Huanglian Jiedu Wan intervened with "Shi-Re Shanghuo" syndrome through regulating immune balance mediated by biomarker succinate.

With increasing stress in daily life and work, subhealth conditions induced by "Shi-Re Shanghuo" syndrome was gradually universal. "Huanglian Jiedu Wan" (HLJDW) was the first new syndrome Chinese medicine approved for the treatment of "Shi-Re Shanghuo" with promising clinical efficacy. Preliminary small-sample clinical studies have identified some notable biomarkers (succinate, 4-hydroxynonenal, etc.). However, the correlation and underlying mechanism between these biomarkers of HLJDW intervention on "Shi-Re Shanghuo" syndrome remained ambiguous. Therefore, this study was designed as a randomized, double-blind, multicenter, placebo-controlled Phase II clinical trial, employing integrated analysis techniques such as non-targeted and targeted metabolomics, salivary microbiota, proteomics, parallel peaction monitoring, molecular docking and surface plasmon resonance (SPR). The results of the correlation analysis indicated that HLJDW could mediate the balance between inflammation and immunity through succinate produced via host and microbial source to intervene "Shi-Re Shanghuo" syndrome. Further through the HIF1α/MMP9 pathway, succinate regulated downstream arachidonic acid metabolism, particularly the lipid peroxidation product 4-hydroxynonenal. Finally, an animal model of recurrent oral ulcers induced by "Shi-Re Shang Huo" was established and HLJDW was used for intervention, key essential indicators (succinate, glutamine, 4-hydroxynonenal, arachidonic acid metabolism) essential in the potential pathway HIF1α/MMP9 discovered in clinical practice were validated. The results were found to be consistent with our clinical findings. Taken together, succinate was observed as an important signal that triggered immune responses, which might serve as a key regulatory metabolic switch or marker of "Shi-Re Shanghuo" syndrome treated with HLJDW.