Study design

This study was a randomized, single-blind, parallel-controlled, single-center clinical trial. The participants were patients with carotid atherosclerosis disease (CAD) who were randomized 1:1 into the RA group (receiving real acupuncture therapy) and the SA group (receiving sham acupuncture therapy). The protocol was approved by the Institutional Ethics Committee of Guangdong Provincial Hospital of Chinese Medicine (approval no.YF2020-195-01). The study was planned in accordance with the Declaration of Helsinki. All participants gave written informed consent before participating in this study. The design and reporting of this trial adhered to the Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA) and Consolidated Standards of Reporting Trials (CONSORT) reporting guidelines. It was registered (ChiCTR2100041762, 04/01/2021) before inclusion of the first participant.

Participants

Participants from Ultrasonography and Acupuncture clinic of the Second Affiliated Hospital of Guangzhou University of Chinese Medicine were enrolled in the study after meeting the following criteria.

Inclusion criteria

(1) Age between 45 and 70 (unrestricted sex); (2) Conformation to the diagnostic criteria of CAD; (3) No susceptible plaque and less than five plaques on both sides; (4) Carotid aortic stenosis < 50%; (5) Cholesterol between 2.59 and 8.0 mmol/L and triglycerides between 1.7 and 10.0 mmol/L; (6) No recent two-week usage of any medication related to the improvement of atherosclerosis, such as medicine for hypertension, heart failure, hyperlipidemia, and diabetes; (7) Volunteer to participate in the trail and cooperate with researchers and understand the content of the trail.

Exclusion criteria

(1) More than 4 risk factors such as age over 60 years old, obesity, diabetes, hypertension, low HDL-C, and smoking; (2) A history of cerebrovascular accident, transient ischemic attack, severe trauma or major surgery; (3) Major cardiovascular, hepatic, renal, or hematopoietic deficiency; (4)A history of mental disorders such as cognitive impairment; (5) A history of skin and bleeding disorders: (6) Intolerance to acupuncture or pregnancy or planning to become pregnant; (7) Participation in other trials.

Dropout criteria

(1) If severe cardiovascular condition or cerebrovascular condition occurred, it was recommended that the patient take appropriate treatment after evaluation by a physician; (2) If aggravated complications occurred after receiving acupuncture treatment, it was recommended that the patient take appropriate treatment after evaluation by a physician; (3) The participant was unable to take part in the trail for personal reasons.

Randomization and blinding

The participants were randomly allocated into the RA and SA groups in a ratio of 1:1. Randomization was created by a mathematician, who was not involved in the study, using SPSS Statistics version 22.0 (IBM). Random numbers and grouping results were saved in opaque envelopes by the mathematician who performed the randomization. The envelopes were opened by the research assistant to assign them in the order in which they are registered.

The participants were blinded, while the acupuncturists in this study were not blinded because of the characteristics of acupuncture. Single-blinded acupuncture intervention using a special acupuncture tool for patients in the 2 groups. The acupuncture device is depicted in Fig. 1. In addition, duration of acupuncture and the acupoints used for the 2 groups were the same. All patients’ evaluations of ultrasound were done by an experienced sonographer who was not aware of the study design and who did not know the patients’ classification.

Fig. 1

An acupuncture tool applied in the RA and SA groups

Interventions

Participants in the RA and SA groups received 30-minute acupuncture once per day, twice per week for a period of 12 weeks, with fixed prescriptions according to the traditional Chinese medicine theory. All participants received acupuncture at ST9 (ren ying), PC6 (nei guan), GB34 (yang lingquan), DU20 (bai hui) and EX-HN3 (yin tang) (Table 1; Fig. 2). The names and locations of acupoints are labeled according to the WHO Standard Acupuncture Locations guideline [20]. Before undergoing acupuncture, patients were asked to lie in the supine position in a quiet environment, and his or her skin near the acupoints was sterilized by the acupuncturists with 0.5% iodine. For RA, acupuncture was performed with a stainless steel needle (Huatuo, Suzhou Medical Appliance Factory, Suzhou, China 25 × 25 mm) at ST9. The depth was between 0.5 and 1.0 cm. And then use specific disposable, sanitized, blunt, sham stainless steel needles (Huatuo, Suzhou Medical Appliance Factory, Suzhou, China 25 × 25 mm) at PC6, GB34, DU20 and EX-HN3, which is a noninsertion procedure applied. For SA, acupuncture was performed using the same sham stainless steel needles (Huatuo, Suzhou Medical Appliance Factory, Suzhou, China 25 × 25 mm) at ST9, PC6, GB34, DU20 and EX-HN3. After acupuncture was started, the needle was kept in place for 30 min in both groups.

Table 1 Acupuncture point positioning criteriaFig. 2

Acupuncture Point Location diagram

Outcome measures

The primary outcomes and the secondary outcome of all participants in 2 groups were assessed at baseline, posttreatment, and follow-up (12 weeks after posttreatment).

Primary outcome

The primary outcomes mainly reflected the changes of carotid artery structure, which included carotid intima-media thickness (cIMT), plaque score (PS), plaque volume (PV) and grey-scale median (GSM). cIMT and PS were examined by Philips EPIQ color Doppler ultrasonography in a two-dimensional grayscale imaging mode. PV and GSM were three-dimensional ultrasound indexes, which were examined by Philips EPIQ color Doppler ultrasonography. The software automatically identifies and outlines the vessel wall and plaque boundary, collects plaque volume information and plaque echo characteristics, and realizes real-time analysis to obtain PV and GSM values. cIMT, PS, PV are positively correlated with the degree of carotid atherosclerosis, and GSM is positively correlated with carotid plaque stability.

Secondary outcomes

The secondary outcome was pulse wave velocity (PWV), which mainly reflected the changes of carotid artery function. PWV was caused by blood flow along the aorta to the peripheral artery during systolic and dilated phases of cardiac ejection, which was used for assessing the stiffness of the carotid artery as well as degree of carotid atherosclerosis. PWV was measured by French Supersonic Aixplorer ultrasonic diagnostic instrument with the SL10-2 probe, frequency 2–10 Hz, vascular PWV mode. The sonographer selected 1–1.5 cm below the carotid bifurcation of the common carotid artery as measurement site. Indicators included PWV at the beginning of the systole (BS, m/s) and PWV at the ending of the systole (ES, m/s). If the standard deviation is more than 20% of the corresponding speed, the operation is deemed invalid. Both sides carotid arteries were measured at least three times and then the researcher recorded the average PWV value. A higher PWV indicates higher degree of carotid atherosclerosis.

Sample size calculation

The sample size was determined by the variation in the PV in pilot study. The mean (SD) PV of patients who received acupuncture was 9.54 (15.74) mm3, whereas that of patients who received the sham acupuncture was − 1 (8.02) mm3. The sample size estimation was calculated by PASS15.0 software, with a two-sided significance level of α = 0.05, power efficiency 1-β = 0.9, and the ratio of the two groups was 1:1. It was calculated that 31 subjects were needed in each group, and a total of 62 subjects were needed.

Adverse events

Acupuncture may cause some adverse events, such as fainting, bleeding and pain. All adverse events need to be recorded. The record of adverse events should include the symptoms, duration, extent, and relationship with acupuncture.

Statistical analysis

SPSS Statistics version 22.0 (IBM) was used to analyze the study data from January 2021 to February 2022. Continuous variables were conveyed as mean and standard deviation (SD) if meet normal distribution, and categorical variables were conveyed as composition ratio. The Continuous variables of the groups were compared using the t test or Mann-Whitney U test. The categorical variables of the groups were compared using the χ2 test or Fisher’s exact test. The primary outcome and secondary outcome were assessed by repeated measure two factorial analysis of variance with interaction effects of time and group. P < 0.05 was accepted as statistically significant.