Abstract

Background: Special education rehabilitation therapy is the primary approach for improving core symptoms of autism spectrum disorder (ASD). However, most children with ASD suffer from comorbid gastrointestinal dysfunction, including constipation, abdominal pain, and nausea, which severely compromise their physical and mental well-being. Press-needle acupuncture can improve gastrointestinal function, while vitamin D (VitD) participates in neurological development and immune regulation and is closely associated with ASD symptoms and functional outcomes. The therapeutic efficacy of combining press-needle acupuncture with VitD for behavioral and gastrointestinal symptoms in children with ASD requires investigation.

Objective: To examine the effects of press-needle acupuncture combined with VitD on behavioral and gastrointestinal symptoms in children with ASD, building upon special education rehabilitation therapy.

Methods: Eighty-four children with ASD undergoing rehabilitation training at Gansu Province Hospital Rehabilitation Center and Affiliated Hospital of Gansu University of Chinese Medicine between April 2022 and June 2024 were enrolled. Participants were randomly divided into two groups (42 in the observation group and 42 in the control group) using a random number table method, with 40 cases ultimately included in each group after applying exclusion criteria. The control group received special education rehabilitation therapy alone, while the observation group received press-needle acupuncture combined with VitD2 in addition to special education rehabilitation therapy. Both groups underwent three months of treatment. The Autism Behavior Checklist (ABC), Childhood Autism Rating Scale (CARS), Social Responsiveness Scale (SRS), Traditional Chinese Medicine (TCM) gastrointestinal symptom scores, and serum 25-hydroxyvitamin D [25-(OH)D] levels were assessed before and after treatment to evaluate intervention efficacy.

Results: Compared with baseline, both groups showed significant reductions in ABC, CARS, SRS, and TCM gastrointestinal symptom scores after treatment (P<0.05). Post-treatment scores in the observation group were significantly lower than those in the control group (P<0.05). Serum 25-(OH)D levels decreased in the control group but increased significantly in the observation group (P<0.05), with post-treatment levels higher in the observation group than in the control group (P<0.05). The total effective rate was 36 cases (90.0%) in the observation group and 30 cases (75.0%) in the control group, with the observation group demonstrating superior efficacy (P<0.05).

Conclusion: Press-needle acupuncture combined with VitD demonstrates significant therapeutic effects in children with ASD, effectively improving social interaction and behavioral capabilities, alleviating gastrointestinal discomfort, enhancing quality of life, and improving rehabilitation outcomes.

Keywords: Autism spectrum disorder; Vitamin D; Acupuncture; Child; Behavioral symptoms; Gastrointestinal tract

1. Subjects and Methods

Autism spectrum disorder (ASD) is a severe, pervasive neurodevelopmental disorder characterized by social communication impairments, restricted interests or activities, and repetitive stereotyped behaviors [1]. Primary interventions include applied behavior analysis (ABA), Treatment and Education of Autistic and Related Communication-Handicapped Children (TEACCH), Relationship Development Intervention (RDI), and other special education rehabilitation therapies [2]. Additionally, children with ASD frequently experience comorbid gastrointestinal dysfunction, including constipation, abdominal pain, and nausea [3], for which no specific treatment exists. Press-needle acupuncture, which involves pressing needles embedded in acupoints on the body surface to provide sustained, effective stimulation, has been shown to improve gastrointestinal function by acting on the skin regions [4]. Vitamin D is a neurosteroid that regulates multiple brain functions and participates in neurological development and immune modulation [5]. Research indicates that VitD levels are closely associated with ASD symptoms and functional outcomes [1]. VitD2 is the most important component of VitD, and 25-hydroxyvitamin D [25-(OH)D] serves as a key intermediate metabolite for assessing VitD status [6]. Currently, rehabilitation physicians and parents have limited options for addressing gastrointestinal symptoms in children with ASD, and research on this issue remains scarce. This study leverages the advantages of integrated traditional Chinese and Western medicine by applying press-needle acupuncture combined with VitD to improve both gastrointestinal symptoms and core ASD symptoms, thereby enhancing quality of life and reducing family and societal burden.

1.1 Study Subjects

We enrolled 84 children with ASD undergoing rehabilitation training at Gansu Province Hospital Rehabilitation Center and Affiliated Hospital of Gansu University of Chinese Medicine between April 2022 and June 2024. Participants were randomly divided into two groups (observation and control groups, 42 cases each) using a random number table method. The final sample included 40 cases per group (80 total) after applying exclusion criteria: one case in the observation group was excluded for discontinuing intramuscular VitD2 injections, one for discontinuing press-needle treatment, and two cases in the control group were excluded for failing to undergo scheduled venous blood sampling for 25-(OH)D testing. This study was approved by the Medical Ethics Committee of Gansu Province Hospital Rehabilitation Center.

Diagnostic Criteria: (1) ASD diagnosis followed criteria from the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) [7]; (2) VitD nutritional status was classified as: deficiency (serum 25-(OH)D ≤ 20 ng/mL), insufficiency (21–29 ng/mL), or sufficiency (≥30 ng/mL) [8].

Inclusion Criteria: (1) Met diagnostic criteria; (2) Age 1–7 years; (3) Presence of gastrointestinal symptoms (stomach pain, nausea, poor appetite, constipation, insomnia, etc.); (4) Parental informed consent provided.

Exclusion Criteria: (1) Bleeding tendency; (2) Severe organ damage; (3) Needle phobia; (4) Allergy to press-needle adhesive tape; (5) Participation in other clinical drug studies.

Treatment Course: Three months per treatment cycle.

Dropout and Termination Criteria: (1) Withdrawal before completing treatment; (2) Incomplete data preventing efficacy determination; (3) Severe adverse reactions preventing continued treatment.

1.2 Sample Size Calculation

Sample size was estimated using the formula for comparing two independent sample rates from Medical Statistics [9]:

n1=n2= 2πc（1-πc） π1（1-π1）+π2（1-π2） π1-π2

Where α=0.05, β=0.20, and c is the correction coefficient. Literature review indicated an effective rate of approximately 72.73% for ASD rehabilitation therapy [10]. Based on clinical observation, the expected effective rate for press-needle combined with VitD treatment was 90%, with a maximum 20% dropout rate considered, yielding 42 cases per group and a total sample size of 84.

1.3 Treatment Methods

1.3.1 Control Group: Special Education Rehabilitation Therapy

The control group received special education rehabilitation therapy. For children with significant training difficulties or behavioral problems, initial one-on-one individualized intervention or parent-child collaborative teaching models were recommended. After adaptation, training gradually transitioned to a combined group and individual intervention system. Group interventions were conducted in units of 4–5 children through diversified training forms, including language ability training, cognitive ability training, motor ability training, self-care ability training, and social adaptation ability training (5 sessions daily, 30 minutes each).

Language Ability Training: For nonverbal children with ASD, training focused on articulation and oral muscle exercises; for verbal children, training emphasized language comprehension, guided verbal expression, and communication skills.

Cognitive Ability Training: This focused on external characteristics and attributes of objects through pairing, classification, and sorting activities to strengthen understanding and discrimination of basic concepts, including color and shape identification and number conservation. Specialized training targeted abstract cognitive abilities such as time and space perception, logical reasoning, and comparative analysis. Mathematical knowledge and reading skills were integrated to help children construct a systematic, comprehensive cognitive framework and enhance comprehensive thinking abilities.

Motor Ability Training: This comprised gross and fine motor training using equipment such as swings, slides, balance boards, and multi-purpose combinations to improve sensory integration function, limb coordination, and hand function.

Self-Care Ability Training: Training included drinking, eating, washing, dressing, toileting, and organizing to enhance self-care capabilities.

Social Adaptation Ability Training: This targeted initiative, communication, and interpersonal social integration skills.

Although these training components had different emphases, various障碍 issues could not be completely separated, and stereotyped behaviors were continuously corrected during training. Parents were advised to conduct life-oriented training at home using daily routines as the main thread, recording children's performance and abnormal behaviors weekly to adjust training protocols. Training was conducted 5 days per week with 2 days of rest, over a 3-month treatment cycle.

1.3.2 Observation Group: Combined Press-Needle and VitD2 Treatment

The observation group received press-needle acupuncture combined with VitD2 in addition to the control group's conventional treatment protocol.

(1) Press-Needle Acupuncture: Selected acupoints included Zhongwan (CV12), Tianshu (ST25, bilateral), Guanyuan (CV4), Zusanli (ST36, bilateral), Xinshu (BL15, bilateral), Ganshu (BL18, bilateral), Pishu (BL20, bilateral), and Shenshu (BL23, bilateral). Point location followed Acupuncture Therapeutics [11]. After routine disinfection and proper positioning, 0.2 mm × 0.9 mm disposable sterile press-needles (Sichuan Yuanquan Medical Devices Co., Ltd.) were fully pressed into the acupoints. Needles were retained for 24 hours with appropriate pressing to enhance needle sensation. Treatment was administered 5 times weekly for 3 months. All procedures were performed by acupuncturists with over 10 years of experience.

(2) VitD2 Intramuscular Injection: VitD2 [Jiangxi Ganhai Xinxin Pharmaceutical Co., Ltd., National Drug Approval No. H20054433, 5 mg (200,000 units)/vial] was administered intramuscularly. For children with deficient or insufficient 25-(OH)D levels, VitD2 was injected at 5 mg/dose (ages 1–2) or 10 mg/dose (ages 2–7) every 2 weeks until 25-(OH)D levels normalized (maximum 12 injections). For children with 25-(OH)D levels between 30–50 ng/mL, VitD2 was injected twice at the same dosage. Treatment duration was 3 months per cycle.

1.4 Outcome Measures

1.4.1 General Information

General data for eligible children with ASD, including gender, age, and disease duration, were collected from electronic medical records in the Hospital Information System (HIS) of both institutions.

1.4.2 Autism Behavior Checklist (ABC) Score [12]

The ABC comprises 57 items covering behavior, social interaction, language, sensation, and self-care abilities, with a total score of 158 points. Higher scores indicate more severe behavioral problems.

1.4.3 Childhood Autism Rating Scale (CARS) Score [13]

The CARS assesses 15 aspects including behavior, language, and emotion, with a total score of 60 points. Higher scores indicate more severe symptoms.

1.4.4 Social Responsiveness Scale (SRS) Score [14]

The SRS includes 65 items focusing on functional development, with a total score of 195 points. Higher scores indicate poorer social competence.

1.4.5 TCM Gastrointestinal Symptom Score [15]

This scale comprises 14 items including stomach pain, nausea, poor appetite, constipation, and insomnia, totaling 24 points. Higher scores indicate more severe gastrointestinal symptoms.

1.4.6 Serum 25-(OH)D Level

Three milliliters of fasting venous blood were drawn in the morning, centrifuged to separate serum, and serum 25-(OH)D levels were measured using high-performance liquid chromatography-tandem mass spectrometry.

1.5 Efficacy Evaluation

Efficacy was evaluated based on changes in ABC, CARS, and SRS scores before and after intervention [10,16]. (1) Markedly effective: Significant improvement in language, social, and emotional abilities post-intervention, with ABC, CARS, and SRS scores all decreasing by ≥10 points; (2) Effective: Improved language, social, and emotional abilities, with scores decreasing by 5–<10 points; (3) Ineffective: No significant improvement, with scores decreasing by <5 points. Total effective rate = (markedly effective cases + effective cases) / total cases × 100%.

1.6 Statistical Analysis

Data were analyzed using SPSS 26.0 statistical software. Normally distributed continuous data were expressed as (x̄±s) and compared between groups using independent samples t-tests; within-group comparisons before and after treatment used paired t-tests. Categorical data were described as cases (%) and compared between groups using χ² tests. P<0.05 was considered statistically significant.

2. Results

2.1 General Information

Among the 84 children with ASD, 50 were male (59.5%) and 34 were female (40.5%), with a mean age of (4.44±1.54) years. No statistically significant differences were observed between the two groups in gender, age, or disease duration (P>0.05) [TABLE:1].

Table 1 Comparison of General Information Between Two Groups of ASD Children

Group Cases Gender (Male/Female) Age (years) Disease Duration (years) t (χ²) value Control 40 24/16 4.43±1.56 2.71±1.38 0.049ᵃ Observation 40 26/16 4.45±1.54 2.58±1.39 2.2

Note: ᵃ represents χ² value.

2.2 Comparison of ABC, CARS, SRS, and TCM Gastrointestinal Symptom Scores

Compared with baseline, both groups showed significant reductions in ABC, CARS, SRS, and TCM gastrointestinal symptom scores after treatment (P<0.05). Before treatment, no significant differences existed between groups in these scores (P>0.05). After treatment, the observation group demonstrated significantly lower scores than the control group (P<0.05) [TABLE:2].

Table 2 Comparison of ABC, CARS, SRS, and TCM Gastrointestinal Symptom Scores Between Two Groups of ASD Children Before and After Treatment (x̄±s, points)

Group Cases ABC Score CARS Score SRS Score TCM Gastrointestinal Symptom Score Control (Before) 40 95.38±19.83 34.33±3.98 96.03±10.12 6.25±1.43 Control (After) 40 80.58±20.80 31.80±3.81 89.08±10.32 5.00±1.32 Observation (Before) 40 95.10±19.29 34.03±3.70 96.45±10.12 6.18±1.01 Observation (After) 40 72.40±21.54 30.05±3.29 84.95±10.22 4.00±0.72 t (paired) <0.001 <0.001 <0.001 <0.001

Note: ABC=Autism Behavior Checklist, CARS=Childhood Autism Rating Scale, SRS=Social Responsiveness Scale.

2.3 Serum 25-(OH)D Levels

Compared with baseline, serum 25-(OH)D levels decreased in the control group but increased significantly in the observation group (P<0.05). No significant difference existed between groups before treatment (P>0.05). After treatment, the observation group showed significantly higher 25-(OH)D levels than the control group (P<0.05) [TABLE:3].

Table 3 Comparison of Serum 25-(OH)D Levels Between Two Groups of ASD Children Before and After Treatment (x̄±s, ng/mL)

Group Cases Before Treatment After Treatment t (paired) P value Control 40 26.28±7.28 24.42±7.41 <0.001 Observation 40 26.30±7.19 62.80±13.94 <0.001 Between-group P value <0.001

2.4 Comparison of Clinical Efficacy

The observation group achieved a total effective rate of 36 cases (90.0%), compared to 30 cases (75.0%) in the control group. The observation group's efficacy was significantly superior (χ²=16.290, P<0.001) [TABLE:4].

Table 4 Comparison of Efficacy Between Two Groups After Treatment [cases (%)]

Group Cases Markedly Effective Effective Ineffective Total Effective Control 40 3 (7.5) 27 (67.5) 10 (25.0) 30 (75.0) Observation 40 6 (15.0) 30 (75.0) 4 (10.0) 36 (90.0)

3. Discussion

ASD is a complex and severe neurodevelopmental disorder with incompletely understood pathogenesis. Current research suggests that multiple mechanisms contribute to its development, including oxidative stress imbalance, genetic factors, neurotransmitter metabolism disorders, inflammatory responses, and autoimmune abnormalities [17]. The human gut microbiota, as a crucial endogenous ecosystem, plays a key role in nutrient metabolism and regulates brain development, gene expression patterns, and behavioral trajectories through the immune, neuroendocrine, and enteric nervous systems. These microorganisms promote dietary fiber fermentation, synthesize essential vitamins, and maintain energy homeostasis and immune balance [18]. Recent studies have revealed that the gut and central nervous system are closely connected through the bidirectional "brain-gut axis," which participates in emotional cognition and behavioral regulation [19]. Abnormal signal transmission along this axis is associated with the onset and progression of ASD and functional gastrointestinal disorders [20]. Clinical investigations demonstrate that most children with ASD experience comorbid gastrointestinal dysfunction such as constipation, abdominal pain, and nausea [3,21], suggesting that gut microbiota dysbiosis may represent an important pathological feature of ASD significantly associated with core symptoms [21-22]. Consequently, brain-gut axis dysfunction has been incorporated into research on potential ASD pathogenic mechanisms [23]. Gut microbiota can influence clinical manifestations in children with ASD, including social interaction, language communication, executive function, and stereotyped behaviors, through metabolite-mediated pathways, immune modulation, neuroendocrine signaling, and vagal nerve pathways [24-25].

This study employed press-needle acupuncture combined with VitD to treat children with ASD. The results demonstrated that both groups showed reduced ABC, CARS, SRS, and TCM gastrointestinal symptom scores after treatment, with the observation group exhibiting significantly greater reductions than the control group, indicating that the combined intervention effectively alleviated both behavioral and gastrointestinal symptoms. Serum 25-(OH)D levels decreased in the control group but increased substantially in the observation group, with post-treatment levels significantly higher in the observation group, demonstrating that the combined treatment effectively regulated serum 25-(OH)D status. The observation group achieved a total effective rate of 90.00%, surpassing the control group's 75.00%, confirming the significant therapeutic efficacy of press-needle acupuncture combined with VitD for children with ASD.

Traditional Chinese medicine does not contain a specific disease term for "autism," but ancient and contemporary practitioners have categorized ASD under "language delay," "lack of intelligence," "fetal weakness," and "childhood dementia." The etiology and pathogenesis primarily result from insufficient essence and marrow, with the disease location in the brain and close relationships to the heart, liver, spleen, and kidney [21]. This study utilized press-needle acupuncture, which combines acupuncture and massage effects through sustained, effective stimulation of embedded needles at acupoints. Minimal pain reduces children's fear and enhances acceptance. As stated in Suwen·Yinyang Yingxiang Dalun, "Skilled practitioners treat the skin." Press-needle stimulation at the skin surface can mobilize defensive qi circulation, which warms the skin, regulates sweat pore opening, and protects against external pathogens. Selected acupoints included Zhongwan (CV12), Tianshu (ST25), Guanyuan (CV4), Zusanli (ST36), Xinshu (BL15), Ganshu (BL18), Pishu (BL20), and Shenshu (BL23). Zhongwan and Guanyuan are important points on the Conception Vessel (Ren Mai). Stimulating Zhongwan regulates Conception Vessel qi and blood, influencing all yin meridians, and as the front-mu point of the stomach, it regulates spleen-stomach function and treats gastrointestinal symptoms. Guanyuan is a key health-preserving point that strengthens the foundation and regulates qi-blood function of the liver, spleen, and kidney. As the front-mu point of the small intestine, it also regulates gastrointestinal function. Tianshu belongs to the Stomach Meridian of Foot-Yangming and serves as the front-mu point of the large intestine, where stimulating it effectively conducts and regulates gastrointestinal qi. Zusanli is a crucial point on the Stomach Meridian of Foot-Yangming and the lower-he-sea point of the stomach. Lower-he-sea points are key locations where qi of the six fu organs converges, and the classic Chinese medicine principle states "abdominal disorders are treated at Zusanli." Since the stomach and intestines belong to the fu organs, stimulating Zusanli can strengthen the spleen-stomach, harmonize qi-blood, and improve discomfort. Xinshu, Ganshu, Pishu, and Shenshu are back-shu points that regulate organ functions and harmonize yin-yang.

VitD is an essential nutrient closely related to central nervous system function [5]. Research by L Bishop et al. [26] demonstrates VitD's immunomodulatory functions. When serum VitD falls below 20 ng/mL, it cannot meet the functional demands of the brain, immune system, and gene regulation. Serum VitD levels above 38 ng/mL are required for neuroimmune function [24], with experts recommending levels ≥40 ng/mL [25]. Studies indicate that neurotransmitter system dysregulation involving serotonin (5-HT), oxytocin, and dopamine is associated with ASD [27], playing critical roles in neural transmission, brain maturation, cortical organization, and social and repetitive behaviors. VitD facilitates transcription of tryptophan hydroxylase 2, thereby promoting synthesis of 5-HT, a monoamine neurotransmitter that importantly regulates emotions in social decision-making [28]. Increasing evidence highlights significant inflammatory pathological features in ASD. VitD demonstrates unique regulatory effects in this process [29-30]: it downregulates pro-inflammatory cytokine levels to rebalance inflammatory-immune responses and optimize protective immune mechanisms, while also inhibiting abnormal autoantibody secretion and enhancing γ-glutamyl transpeptidase activity to promote biosynthesis of the antioxidant glutathione, thereby enhancing neurotissue detoxification and reducing oxidative stress-induced brain damage. VitD can alleviate ASD severity through anti-inflammatory effects, promotion of T-regulatory cell proliferation, autoimmune modulation, and enhancement of glutathione system activity [23]. Yin Heng et al. [5] demonstrated that VitD treatment can improve core ASD symptoms. Gould et al. [31] found that higher cord blood VitD levels correlated with more favorable early childhood language development. Huang Zhaohua [1] identified VitD as an important nutrient affecting ASD clinical symptoms.

VitD plays a key role in regulating intestinal health. By modulating gut microbiota composition, repairing intestinal barrier function, and inhibiting local inflammatory responses, VitD effectively improves the intestinal immune microenvironment and alleviates various gastrointestinal symptoms. Its mechanisms primarily involve two aspects: optimizing intestinal mucosal barrier function and regulating immune cell behavior to reshape microbiota ecology [22]. Regarding barrier function, the active metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃] specifically binds to VitD receptors to regulate tight junction protein expression and distribution, maintaining intestinal epithelial barrier integrity [22]. In immune regulation, 1,25(OH)₂D₃ activates various immune cells including epithelial cells and monocytes, promoting antimicrobial peptide synthesis and secretion while enhancing macrophage phagocytic activity. Additionally, this active component bidirectionally modulates adaptive immunity by promoting directional differentiation of immune cells and inhibiting excessive cytokine release and cell proliferation to maintain intestinal immune homeostasis [32-34]. Thus, VitD plays an indispensable role in maintaining intestinal barrier function and immune regulation.

In summary, children with ASD and gastrointestinal symptoms commonly exhibit VitD deficiency, indicating that VitD levels are closely associated with ASD and its gastrointestinal manifestations. This study integrated traditional Chinese medicine's "zang-fu meridian" theory (press-needle acupuncture) with Western medicine (VitD) to treat behavioral and gastrointestinal symptoms in children with ASD, demonstrating the advantages of combined therapy. This approach effectively improved children's social interaction and behavioral capabilities, alleviated gastrointestinal discomfort, and enhanced rehabilitation training outcomes. However, this study has limitations, including a relatively small sample size. Future research should expand the sample size and further investigate the specific effects of press-needle combined with VitD on individual gastrointestinal symptoms and ASD severity levels to provide more refined treatment approaches for children with ASD.

Author Contributions: Gu Jianhui was responsible for study implementation, manuscript writing, and statistical analysis; Jing Yuzhen and Wei Qinglin provided technical guidance and supervision; Lu Junfeng and Yang Lina were responsible for data collection and management; Jia Yongnan designed the study and ensured quality control.

Conflict of Interest: The authors declare no conflict of interest.

ORCID IDs:
- Gu Jianhui: https://orcid.org/0009-0003-7471-1489
- Jia Yongnan: https://orcid.org/0009-0008-2419-7393

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(Received: June 9, 2025; Revised: July 12, 2025)