The mechanism of indigo naturalis and its active ingredients against ulcerative colitis.

Indigo Naturalis (IN), utilized for thousands of years in China, is known for its effects in clearing heat, removing toxins, and alleviating diarrhea. Studies indicate that IN possesses anti-ulcerative colitis (UC) effects. However, the molecular processes that explain its therapeutic outcomes are still not fully understood. This study was to clarify the mechanism of IN against UC using both in vitro and vivo research methods. RAW264.7 cells were activated by lipopolysaccharide (LPS) (1 µg/mL) to found an inflammatory model; MTT and NO test was conducted to examine the influence of IN, indigo, indirubin, isatin, and MIX (indigo, indirubin, and isatin) on cell viability and anti-inflammatory activity. Inflammatory cytokines expression was evaluated with ELISA. Western Blotting was employed to analyze the MAPK, STAT3, and NF-κB pathways. UC model was generated by 2.5% DSS in C57BL/6 mice. Disease activity index (DAI) scoring , myeloperoxidase (MPO), and H&E staining are all for mice status. We demonstrated that IN, indirubin, and isatin can all regulate the expression of inflammatory cytokines. NF-κB, STAT3, and MAPK pathways was closely associated with IN, whereas indirubin acted to block the activation of both the NF-κB and STAT3 pathways. Contrastingly, the isatin mechanism of action was tightly connected with the regulation of the NF-κB and MAPK pathways. Additionally, MIX inhibited the activation of STAT3 pathway while suppressing the level of p-P38 expression in the MAPK pathway. MIX can enhance multiple indicators and conditions in UC mice. The active ingredients in IN (indigo, indirubin, and isatin, MIX) can significantly improve the condition of UC mice. MIX exerts anti-inflammatory effects by inhibiting the activation of the STAT3 pathway and expression of p-P38 in the MAPK pathway.