Huanglian Ganjiang Decoction alleviates DSS-induced colitis through suppressing inflammation and protecting intestinal barrier: from the perspective of disassembling prescriptions

Ethnopharmacological relevance: Huanglian Ganjiang decoction (HGD), which is composed of Chinese medicines with cold, warm, and astringent properties, has demonstrated significant therapeutic efficacy in ulcerative colitis (UC). However, the underlying mechanisms remain unclear, highlighting the need for a multi-faceted investigation. Disassembling prescriptions is a crucial approach for investigating compatibility mechanisms. Analyzing the interactions among Chinese herbs through this method can refine treatment focus and provide novel insights into TCM prescription compatibility research. Therefore, HGD was disassembled into three groups: Cold Medicine Removed (C-R), which warm medicine and astringent medicine are combined; Warm Medicine Removed (W-R), which cold medicine and astringent medicine are combined; and Astringent Medicine Removed (A-R), which cold medicine and warm medicine are combined.

Aim of the study: To elucidate the therapeutic effects of HGD and its three disassembled prescriptions against UC and to uncover their compatibility mechanisms.

Materials and methods: The chemical composition of HGD and its disassembled prescriptions (HGDADPs) was qualitatively analyzed using UPLC-MS/MS. 3% dextran sulfate sodium (DSS) was used to induce a UC mouse model. The efficacy in treating UC was evaluated by body weight loss, disease activity index (DAI), colon length, spleen index, thymus index, and histopathological score. A compound-UC target network was established utilizing the network pharmacology. The underlying mechanisms were then investigated by assessing intestinal barrier function and inflammatory responses, as well as the APOC1/P38 MAPK and TLR4/NF-κB signaling pathways through qRT-PCR, Western blotting, and immunofluorescence. Subsequently, anisomycin, a P38 MAPK agonist, was used to confirm whether C-R protects the gut barrier via the APOC1/P38 MAPK pathway. Monophosphoryl lipid A (MPLA), a TLR4 agonist, was employed to investigate whether W-R mediates its anti-inflammatory effects via the TLR4/NF-κB signaling pathway.

Results: HGDADPs improved colitis symptoms, increasing ZO-1, Occludin, Claudin-1, and E-cadherin levels while reducing blood cell counts and IL-6 and IL-1β levels. HGD was the most effective in reducing inflammation and repairing the intestinal barrier, with A-R showing similar effects. C-R excelled in repairing the barrier, while W-R was better at reducing inflammation. Network pharmacology indicated C-R inhibits the APOC1/P38 MAPK pathway, and W-R suppresses the TLR4/NF-κB pathway, aligning with Western blotting results. ANI and MPLA reversed the effects of C-R and W-R, respectively.

Conclusion: The mechanism by which HGDADPs treat colitis is demonstrated for the first time in our study. In HGD, cold medicine serves as the "Jun", primarily exerting anti-inflammatory effects; Warm medicine acts as the "Chen", mainly protecting the intestinal mucosal barrier; And astringent medicine functions as the "Zuo", playing a synergistic role in the treatment of colitis. In addition, the combination of cold and warm medicines (A-R) was the most crucial for HGD' s compatibility. The pairing of warm and astringent medicines (C-R) significantly contributed to the restoration of the gut barrier and the inhibition of the APOC1/P38 MAPK signaling pathway. Meanwhile, the combination of cold and astringent medicines (W-R) primarily contributed to alleviating inflammation and inhibiting the TLR4/NF-κB pathway.