Research on mechanism of moxibustion with seed-sized moxa cones for improving ulcerative colitis based on cuproptosis

To explore the mechanism by which moxibustion with seed-sized moxa cones improves colonic tissue damage by regulating the cuproptosis signaling pathway in mice with ulcerative colitis (UC).

Thirty male C57BL/6 mice were randomly and equally divided into a control group, a model group, and a seed-sized moxa cone moxibustion (moxibustion) group (n=10 per group) using a random number table. The UC model was established by feeding the mice with 3% dextran sulfate sodium (DSS) solution for 7 consecutive days. After successful establishment of the UC model, the mice of the moxibustion group received moxibustion at “Zhongwan” (CV12), bilateral “Tianshu” (ST25) and bilateral “Shangjuxu” (ST37) for 3 cones, once daily for 7 consecutive days. The disease activity index (DAI) was recorded, fecal occult blood assessed, colon length measured, and the serum contents of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-10 were determined by ELISA. Histopathological changes of the colon tissue were observed after HE staining, changes of colonic ultrastructure observed by using a transmission electron microscopy (TEM), and the mitochondrial membrane potential in the colonic epithelial cells measured after JC-1 staining. The reactive oxygen species (ROS) level was detected using flow cytometry, and the copper ion (Cu²⁺) content in the colon tissue was detected by colorimetry. The mRNA and protein expression levels of ferredoxin 1 (FDX1), dihydrolipoamide acetyltransferase (DLAT), dihydrolipoamide succinyltransferase (DLST), lipoic acid synthetase (LIAS), and heat-shock protein 70 (HSP70) in the colon tissue were detected by using quantitative real-time PCR and Western blot, separately. The immunofluorescence intensity of DLAT in the colon tissue was evaluated by immunofluorescence staining.

Compared with the control group, the model group showed a significant increase in the DAI score, serum TNF-α and IL-1β contents, mitochondrial low-potential ratio, ROS level in colonic epithelial cells, Cu²⁺ content, and expressions of HSP70 mRNA and protein (P<0.01), and a considerable decrease in the colon length, serum IL-10, expression levels of FDX1, DLAT, DLST and LIAS mRNAs and proteins and immunofluorescence intensity of DLAT immunoactivity (P<0.01). In comparison with the model group, both the increased and decreased levels of the indexes mentioned above were all reversed in the moxibustion group (P<0.01, P<0.05). In addition, the fecal occult blood test was positive in the model group and weakly positive in the moxibustion group. Results of HE staining and TEM exhibited obvious damage of the intestinal mucosal epithelium with inflammatory cell infiltration, irregular mitochondrial morphology, varying degrees of swelling and vacuolization, and blurred or even broken and disappeared cristae structure in the model group； and alleviation in the pathological damage of colonic mucosal epithelium and inflammatory infiltration, improved internal structure of the colon epithelial cells, and reduction in the degree of mitochondrial swelling and vacuolization in the moxibustion group.

Moxibustion with seed-sized moxa cones can alleviate symptoms and colonic tissue injury in UC mice, which may be associated with its functions in up-regulating the expressions of cuproptosis-related proteins and genes and improving mitochondrial function and oxidative stress.