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).
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