Purified Indigo naturalis alleviated ulcerative colitis by protecting colon barrier and inhibiting NLRP3 inflammasome pathway

Indigo naturalis Baphicacanthus cusia (Nees) Bremek., QD, as a traditional Chinese medicine, has exhibited efficacy in the ulcerative colitis (UC). Cu Dian (CD), the current form of Indigo naturalis, has been regarded as the mainstream form of medicine today. Dian Hua (DH) is the traditional purified form of QD, in which the content of indigo and indirubin is higher than that of CD. The study evaluated the efficacy of DH and CD in UC and explored their mechanism. Male BALB/c mice were subjected to an 8-day regimen of 3% dextran sodium sulphate (DSS) drinking water to induce UC. The experiment was approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (approval number: 2024075). Concurrently, the mice received intragastric administration of CD (400, 200, and 100 mg·kg^-1) and DH (400, 200, 100, and 50 mg·kg^-1) for the same duration. The anti-inflammatory properties of CD and DH were evaluated by quantifying levels of myeloperoxidase (MPO), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-18 in colon tissue. Western blot and immunofluorescence assays were employed to assess the protein levels of zonula occludens 1 (ZO-1) and occludin. Additionally, Western blot and RT-qPCR were utilized to analyze the protein and gene expression levels of AMP-activated protein kinase (AMPK), nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3), and related factors in colon tissue. CD and DH demonstrated a capacity to alleviate inflammatory responses in mice with UC. The protective impact of both CD and DH on the intestinal mucosal barrier was associated with an elevation of ZO-1 and occludin. Furthermore, the anti-inflammatory effects of CD and DH were attributed to the inhibition of the NLRP3 inflammasome through the activation of the AMPK/silent information regulator of transcription 1 (SIRT1) pathway. Notably, DH exhibited a more pronounced improvement in UC compared to CD, particularly at the dosage of DH-M (200 mg·kg^-1). Our investigation substantiates the effectiveness of CD and DH in mitigating DSS-induced UC in mice. They demonstrated a capacity to diminish the production of inflammatory cytokines and safeguard the integrity of the intestinal epithelial barrier, notably by elevating level of tight junctions. The anti-colonic inflammatory effects of CD and DH were elucidated through the inhibition of both the formation and activation of the NLRP3 inflammasome, mediated by the AMPK/SIRT1 pathway.