Bloodletting Acupuncture at Sifeng Points (EX-UE-10) for Regulating Th1/Th2 Balance in Pediatric Mycoplasma Pneumonia

Summary

This protocol describes a randomized controlled trial to evaluate the immunomodulatory effects of adjunctive Sifeng point (EX-UE-10) acupuncture on the Th1/Th2 balance in children with Mycoplasma pneumoniae pneumonia receiving standardized antibiotic treatment.

Abstract

Mycoplasma pneumoniae is a common cause of pneumonia in children, and the emergence of macrolide-resistant strains underscores the need to explore effective adjunctive therapies. This article introduces a randomized controlled trial protocol to evaluate the clinical and immunological effects of acupuncture as an adjunctive therapy. The study includes children diagnosed with Mycoplasma pneumoniae pneumonia who are receiving standardized azithromycin treatment. The primary objective is to determine whether a specific acupuncture procedure applied to the fingers can modulate the host's immune response by altering serum levels of interferon-γ (IFN-γ) and interleukin-4 (IL-4), which reflect the Th1/Th2 cell balance. This method describes the case selection criteria, randomization grouping, a detailed acupuncture intervention protocol, and assessment of clinical symptoms and immunological markers. This study aims to provide scientific evidence for the application of acupuncture as a non-pharmacological intervention, with the expectation of shortening the course of pediatric pneumonia and reducing its inflammatory response.

Introduction

Community-acquired pneumonia in children remains a significant cause of morbidity, with Mycoplasma pneumoniae being one of the primary pathogens. The increasing prevalence of macrolide-resistant Mycoplasma pneumoniae complicates treatment, often leading to prolonged disease courses^1,2, thereby heightening the need for alternative or adjunctive therapies. Acupuncture is a technique involving the insertion of fine needles into specific body points for therapeutic purposes and is currently being investigated as a non-pharmacological intervention for various inflammatory diseases^3,4.

In traditional Chinese medicine (TCM) theory, the Sifeng points (EX-UE-10) are important acupoints in pediatric massage and acupuncture, with a history spanning several centuries⁵. Traditionally, they are primarily used to treat "gan ji," a condition characterized by indigestion, nutritional disorders, and developmental delays, often accompanied by recurrent respiratory infection symptoms such as cough and sputum production. In pediatric Traditional Chinese Medicine (TCM), Sifeng points are specifically favored for "strengthening the Spleen to support the Lungs" (Pei Tu Sheng Jin), a theory suggesting that clinical recovery from respiratory infections is enhanced by improving digestive and systemic health. Unlike common body points like Feishu (BL 13) or Zusanli (ST 36), which often require long needle retention, Sifeng points are uniquely suited for children due to the efficacy of the quick-prick bloodletting technique. Compared to standard supportive care alone, this adjunctive approach offers a targeted, non-pharmacological means to modulate host immune dysregulation without the risk of additional drug interactions or gastric distress common in pediatric pharmacology⁵. However, the direct application of these points for Mycoplasma pneumoniae infection requires further explanation. While traditionally applied for digestive and respiratory symptoms, it is hypothesized that these points may stimulate peripheral nerves, eliciting systemic anti-inflammatory and immunomodulatory responses, potentially balancing Th1/Th2 cytokine levels^6,7. This protocol is most appropriate for children with mild-to-moderate MPP in a stable inpatient setting to facilitate recovery; it may be less applicable in critical care settings requiring emergency respiratory stabilization.

The immunopathology of Mycoplasma pneumoniae infection involves significant inflammatory damage, which is associated with an imbalance in T helper cell (Th) responses. Specifically, the infection often leads to a shift toward Th2 dominance, characterized by elevated levels of cytokines such as interleukin-4 (IL-4), while the Th1 response is suppressed, manifesting as reduced interferon-γ (IFN-γ) levels^8,9,10. This Th2 shift is linked to persistent inflammation and pathological damage. Restoring a balanced Th1/Th2 ratio is considered a key therapeutic target.

This protocol aims to rigorously test the hypothesis that acupuncture can modulate immune imbalance in children with Mycoplasma pneumoniae pneumonia. By applying peripheral stimulation at specific sites on the fingers, this study seeks to determine whether the intervention can shift the cytokine profile toward a more balanced state (i.e., increasing IFN-γ and decreasing IL-4). Thus, the primary objective of this study is to determine whether the intervention can shift the cytokine profile, while secondary objectives include evaluating its impact on clinical symptom resolution-specifically the time to defervescence and relief of cough-and systemic inflammatory markers such as high-sensitivity C-reactive protein (hs-CRP), thereby improving clinical outcomes. This method provides a framework for evaluating the potential of acupuncture as a safe and effective adjunctive therapy for infectious diseases in children.

Protocol

This study was approved by the Ethics Committee of Jiangning Hospital Affiliated to Nanjing Medical University (approval number: 2021-03-057-k01). The study will adhere to the Declaration of Helsinki. Written informed consent was obtained from the guardians of all participants prior to enrollment. This study was registered with the Chinese Clinical Trial Registry, registration number: ChiCTR2200064052.

1. Patient screening and enrollment

1. Refer to the diagnostic criteria for Mycoplasma pneumoniae pneumonia¹¹ and confirm a diagnosis of Mycoplasma pneumoniae (MP) pneumonia if the following clinical and imaging manifestations are present along with positive serum MP antibodies or positive MP-DNA detection.
   1. Look for fever and cough symptoms, with fever mainly moderate to high, and cough being relatively severe, potentially resembling pertussis-like cough, which may be accompanied by headache, runny nose, sore throat, ear pain, etc.
   2. Perform auscultation of the lungs to look for reduced breath sounds and dry or wet rales.
   3. Look for thickened and increased peribronchovascular markings, with segmental or lobar pneumonia on one side in the chest X-ray, and large areas of consolidation, ground-glass opacities, "tree-in-bud" sign, and thickened interlobular septa in the chest CT.
2. Inclusion criteria
   1. Ensure that the child meets the diagnostic criteria for Mycoplasma pneumoniae pneumonia.
   2. Confirm that the child's age is between 2 and 12 years.
   3. Verify that the child has not used other experimental drugs or therapies.
   4. Ensure that the child has no history of hemophobia or needle phobia.
   5. Confirm that the disease duration is ≤7 days.
   6. Obtain a signed informed consent form from the child's legal guardian.
3. Exclusion criteria
   1. Exclude children with severe pneumonia, or those with underlying conditions such as primary immunodeficiency diseases, congenital respiratory malformations, or pulmonary malignancies.
   2. Exclude children with bleeding tendencies, severe liver or kidney dysfunction, or known drug allergies.
   3. Exclude those treated with other TCM methods that may affect efficacy assessment.
   4. Exclude those with a history of allergy to the drugs used in this study.
4. Set withdrawal and dropout criteria: patients who do not comply with the study protocol for treatment and those who experience serious adverse events and are unsuitable to continue treatment.

2. Randomization and group allocation

1. Open the statistical software. Select Transform | Random Number Generators | Set Starting Point to ensure the sequence is reproducible. Use the Compute Variable function to generate a random allocation sequence for 60 participants.
2. Place the generated allocation numbers into individual, sealed, opaque envelopes.
3. Assign enrolled children to the control or treatment group by opening the envelopes in the chronological order of patient visits.

3. Baseline data and sample collection

1. Record baseline demographic and clinical information for each participant.
2. Assess and record baseline clinical severity scores for fever, cough, and dyspnea. Assign 0 points for 'none', 2 points for 'mild', 4 points for 'moderate', and 6 points for 'severe' for each symptom.
3. Collect 3 mL of venous blood samples into EDTA-2K anticoagulant tubes before initiating treatment.
4. Centrifuge the blood samples at 1,000 × g for 10 min at 4 °C using a refrigerated benchtop centrifuge. Separate the supernatant (serum) and store it at -80 °C for subsequent cytokine analysis.

4. Control group: standard therapy

1. Administer azithromycin via intravenous infusion at a dose of 10 mg/kg once daily for 5 days according to standardized clinical guidelines.

5. Treatment group: standard therapy plus acupuncture

1. Supplement the standard therapy (Step 4.1) with a daily acupuncture procedure for 5 consecutive days.
2. Position the child in a comfortable sitting or lying position with a guardian present for comfort. Identify the four Sifeng points (EX-UE-10) on the palmar side of the 2nd, 3rd, 4th, and 5th fingers at the center of the proximal interphalangeal joint creases. Alternate between left and right hands daily (Figure 1).
3. Wear disposable sterile gloves. Instruct the guardian to hold the child's finger steady. Disinfect the points with iodized cotton swabs.
4. Continuously monitor the child for signs of pain, dizziness, or syncope throughout the procedure.
5. Using a disposable sterile filiform needle (0.30 mm × 13 mm), quickly insert the needle straight into each point to a depth of 1-2 mm, then immediately withdraw the needle (Figure 2).
6. Gently squeeze the tissue around the puncture site to extrude a small amount of blood or yellowish-white transparent mucus (Figure 3).
   NOTE: Confirm correct execution by observing the visual extrusion of these fluids.
7. Wipe the sites with a sterile dry cotton ball and perform secondary disinfection with iodized swabs.
8. Safety and disposal: Immediately place used needles into a puncture-resistant sharps container. Dispose of all blood-contaminated cotton balls and gloves in a designated biohazard waste bag.
9. Ensure the puncture sites remain dry for 24 h to prevent infection. Instruct the guardian to avoid submerging the child's hands in water; if cleaning is necessary, use only mild soap and water followed by immediate, gentle drying.
10. If signs of needle fainting occur, immediately stop the procedure, place the child in a flat resting position, and provide warm water.
11. Safety monitoring: Monitor and record local adverse events using the Common Terminology Criteria for Adverse Events (CTCAE) version 5.0¹².

6. Outcome assessment

1. Measure the child's axillary temperature four times daily (06:00, 10:00, 14:00, and 18:00) using a calibrated electronic clinical thermometer.
   NOTE: All measurements must be performed by trained nursing staff in the inpatient ward to ensure accuracy.
2. Determine the "time to defervescence," defined as the period (in days) from treatment initiation until the axillary temperature remains below 37.3 ℃ for at least 24 h without the use of antipyretics.
3. Evaluate the "time to relief of cough and dyspnea." Record this as the period until symptoms are graded as "none" or "mild" on the clinical severity scale for two consecutive evaluations by the nursing staff.
4. Collect 3 mL venous blood samples 24 h (±2 h) after the final (fifth) treatment session. Centrifuge at 1,000 × g for 10 min at 4 °C to separate serum.
   NOTE: Venous blood samples are collected both before initiating treatment and 24 h after the final (fifth) treatment session.
5. Detect serum IFN-γ, IL-4 and and hs-CRP levels using an enzyme-linked immunosorbent assay (ELISA) kit. Follow the manufacturer's protocol, ensuring the plates are incubated at 37 °C for 60 min during the primary antibody binding stage.
6. For statistical analysis, import the data into the referenced software.
   1. Select Analyze | Descriptive Statistics | Explore to verify the normality of continuous data.
   2. Select Analyze | Compare Means| Paired-Samples T Test for intragroup comparisons and select Analyze | Compare Means | Independent-Samples T Test for between-group comparisons. Express results as mean ± standard deviation (X̅ ± s).
   3. Select Analyze | Descriptive Statistics | Crosstabs and choose the Chi-square option to analyze categorical efficacy data. Consider P-values < 0.05 statistically significant.

Results

Baseline characteristics

Random allocation sequences were generated using statistical software and distributed via sealed, opaque envelopes. A total of 60 participants were randomly assigned to either the control group (n = 30) or the treatment group (n = 30). Statistical analysis confirmed no significant differences in baseline demographic or clinical data between the two groups (P > 0.05), ensuring comparability (Table 1). The control group inclu...

Discussion

This study demonstrates that combining bloodletting therapy at the Sifeng points (EX-UE-10) with standard antibiotic treatment can significantly accelerate the clinical recovery process in children with Mycoplasma pneumoniae pneumonia (MPP). Compared to the control group receiving antibiotics alone, the treatment group exhibited faster resolution of fever and cough, as well as more pronounced reductions in clinical severity scores. These improvements were accompanied by the anticipated immunomodulatory effects: ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Opaque sealed envelopes	Nanjing Kongdu Printing Co., Ltd.	201701	For randomization and group allocation
Puncture-resistant sharps container	Lixinyuan Plastic Industry Co., Ltd.	KMT-15Y	For safe disposal of used needles
Refrigerated benchtop centrifuge	Thermo Fisher Scientific	75002476	Used for serum separation (1,000 x g, 10 min, 4 °C)
Sterile dry cotton balls	Zhende Medical Co., Ltd.	202203121B	For post-procedure pressure and cleaning
Ultralow temperature freezer	Haier Biomedical	DW-86L578J	For long-term storage of serum samples (-80 °C)
Venous blood collection needles	Guangzhou Improve Medical Instruments Co., Ltd.	220123	For obtaining 3 mL venous blood samples