Mechanism study on electroacupuncture at "Changbing Formula" regulating the Nrf2/HO-1 signaling pathway to ameliorate colonic mucosal barrier injury in mice with ulcerative colitis

To investigate the regulatory effect of electroacupuncture at "Changbing Formula" on the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway in the colon of ulcerative colitis(UC) mice, and to elucidate the potential mechanism of alleviating colonic mucosal barrier injury.

According to the random number table method, 40 C57BL/6 male mice were divided into the blank group (n=10) and modeling group (n=30). The UC model was established by free drinking of 3% dextran sulfate sodium solution for 7 days. According to the random number table method, the successfully modeled mice were divided into model, electroacupuncture, and mesalazine groups, with 10 mice per group. The mice in the electroacupuncture group were treated with acupuncture at "Changbing Formula," including "Zhongwan" (CV12), bilateral "Tianshu" (ST25), and bilateral "Shangjuxu" (ST37), and the ipsilateral "Tianshu" (ST25) and "Shangjuxu" (ST37) were connected with electroacupuncture, once a day, 20 min each time. The mesalazine group was administered mesalazine solution (300 mg/kg) by gavage. The intervention was administered for 7 days. The general state of mice (body weight, stool characteristics, and hematochezia) was observed before modeling, before intervention, and after intervention. After intervention, the colon length of mice was recorded, and the disease activity index (DAI) score was calculated. Hematoxylin-eosin and alcian blue-periodic acid Schiff stainings were used to observe the pathological morphology of colon tissue. The contents of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-6 in serum and glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA) in colon tissue were detected using enzyme-linked immunosorbent assay. The average fluorescence intensity of reactive oxygen species (ROS) in colon tissue was detected using immunofluorescence. The positive expressions of ZO-1, Occludin, and Claudin 1 in colon tissue were detected using immunohistochemistry. The protein expressions of Kelch-like epichlorohydrin-related protein 1 (Keap1), Nrf2, NAD (P) H quinone dehydrogenase 1 (NQO1), and HO-1 in colon tissue were detected using Western blotting.

Before the intervention, the mice in the model, electroacupuncture, and mesalazine groups showed varying degrees of soft to loose stool, positive fecal occult blood test or visible blood in the stool. After intervention, compared with the blank group, the mice in the model group still showed loose stool, positive fecal occult blood test, weight loss, shortened colon length, and increased DAI score (P<0.05); inflammatory cell infiltration was observed in colon tissues, along with a decrease in goblet cell number; serum contents of IL-1β, TNF-α, and IL-6, as well as MDA content in colonic tissue were increased, whereas SOD and GSH contents in colon tissue were decreased (P<0.05); the average fluorescence intensity of ROS in colon tissue was increased (P<0.05); the positive expressions of ZO-1, Occludin, and Claudin 1 in colon tissue were decreased (P<0.05); Keap1 protein expression in colon tissue was increased, whereas Nrf2, HO-1, and NQO1 protein expressions were decreased (P<0.05). Compared with the model group, the stool of the mice in the electroacupuncture and mesalazine groups were basically formed, the fecal occult blood test was weakly positive, the body weight was increased, the colon length was increased, and the DAI score was decreased (P<0.05); the inflammatory cell infiltration in colon tissue was reduced and the goblet cell number was increased; the serum contents of IL-1β, TNF-α, IL-6, and MDA content in colon tissue were decreased, whereas SOD and GSH contents in colon tissue were increased (P<0.05); the average fluorescence intensity of ROS in colon tissue was decreased (P<0.05); the positive expressions of ZO-1, Occludin, and Claudin 1 in colon tissue were increased (P<0.05); Keap1 protein expression in colon tissue was decreased, whereas Nrf2, HO-1, and NQO1 protein expressions were increased (P<0.05). Compared with the electroacupuncture group, there was no significant difference in the indexes of the mesalazine group.

Electroacupuncture at "Changbing Formula" can reduce the damage of the colonic mucosal barrier in UC mice. The mechanism may be related to regulating the protein expressions involved in the Nrf2/HO-1 signaling pathway, enhancing its antioxidant defense ability, and reducing the release of pro-inflammatory factors.