Electroacupuncture Combined with Press Needles Alleviates Simple Obesity via VEGF-C/VEGFR-3/PI3K/AKT Signaling Pathway

The incidence of obesity has been consistently increasing due to improvements in living standards, shifts in dietary habits, and environmental influences, making weight loss a considerable challenge [1‒4]. Obesity is classified into two categories: simple obesity and secondary obesity, with simple obesity accounting for approximately 95% of all cases. Simple obesity is defined by the absence of identifiable causes related to endocrine or metabolic disorders. This condition arises from an energy imbalance in which caloric intake exceeds caloric expenditure, leading to excessive fat accumulation and increased body weight (BW) [5‒7]. Traditional weight loss approaches, including dietary therapy, pharmacotherapy, and bariatric surgery, often come with complications such as weight regain, gastrointestinal issues, and oily stool [4].

Accumulating studies have demonstrated the potential effectiveness of electroacupuncture (EA) therapy in treating obesity, showing promising outcomes like weight reduction, decreased abdominal fat, and hormone regulation [8‒11]. However, factors such as intermittent acupuncture stimulation and patient adherence may influence the efficacy of EA treatment. To address these challenges, press needles therapy can offer continuous stimulation through needle retention, which could improve patient compliance and enhance therapeutic outcomes for obesity. Despite the promise of these therapies, there remains a gap in understanding whether combining EA with press needles provides superior efficacy compared to EA alone in treating obesity. Additionally, the underlying mechanisms of action for this combined approach warrant further exploration.

Lymphatic vessels played a crucial role in the pathology of obesity by affecting lipid absorption and metabolism. Even minor damage to the lymphatic system could trigger gene activation in adipose tissue, leading to hypertrophy and proliferation [12‒14]. The VEGF-C/VEGFR-3 signaling pathway was essential for lymphangiogenesis [15‒17], with VEGF-C being a critical factor during embryonic development and VEGFR-3 promoting the proliferation and migration of lymphatic endothelial cells [18, 19]. Dysregulation of the VEGF-C/VEGFR-3 pathway contributed to lymphatic vascular dysfunction, resulting in fat accumulation and abnormal metabolic processes that could lead to obesity [20]. Therefore, targeting intestinal lymphatic function may represent a new strategy for obesity treatment.

In light of these observations, our study aimed to investigate, for the first time, the combined effect of EA and press needles on simple obesity. We hypothesized that this combination would demonstrate greater therapeutic efficacy than EA alone, potentially by modulating the VEGF-C/VEGFR-3/PI3K/AKT signaling pathway, which may lead to improvements in lymphatic vessel structure and function. This research endeavored to provide valuable insights into a novel approach for treating simple obesity, focusing on both the efficacy of the combined therapies and the underlying mechanisms involved.

A total of 80 patients with simple obesity were recruited in this study from December 2021 to January 2023. The age range of the patients included in this study spanned from 18 to 53 years. The diagnostic criteria for simple obesity were formulated with reference to the “Evidence-based Clinical Practice Guidelines: Obesity” and the “Waist Circumference and Waist-Hip Ratio: Report of a WHO Expert Consultation.” The criteria are as follows: (1) body mass index (BMI) ≥25.0 kg/m²; (2) waist circumference (WC): ≥90 cm for males, ≥80 cm for females; and (3) standard BW: for males, [height (cm) − 80] × 70%; for females, [height (cm) − 70] × 60%, exceeding 20% of the standard BW. Diagnosis of simple obesity is made if the patient meets two out of the three above criteria, and secondary causes are excluded. This study was approved by the Ethics Committee of Nantong University Hospital (Ethics Approval No. 2020-K032). All participants were fully informed and signed written informed consent forms.

In this study, 80 simple obese patients were randomly divided into two groups: the control group and the observation group. The control group received EA treatment at specific acupoints, including RN12 (Zhong Wan), LI11 (Qu Chi), ST36 (Zu San Li), ST37 (Shang Ju Xu), ST44 (Nei Ting), ST25 (Tian Shu), SP15 (Da Heng), BL20 (Pi Shu), and BL21 (BL21). The pulse acupuncture treatment instrument (KWD-808I, Yingdi Electronic Medical Equipment Co., Ltd., Changzhou, China) was used, with ipsilateral ST36 and ST37 and bilateral ST25 connected to electrodes. The treatment involved using a sparse-dense wave with a frequency of 2 Hz/15 Hz for a duration of 30 min. The observation group, after receiving the same EA treatment as the control group, underwent press needle therapy. Press needles were inserted into selected acupoints including ST25, SP15, ST36, ST37, LI11, BL20, and BL21. Before each meal, patients pressed their hands on the embedded needles 50 times for a period of 30 min. Both groups received treatment every other day, three times a week, for a total of 3 months. During the treatment period, patients were instructed not to consume any food after 20:00, except for drinking water.

For BW measurement, patients were asked to be fasting, barefoot, and wearing light clothes. The WC was measured at the level of the midpoint of the line between the iliac crest and the lower edge of the 12th rib at the end of natural expiration using a soft ruler with a minimum scale of millimeters. The sebum thickness of the right triceps brachii and the right subscapularis was measured, and the average value was taken three times. BMI, body fat percentage (F%), and abdominal circumference (A) were calculated separately. BMI = weight [kg] ÷ (height [m])², F% = (4.570 ÷ D - 4.142) × 100%, and A = ([measured BW - standard body weight] ÷ standard body weight) × 100%. D is the body density, and for males D = 1.093-0.0016X, female D = 1.097-0.0013X, where X = right subscapular horn sebum thickness (mm) + right triceps sebum thickness (mm).

Serum levels of factors related to the function of intestinal lymphatics: 5 mL of fasting venous blood was collected from patients, serum was centrifuged, and serum levels of VEGF-C, delta-like ligand 4 (DLL4), and adrenomedullin (ADM) were detected by using enzyme-linked immunosorbent assay (ELISA) kits (CUSABIO Life Science, Wuhan, China) following the manufacturer’s instructions.

A total of 55 male Sprague-Dawley rats, aged around 3 weeks and weighing between 40 and 60 g, were obtained from Nantong University Experimental Animal Center. The study was approved by the Nantong University Institutional Animal Care, and the animal license number was SCXK (Su) 2019-0001. After 1 week of acclimatization on regular chow, the rats were randomly divided into two groups: a blank control group consisting of 10 rats, and an HFD group consisting of 45 rats. Following a 12-week feeding period, the rats in the HFD group with a body mass exceeding 20% of the mean body mass of the blank control group were identified as obese rats. These obese rats were then further randomized into three groups: the obesity control group, EA treatment group, and EA combined with press needles group, each consisting of 10 rats. Throughout the modeling period, the rats’ BW and length were measured and recorded weekly, and Lee's index was calculated. This study received approval from the Animal Ethics Committee of Nantong University, with an ethics approval number of S20200905-001.

After isoflurane gas anesthesia, the rats were disinfected with local iodine volt, six 13 mm stainless steel acupuncture needles (Hwato, Suzhou, China) were stabbed directly into the bilateral ST36 (Zu San Li) and ST37 (Shang Ju Xu) with the depth of needle penetration at about 6 mm, and the bilateral ST44 (Nei Ting) with the depth of needle penetration at about 2 mm. EA instrument (KWD-808I, Yingdi Electronic Medical Equipment Co., Ltd., Changzhou, China) was used to connect the ipsilateral ST36-ST37 (frequency: 2/15 Hz; current: 2 mA; duration: 30 min). After the EA operation, the hair was scraped off at the acupoints of BL20 (Pi Shu) and BL21 (Wei Shu) of the rats, and after local routine disinfection, the press needles (DIG INTO CREATOR, Suzhou, China) were inserted into the selected acupoints, fixed with tape for 120 min, and placed in a single cage. The acupoint area was pressed with finger pressure for 1 min every 30 min. The treatment was taken every other day, 3 times a week, 4 weeks for continuous treatment. Meanwhile, the rats in the EA group for EA operation only and the rats in the obesity control group were subjected to the same anesthesia but no EA and press needles, and the rats in the blank control group were fed and moved freely without any treatment.

After a 4-week intervention period of EA combined with press needles, the four groups of rats were subjected to a 12-h fasting period. Following this, the rats were anesthetized with intraperitoneal injection of 3% sodium pentobarbital (0.2 mL/100 g). Approximately 3–4 mL of blood was collected from the abdominal aorta and centrifuged at 4,000 r/min for 8 min at 4°C. The resulting serum was isolated and stored at −80°C for lipid testing. The levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) in the serum were measured using enzyme assay kits (Beckman Coulter, Inc., CA, USA). After blood collection, the duodenum and part of the liver were removed from each group of rats. They were rinsed 2–3 times with saline, and a portion of the duodenum and liver was dissected and fixed with 4% paraformaldehyde. The remaining duodenum was rapidly stored in a −80°C liquid nitrogen tank. Additionally, the white adipose tissue (WAT) from the groin and epididymis on one side of the rats in each group was dissected, washed 2–3 times with saline, dried quickly with filter paper, weighed, and fixed with 4% paraformaldehyde.

RNA samples were isolated from rat intestinal lymphatic tissues using RNA-Quick Purification Kit (Yeasen Biotechnology, Shanghai, China) and reversely transcribed using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). The qRT-PCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China) with specific primer pairs on a StepOne Real-Time PCR System (Applied Biosystems, CA, USA). The primers sequences used in the experiment are shown in Table 1.

Total proteins were extracted from rat intestinal lymphatic tissues using RIPA lysis buffer (Beyotime, Shanghai, China) and quantified with the BCA Protein Assay Kit (Beyotime, Shanghai, China). Briefly, equal amounts of protein samples were loaded onto 8% or 12% SDS-PAGE gels (Beyotime, Shanghai, China) and electrotransferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% BSA (Beyotime, Shanghai, China) at room temperature and incubated overnight at 4°C with primary antibodies. Following this, they were probed with HRP-conjugated secondary antibodies (Beyotime, Shanghai, China) at room temperature and treated with an ECL Detection Kit (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was determined and quantified using ImageJ software (NIH, MD, USA). The following primary antibodies were used in the experiments: anti-AKT (1:1,000) and anti-p-AKT (1:1,000) from Abclonal Technology; anti-PI3K (1:500), anti-VEGF-C (1:500), and anti-VEGFR-3 (1:500) from Santa Cruz Biotechnology; anti-p-PI3K (1:1,000) from Affinity Biosciences; and anti-β-tubulin from Signalway Antibody.

The fixed liver and adipose tissues were embedded in paraffin separately. Then, 8-μm-thick sections were obtained by using a rotary slicer and mounted on slides. Hematoxylin-eosin staining was performed according to a standard histological protocol [21]. Then the tissue slides were observed under a light microscope (Olympus, Tokyo, Japan) to study pathological changes.

Intestinal tissues were fixed and sectioned, followed by dewaxing and dehydration. Antigen retrieval was performed through heating to enhance antibody binding efficiency. The samples were then treated with a blocking solution to minimize nonspecific interactions. Subsequently, sections were incubated with primary antibodies specific to the target proteins, followed by washing and incubation with secondary antibodies for detection. Cell nuclei were stained with DAPI (Southern Biotech, Birmingham, AL, USA), and the samples were examined using a Zeiss Axio Imager M2 microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) to capture detailed images, which were quantified using ImageJ (NIH, MD, USA). The following primary antibodies were used: anti-AKT (1:200) and anti-p-AKT (1:200) from Abclonal Technology; anti-PI3K (1:100), anti-VEGF-C (1:100), and anti-VEGFR-3 (1:100) from Santa Cruz Biotechnology; anti-p-PI3K (1:100) from Affinity Biosciences; and anti-β-Tubulin from Signalway Antibody. Secondary antibodies used were goat anti-mouse IgG-Alexa Fluor 488 (1:500) from Abcam and sheep anti-rabbit IgG-Cy3 (1:1,000) from Sigma-Aldrich.

Statistical analyses were performed using IBM SPSS Statistics software version 22.0 (Chicago, IL, USA) and GraphPad Prism 9 (CA, USA). Data were first assessed for normality using the Kolmogorov-Smirnov test, with normally distributed continuous variables reported as mean ± standard deviation. For clinical data, statistical methods included one-sample t tests and independent samples t tests, while effectiveness was evaluated using chi-squared tests. Significant differences in the results of animal experiments were analyzed using one-way ANOVA with Tukey’s multiple comparisons test or Student’s t test. The significance levels were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Patients meeting the inclusion criteria for simple obesity were randomly assigned to two groups: the observation group (treated with EA combined with press needles) and the control group (treated with EA alone). The specific treatment methods are illustrated in Figure 1. Compared to baseline measurements, both the EA treatment group and the combined treatment group exhibited significant reductions in BW, BMI, F%, A, and WC (all p < 0.05). Additionally, the serum levels of intestinal lymphatic-related factors, including VEGF-C, DLL4, and ADM, were also significantly lower than those measured before treatment (all p < 0.05). Notably, the combined treatment group showed significantly greater effectiveness compared to the EA treatment group alone (all p < 0.05) (Table 2). These findings indicated that the combination of EA and press needle therapy could improve obesity-related metrics and modulate levels of intestinal lymphatic function-related factors, highlighting the therapeutic potential of this integrated approach for managing simple obesity.

To further explore the efficacy and underlying mechanisms of EA combined with press needles therapy in the treatment of simple obesity, we established an obesity rat model by subjecting the rats to an HFD. Subsequently, the rats were randomly assigned to different treatment groups: one group received EA treatment, while another group underwent combined therapy involving both EA and press needles (Fig. 2a). Following the modeling phase and prior to intervention, BW (Fig. 2b) and Lee’s index (Fig. 2d) in rats fed an HFD were significantly higher than those of the control group on a normal diet (all p < 0.0001). No significant differences were observed among the other three groups. After 4 weeks of intervention, both EA alone and EA combined with press needles resulted in a significant reduction in BW (Fig. 2c) compared to the obesity control group (all p < 0.0001). Notably, the group receiving the combined treatment exhibited a more substantial weight reduction than those treated with EA alone (p = 0.0402). Additionally, both EA alone (p = 0.0419) and EA combined with press needles (p < 0.0001) led to a significant decrease in the Lee’s index (Fig. 2e) compared to the obesity control group, with the combined treatment group showing a more pronounced reduction than the EA alone group (p = 0.0493). These findings suggested that EA significantly alleviated obesity symptoms. Moreover, the addition of press needles treatment further amplified this therapeutic effect.

To assess the impact of acupuncture on the metabolic profile of obese rats, we analyzed lipid profiles and the total wet weight of WAT following the intervention. The obesity control group exhibited significant elevations in TC (Fig. 3a), TG (Fig. 3b), and LDL-C (Fig. 3c) levels compared to the control group (all p < 0.0001). In comparison to the obesity control group, EA treatment resulted in significant reductions in TC (p = 0.0052), TG (p = 0.0007), and LDL-C (p = 0.0007) levels, with even more pronounced decreases observed in the group that received EA combined with press needles (all p < 0.0001). The levels of TC (p = 0.0458), TG (p = 0.048), and LDL-C (p = 0.0469) in the mice receiving combined treatment were lower than those observed in the EA alone group.

Additionally, the total wet weight of WAT (Fig. 3d), encompassing both epididymal and groin fat, as well as the individual weights of epididymal fat (Fig. 3e) and groin fat (Fig. 3f), was significantly elevated in the obesity control group compared to the control group (all p < 0.0001). In contrast, EA treatment resulted in a significant reduction in the total wet weight of WAT (p = 0.0323), epididymal fat (p = 0.0107), and groin fat (p = 0.0227) when compared to the obesity control group. Notably, the combined treatment group exhibited even more pronounced reductions in these parameters (all p < 0.0001). The total wet weight of WAT (p = 0.0464), as well as the weights of epididymal fat (p = 0.0465) and groin fat (p = 0.0481), in mice receiving the combined treatment was significantly lower than those observed in the EA alone group.

Obesity can significantly alter the morphology of both adipose tissue and the liver [22]. Before treatment, epididymal adipocytes in obese rats displayed irregular sizes, indistinct borders, uneven areas, and loose packing compared to the control group. After treatment with EA alone and in combination with press needles, these adipocytes showed significant improvements, becoming more regular and fuller, albeit with slightly less distinct borders. Cell areas became more uniform, and the adipocytes appeared densely packed, resembling those in the control group. Similarly, the liver tissue of obese rats exhibited disrupted structures with irregular, disorganized cells, increased fat droplets, and a looser cellular arrangement. Following treatment, there was a notable reduction in intercellular fat droplets, and the cells exhibited a more regular, uniform, and closely arranged pattern, resembling the morphology of normal rat liver tissue (Fig. 4a).

The intestinal lymphatic vessels, integral components of the lymphatic drainage system within the intestinal wall, played a crucial role in immune response and detoxification. Obesity impacted the structure and function of these vessels [12]. In obese rats, both the number (p = 0.0012) and diameter (p = 0.0057) of intestinal lymphatic vessels were significantly higher than those in the control group. In comparison to the obesity control group, the EA treatment group demonstrated significant reductions in both the number (p = 0.0003) and diameter (p = 0.0172) of lymphatic vessels. Notably, the combined treatment group exhibited even more pronounced effects in terms of both the number (p < 0.0001) and diameter (p = 0.0001) of these vessels. Furthermore, the number (p = 0.0498) and diameter (p = 0.0477) of lymphatic vessels in the combined treatment group were significantly lower than those in the EA treatment group (Fig. 4b, c).These results indicated that both EA alone and the combination of EA with press needle treatment significantly enhanced lymphatic vessel function, with the combined treatment showing superior efficacy.

VEGF-C and VEGF-D interacted with the VEGFR-3 receptor under both normal and pathological conditions to promote lymphatic proliferation, migration, and survival by activating the PI3K-Akt and MAPK-ERK signaling pathways [23]. Our findings showed that expression levels of VEGF-C and VEGFR-3, at both the mRNA (Fig. 5a–b) and protein (Fig. 5c) levels, were significantly elevated in the intestinal tissues of rats on an HFD. EA treatment resulted in substantial reductions in these factors, with the combination of EA and press needles leading to even greater decreases. Furthermore, we found that protein levels of p-PI3K and p-AKT were significantly elevated in the intestinal tissue of HFD rats. EA effectively reduced the expression of both p-PI3K and p-AKT, and their levels were significantly lower in the group receiving combined EA and press needle treatment compared to EA treatment alone (Fig. 5c). We further validated our findings through immunofluorescence staining, which yielded results consistent with our previous observations (Fig. 6). Collectively, these findings supported the idea that VEGF-C signaling via VEGFR-3 regulated the PI3K-Akt pathway during lymphangiogenesis. Moreover, the dysregulation of this signaling in HFD-induced conditions can be alleviated by EA treatment, particularly when combined with press needles.

Obesity can be classified into two categories based on its pathogenesis and etiology: simple obesity and secondary obesity. Simple obesity is primarily associated with excessive caloric intake and genetic predisposition, while secondary obesity results from endocrine disorders and other contributing factors [5‒7]. The implications of obesity are profound, affecting both quality of life and overall health. This condition is closely linked to reduced life expectancy and an increased risk of various diseases, including cardiovascular disorders, diabetes, depression, and cancer [24]. Increasing evidence supported the safe and effective use of EA in treating abdominal obesity. EA achieved this by stimulating the body's meridians and acupoints, promoting overall improvement in body condition, regulating intake, and enhancing metabolic processes [25, 26]. In this study, we aimed to evaluate the effects of combining EA with press needles on simple obesity. Following treatment, we assessed the patients’ physical parameters to validate the efficacy of this approach. Our findings indicated that the combined therapy of EA and press needles significantly reduced BW, BMI, F%, A, and WC compared to their pretreatment values. Furthermore, patients receiving the combination therapy exhibited greater reductions in these physical parameters than those who underwent EA treatment alone. These results underscored the feasibility and potential effectiveness of the combined EA and press needle approach in managing simple obesity.

Following our promising results from human studies on the efficacy of combining EA with press needles in treating primary obesity, we further explored the underlying mechanisms by establishing a mouse model of this condition, which yielded consistent results. In this model, treatment with EA combined with press needles led to significant reductions in BW and Lee’s index. TG and TC, crucial elements in glycolipid metabolism, were typically elevated in individuals with obesity compared to healthy individuals. LDL, a small-diameter lipoprotein, served as the primary carrier of cholesterol during transport, and abnormal LDL levels were indicative of dyslipidemia, a common condition observed in obese patients [27, 28]. We observed substantial decreases in serum levels of TG, TC, and LDL-C, along with a reduction in the WAT. These findings highlighted the efficacy of the EA and press needle combination therapy for managing simple obesity by effectively reducing key physical indicators and improving metabolic parameters.

Obesity induced significant changes in the biology, morphology, and function of adipose tissue [29]. Excessive fat accumulation resulted in an influx of macrophages and immune cells into adipose tissue, liver, and muscle [30, 31]. Inflammation within adipose tissue played a pivotal role in the development of obesity [32]. Research had shown that EA at the ST36 acupoint in Sprague-Dawley rats improved the inflammatory profile of adipose tissue by reducing circulating levels of inflammatory hormones, suppressing the expression of inflammatory cytokines, and decreasing macrophage infiltration, which consequently leads to reduced BW [33]. In alignment with these findings, our study demonstrated that the combination of EA with press needles significantly ameliorated the inflammatory state of WAT and the liver in obese rats, resulting in reductions in both weight and fat mass.

Studies indicated that increased fat cell accumulation was linked to impaired lymphatic vessel function [34]. Patients with obesity demonstrated heightened lymphatic vessel proliferation and increased permeability in the intestinal lymphatics, accompanied by impaired lymphatic function and a significant rise in serum levels of associated factors VEGF-C, DLL4, and ADM [35‒37]. Our clinical trial results demonstrated that, after treatment, the serum levels of VEGF-C, DLL4, and ADM significantly decreased in both the EA combined with press needles and the EA alone group compared to baseline levels. Notably, the treatment effectiveness in the EA combined with press needles was superior to that of the EA alone group. Additionally, in obese rats, the number and diameter of intestinal lymphatic vessels were significantly greater than in the control group. The EA treatment group demonstrated significant decreases in both the number and diameter of these vessels compared to the obesity control group. The combined treatment group showed even more pronounced reductions. Moreover, the number and diameter of lymphatic vessels in the combined treatment group were significantly lower than those in the EA treatment group.

The VEGF-C/VEGFR-3 signaling pathway played a crucial role in the development of lymphatic vasculature; abnormalities in this pathway led to lymphatic dysfunction, resulting in fat accumulation and metabolic disturbances that contributed to obesity [20]. VEGF-C was essential for lymphangiogenesis during embryonic development, as evidenced by perinatal VEGF-C deficiency in mice, which caused degeneration of celiac ducts and impaired fat absorption. Notably, these VEGF-C-deficient mice exhibited resistance to obesity and improved glucose metabolism under a HFD [19]. VEGFR-3, an early-identified receptor specific to lymphatic endothelial cells, promoted their proliferation, migration, and neogenesis by binding to VEGF-C ligands [18]. A significant downstream pathway associated with VEGF-C/VEGFR-3 signaling was the PI3K/AKT pathway, which appeared to play a key role in lymphatic endothelial cell migration [38]. PI3K was known to interact directly with phosphorylated VEGFR-3, facilitating the formation and migration of lymphatic endothelial cells [39]. Furthermore, studies have demonstrated that specifically targeted lymphatic vessel inhibitors effectively reduced VEGF-C release, decreased lymphatic vessel branching and leakage, reversed metabolic abnormalities caused by lymphatic leakage, and ultimately led to reductions in BW [40]. In our study, we found that the combination of EA and press needles significantly reduced both the quantity and diameter of intestinal lymphatic vessels. Additionally, we observed decreased expression levels of VEGF-C and VEGFR-3 mRNA. These findings were corroborated by Western blot and immunofluorescence analyses, which revealed significant reductions in the protein levels of VEGF-C and VEGFR-3. Notably, the phosphorylation levels of PI3K and AKT were also significantly diminished.

The combination of EA with press needles demonstrated significant efficacy in the treatment of simple obesity, outperforming EA treatment alone. This enhanced therapeutic effect was mediated through the modulation of the VEGF-C/VEGFR-3/PI3K/AKT signaling pathway, resulting in marked improvements in lymphatic vessel structure and function.

The clinical study was reviewed and approved by the Ethics Committee of Nantong University Hospital (Approval No. 2020-K032). Informed written consent was obtained from all participants. The experimental animal research protocol was reviewed and approved by the Animal Ethics Committee of Nantong University (Approval No. S20200905-001).

The authors declare that they have no conflicts of interest.

This work was funded by the National Natural Science Foundation of China: Exploring the Specificity of the “Acupuncture Point-autonomic Nerve-fat” Association from Electroacupuncture Weight Loss Effect Specificity (No. 81873238).

Zhi Yu and Bin Xu conceived the study and designed the research methodology. Minghui Xia conducted the experiments and collected the data. Yuhang Wang and Donghua Liu performed the statistical analysis and interpreted the results. Yan Wang and Shuang Wu contributed to the writing and editing of the manuscript. All authors reviewed and approved the final version of the manuscript, ensuring its accuracy and integrity.

The data that support the findings of this study are not publicly available as they contain information that could compromise the privacy of research participants. However, these data are available from the corresponding author, Xu Bin, upon reasonable request.