterally on the leg over the fibular head to target the common peroneal nerve, corresponding to the conduction block site inferior to the knee joint. The indifferent electrode was situated on the foot dorsum to complete the electrical circuit.

Outcome Measures

Laboratory methods: Serum NO levels measured via laboratory assay.

Nerve Conduction Study (NCS): Electrodiagnostic studies are reliable, precise, and sensitive methods for detecting polyneuropathy. 19 Nerve conduction studies (NCSs) are commonly utilized in electrodiagnostic evaluations. Among these tests, NCSs provide the most significant insights for diagnosing polyneuropathy. 20

The NCS protocol was carried out by one of the authors as follows:

NCSs were conducted on one lower limb. The combination of these methods represents the most sensitive electrodiagnostic approach for identifying DSPN. Normal bilateral findings exclude the diagnosis of DSPN, necessitating no further NCS.

It is advised to perform further NCSs if the peroneal motor or sural sensory NCSs are aberrant. An NCS of one upper extremity is recommended, preferably assessing the ulnar motor, median sensory, and ulnar sensory nerves. A contralateral sural sensory and one tibial motor nerve conduction study may be performed at the examiner’s discretion. Interpreting the results of the median and ulnar examination requires caution, as abnormalities may arise from nerve compression at the wrist or ulnar neuropathy at the elbow.

Absent response in any nerve, whether motor or sensory, necessitates contralateral NCS.

In the absence of a detectable peroneal motor response, it is recommended to conduct an NCS of the ipsilateral tibial motor nerve. Undetectable peroneal motor response requires ipsilateral tibial motor NCS.

Electrodiagnostic DSPN confirmation adheres to established criteria. The Numerical Rating Scale (NRS) is validated for individuals ≥9 years. 21 , 22

3. Laser Doppler qualification: (blood flow) The technique can be used to monitor microcirculatory activity in both healthy and pathological tissues, providing critical data on microvascular responses to physiological stimuli.

Sample Size Calculation

Sample size was determined using G*Power software (version 3.1.9.7). We calculated the power for a mixed-factor MANOVA (Global effects) to account for the interaction between the two groups (Experimental vs Control) and the repeated measurements (Pre vs Post) across multiple dependent variables. Based on a pilot study or similar literature (Zaid et al, 2018), we anticipated a Pillai’s V (Effect Size) of 0.15, which corresponds to a Cohen’s f 2 (V) of 0.18. With a significance level (α) of 0.05 and a power (1-β) of 0.90, the required sample size was 68 participants (34 per group). To account for a potential 15% attrition rate, the final enrollment was set at 80 participants (40 per group).

The choice of a mixed-model MANOVA was justified to control for Type I error inflation that occurs when conducting multiple independent t -tests or ANOVAs on highly correlated physiological variables (NO levels, blood flow, and nerve conduction). MANOVA accounts for the inter-correlations between these outcomes, providing a more robust assessment of the intervention’s overall physiological impact before performing univariate follow-up tests.

Statistical Analysis

An unpaired t -test was employed to compare participant characteristics between the two groups. The Shapiro–Wilk test was utilized to determine if the data adhered to a normal distribution, whereas Levene’s test was applied to evaluate the homogeneity of variances among groups. A two-way mixed MANOVA was performed to investigate within-group and between-group effects on variables including nitric oxide (NO), latency, amplitude, and velocity of peripheral nerves. Bonferroni corrections were implemented to address multiple comparisons. The significance threshold for all statistical tests was established at p < 0.05. All analyses were conducted utilizing the Statistical Package for Social Sciences (SPSS) software, version 25, for Windows (IBM SPSS, Chicago, IL, USA).

Results

Subject Characteristics

Eighty patients diagnosed with peripheral neuropathy were enrolled in this study. The characteristics of participants in groups A and B were compared, as presented in Table 1 . No significant differences were observed between the two groups regarding age, weight, height, and BMI (p > 0.05).

Table 1 Basic Characteristics of Participants Group A Group B MD t-value p-value Mean ± SD Mean ± SD Age (years) 49.97 ± 3.82 50.85 ± 4.07 −0.99 −0.33 0.33 Weight (kg) 74.13 ± 6.64 74.35 ± 6.74 −0.15 −0.63 0.88 Height (cm) 160.35 ± 6.18 159.93 ± 5.98 0.31 −0.02 0.75 BMI (kg/m 2 ) 28.96 ± 3.55 29.20 ± 3.49 −0.29 −0.03 0.76 Abbreviations : SD, standard deviation; MD, mean difference; p-value, level of significance.

Basic Characteristics of Participants

Abbreviations : SD, standard deviation; MD, mean difference; p-value, level of significance.

Mixed MANOVA showed a significant treatment × time interaction (F = 266.70, p = 0.001) and significant main effects of time and treatment. Study group A exhibited a 25.12% increase in nitric oxide levels post-treatment (p < 0.001), whereas control group B showed no change. No significant differences were observed in peripheral nerve conduction parameters in either group.

Table 2 presents baseline and acetylcholine iontophoresis-induced perfusion values at room temperature (30°C) for both groups. In the experimental group, baseline perfusion increased from 7.6 to 24.6, and perfusion with acetylcholine iontophoresis increased from 25.2 to 69.7 post-treatment. In the control group, baseline perfusion increased from 4.0 to 11.7, and perfusion with sham acetylcholine iontophoresis increased from 15.0 to 33.1 post-treatment. These results indicate a greater enhancement of microcirculatory perfusion in the experimental group following treatment.

Table 2 Mean and Standard Deviation of Blood Flow (Base Line, Acetylcholine Iontophoresis) Pre and Post-Treatment of Groups Experimental and Control Group Mean SD SdEr Experimental Group (No=40) BaselinePre 7.6 2.2 0.3 BaselinePost 24.6 7.4 1.2 IontophPre 25.2 9.2 1.4 IontophPost 69.7 18.6 2.9 Control Group (No.=40) BaselinePre 4.0 1.4 0.2 BaselinePost 11.7 4.2 0.7 Sham IontophPre 15.0 5.4 0.8 Sham IontophPost 33.1 15.6 2.5 Abbreviations : SD, Standard deviation; MD, Mean difference; SdEr, Standard error.

Mean and Standard Deviation of Blood Flow (Base Line, Acetylcholine Iontophoresis) Pre and Post-Treatment of Groups Experimental and Control

Abbreviations : SD, Standard deviation; MD, Mean difference; SdEr, Standard error.

Discussion

Our study differs from previous studies, which primarily evaluated the skin microvasculature response to iontophoretically applied acetylcholine (ACh) and local warming using a Laser Doppler imaging system. In contrast, we directly quantify nitric oxide (NO)—the primary mediator of microvascular vasodilation—and demonstrate a statistically significant intergroup difference in endothelium-dependent vasodilatory NO, favoring the experimental cohort. This finding suggests a pathophysiological nexus between neuropathy and microcirculatory impairment. This study revealed that the improvement in NO levels was significantly greater in Group A (25.12%) compared to Group B (0.20%). Enhancements in vasodilatory mechanisms were observed, including increased NO bioavailability and reduced oxidative stress. Diminished bioavailability indicates impaired endothelial nitric oxide synthesis and utilization, resulting from oxidative stress, the expression of proinflammatory factors, and dysregulation of vascular tone, which are key factors in endothelial dysfunction. Increased bioavailability of nitric oxide enhances vasodilation and tissue perfusion, leading to improved oxygenation and nutrient delivery, which in turn improves tissue vitality. 12 The study revealed no improvement in latency, amplitude, or nerve conduction compared to the normal ranges for healthy individuals. Only improvement was found in NO, which yielded a mean of 55.8 mmol/L.

Patients with diabetes often exhibit compromised endothelium-dependent vasodilation, leading to microcirculatory dysfunctions linked to diabetic neuropathy. This impairment highlights the significance of addressing vascular health in the management of neuropathic complications. Studies have revealed no statistically significant differences in NCS evaluations of peripheral nerves pre- and post-treatment. The flare response, dependent on a functional axon reflex in peripheral C fibers, is significantly reduced in individuals with neuropathy. The reduction is especially pronounced in neuropathic feet, underscoring the severity of nerve dysfunction associated with diabetic neuropathy. The flare response functions as a protective mechanism, while compromise highlights the importance of early intervention and management strategies. This is related to the dependence of acetylcholine iontophoresis on intact C-nociceptive fibers. Li et al (2022) indicate that acetylcholine influences C-nociceptive pathways, leading to the Lewis triple-flare response, which is characterized by nerve axon reflex-mediated vasodilatation. 13 C-nociceptive fibers exhibit heightened sensitivity due to acetylcholine, which enhances the release of substance P and calcitonin gene-related peptide. Substance P induces histamine release from mast cells, while calcitonin gene-related peptide directly causes vasodilation. This induces vasodilation autonomously. Local anesthetic blockade reduced vasodilation in controls but had minimal additional effect in neuropathy patients, indicating pre-existing C-fiber impairment. This fact influences the lack of statistically significant differences in nerve conduction velocity before and after treatment in this study. Functional vascular improvement in this study likely reflects enhanced endothelial responsiveness and microvascular regulation, potentially improving tissue perfusion and reducing oxidative stress. However, such endothelial changes should not be interpreted as evidence of immediate structural neural recovery. Neural regeneration and functional restoration involve complex, time-dependent remodeling processes; therefore, vascular enhancement may provide a supportive biological environment without necessarily translating into short-term structural or clinical improvement.

Caselli et al (2003) indicated that impaired microcirculation correlates with decreased expression of endothelial Nitric Oxide Synthase (eNOS) and poly (ADP-ribose) polymerase (PARP). 11 A notable decrease in eNOS expression is observed in individuals with peripheral neuropathy, suggesting a correlation between neuropathy and endothelial dysfunction. Under stressful conditions, including pain or trauma, C fibers release neuropeptides such as Substance P, which promote vasodilation and enhance blood vessel permeability. 23 This response functions as a protective mechanism; however, it is notably compromised in diabetic patients, irrespective of neuropathy, with the most pronounced dysfunction occurring in neuropathic feet. This study found a significant decrease in the nerve-axon response among diabetic patients when C fibers were stimulated with acetylcholine. This finding supports the hypothesis that neuropathy contributes to functional ischemia in diabetic feet, as blood flow fails to adequately increase during physiological stress, thereby exacerbating the condition.

Vassalle et al (2003) demonstrated that training induces the release of nitric oxide (NO), which is associated with an enhanced Laser Doppler flowmetry (LDF) response. Elevated levels of NO may arise from either the upregulation of NOS or increased NOS activity. 24 Tew et al (2011) observed that aerobic fitness influences the function of noradrenergic sympathetic fibers in heat-induced NO production by increasing NOS activity. 25 This effect is likely due to repetitive shear stress applied to the endothelium during aerobic exercise. Alongside endothelial adaptations, additional factors may play a role, including heightened sensitivity to endothelial vasodilators and alterations in neural vascular control mechanisms. Local heating acts as a stimulus for vasodilation, further promoting these effects.

The neurovascular response and activation of C-fibers occur when peripheral C-fibers are stimulated by noxious stimuli or experimentally via acetylcholine. This antidromic conduction results in the release of vasodilatory neuropeptides, which leads to a vasodilatory response. This mechanism is integral to Lewis’s triple response, which is linked to its anti-inflammatory effects. Individuals with diabetic neuropathy exhibit a marked impairment in neurovascular response. In patients without diabetic peripheral neuropathy, the neurovascular response is significantly diminished following the application of local anesthesia. In individuals with diabetic peripheral neuropathy, the compromised neurovascular response in the foot is not influenced by local anesthesia, highlighting the severity of the impairment. 24

Berghoff et al (2002) investigated the prolonged vasodilatory effects of acetylcholine (ACh), highlighting the roles of nitric oxide (NO) and prostaglandins (PGs) in this mechanism. 26 The study examined the long-term effects of ACh administered through iontophoresis on cutaneous microcirculation, evaluated using laser Doppler flowmetry (LDF), a method for quantifying skin blood flow. This study sought to enhance the comprehension of the impact of endothelial regulation on microcirculation. Veves et al (1998) investigated the relationship between eNOS expression and microvascular blood flow in diabetic patients with neuropathy. The study investigated endothelium-dependent vasodilation in the dorsal foot, employing cholinergic iontophoresis and heating as stimuli. 27 The findings emphasized the significant role of endothelial mechanisms in the development and progression of diabetic neuropathy.

Limitations

The intervention period was only 4 weeks, followed by an evaluation conducted 6 weeks later. No long-term effect was evaluated. Therefore, future longitudinal studies are required to bridge the gap between these acute physiological changes and sustained functional recovery.

Conclusion

Repeated acetylcholine iontophoresis significantly improved microvascular function, increasing nitric oxide release and tissue perfusion in patients with type 2 Diabetes. While no immediate changes in peripheral nerve conduction were observed, the intervention shows promise as an adjunctive strategy for supporting microvascular health in diabetic neuropathic complications.

Funding Statement

The authors declare that no funding was received for this study.

Abbreviations

NOS, nitric Oxide Synthase; Ach, acetylcholine; DM, diabetes mellitus; NO, nitric Oxide; PAI, plasminogen activator inhibitor-1; t-PA, tissue plasminogen activator; u-PA, urokinase plasminogen activator; cGMP, cyclic guanosine monophosphate; CGRP, calcitonin gene-related peptide; NCS, nerve conduction study; DSPN, diabetic sensorimotor polyneuropathy; LDF, laser doppler flowmetry; PGs, prostaglandins; NRS, numerical rating scale.

Data Sharing Statement

Data available upon request from corresponding author.

Author Contributions

Ibrahim ismail Abuzaid and Hassan Abdelnour: Methodology, supervision, formal analysis, writing, and visualization. Nermeen Bieedy: Investigation, data curation, resources, and reviewing. Abdulmagid Almalty: Investigation, data curation, writing, formal analysis, and resources. Sobhy Mahmoud Aly: Conceptualization, investigation, data curation, and investigation. Mahmoud Sabry Ahmed: Methodology, investigation, formal analysis, and supervision. Reham Abdelrazek: Methodology, data curation, investigation, and resources. Abdelrazak A Ahmed: Formal analysis, writing, visualization, and supervision. All authors gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

No conflict of interest relevant to this article was reported.

References

Associated Data

Data Availability Statement

Data available upon request from corresponding author.