Electroacupuncture

This is a two-armed, parallel-design, patient-assessor blinded, multicenter, randomized, sham-controlled pilot clinical trial. Two clinical research centers in South Korea will conduct the trial: Daejeon Oriental Hospital of Daejeon University and Kyung Hee University Oriental Hospital at Gangdong.
To recruit participants, flyers, daily local newspapers, and advertisement boards at research centers will be utilized. Potential candidates will visit the research centers for screening assessment and to sign informed consent for trial participation after being informed about the aim and details of the study. Eligible participants will be randomly allocated at a 1:1 ratio to one of two arms: electroacupuncture plus moxibustion (treatment group) or sham acupuncture with mock electrical stimulation plus sham moxibustion (control group). Participants will receive treatment for 8 weeks and then visit the research centers twice during a 4-week follow-up period (Fig. 1).Fig. 1

Flow chart of the trial

All experiments were approved according to the Ethics Committee the Institutional Animal Welfare and Use Committee of the Institute of Acupuncture-Moxibustion, China Academy of Chinese Medicine (no. 20170313). 32 Sprague-Dawley (SD) male rats (SPF grade, 12 weeks, 180-200 g) were purchased from Chengdu Dossy Experimental Animals Co., Ltd. (Chengdu, Sichuan; SCXY (Chuan) 2020-034). The feeding environment was 23 ± 1°C, relative humidity 50 ± 5%, and light/darkness for 12 h circulation. SD rats are allowed to eat and drink freely. The SD rats were randomly divided into 4 groups (n = 8), namely, the control group, colitis model group, Zusanli electroacupuncture (Zusanli-EA) group, and sham electroacupuncture (sham-EA) group. For the colitis model group, rats were gavaged with 5% (w/v) dextran sulfate sodium (DSS) saline solution (MP Biomedicals, Santa Ana, California, USA) for 4 days (50 mL/d) as previously described [15 (link), 16 (link)]. The status of rats was monitored using the disease activity index (DAI) [17 (link)]. Meanwhile, the control group rats were gavaged an equal volume of saline solution. For the Zusanli-EA group, electroacupuncture was immediately performed under isoflurane inhalation anesthesia after modeling. Rats received electroacupuncture treatment at Zusanli acupoint (ST36, bilateral) using 1.0-inch filigree needles (0.25 mm × 13 mm, Huatuo Brand, depth of about 7 mm). An electroacupuncture treatment device (G6805-2A, Huatuo Brand) was from Suzhou Medical Appliance Factory, China. The electroacupuncture parameter is a dilute wave with 2/100 Hz, the intensity of 1 mA, and performed for 15 minutes, once a day, for 21 consecutive days as previously described [18 (link)]. For the sham-EA group, rats were anesthetized by inhalation of 3–4% isoflurane and then received sham electroacupuncture with a pragmatic placebo needle on sham acupoints. The neurological function and DAI score of rats were tested per week. At the end of the 3-week administration, all rats were anesthetized with 1% sodium pentobarbital (50 mg/kg) and euthanized. The colon tissues, serum, ipsilateral lumbar 6 (L6) dorsal root ganglia (DRG), and nearby skin were removed and kept at -80°C for subsequent analysis. The flow of subjects through the experimental procedure is described in Figure 1(a).

Optogenetic activation or inhibition experiments were performed 3 weeks after viral expression. Chronically implantable fibers were connected to a laser generator using optic fiber sleeves. A Master-8 pulse stimulator (Shanghai Fiblaser Technology Co., Ltd., China) was used to deliver a 5-min pulse of blue (473 nm, 10 Hz, 5–8 mW) or yellow light (594 nm, 5–8 mW, constant). In optogenetic inhibition experiments, yellow light was given immediately after establishing the GD model and gastric motility was recorded for 20 min; The control group was not given yellow light after establishing the GD model. In the optogenetic activation experiment, gastric motility was recorded for 20 min immediately after the application of blue light in the light group and in the absence of light in the control group; in the EA group, electroacupuncture was performed for 20 min immediately after the blue light and gastric motility was recorded at the end of the electroacupuncture treatment.

The site of animal electroacupuncture intervention was consistent with those used in previous experiments (Wang et al., 2015 (link)). Zhong-wan-acupoint (RN12) was located on the intersection of the upper 1/3 and lower 2/3 of the line connecting the xiphoid process and the upper border of the pubic symphysis, wei-shu-acupoint (BL21) was 2 mm adjacent to the spinous process of the 12th thoracic vertebra. Using disposable sterile acupuncture needles (0.25*13 mm, Yunlong Medical Co., Ltd., China) and electrical stimulator (G6805, Qingdao Xinsheng, China). A 20 min EA procedure was performed at a frequency of 2/100 Hz by using current with an intensity of 0.1–1 mA.