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For numerous years, the Xiehuo Xiaoying decoction (XHXY), a traditional Chinese medicine formula, has demonstrated substantial promise in treating Graves' disease (GD) in clinical settings, showcasing significant potential. However, the therapeutic mechanism and efficacy material basis of XHXY remains obscure. This work aims to investigate the underlying mechanisms and to study the efficacy material basis of XHXY in anti-GD effect using a combination of TMT quantitative proteomics and molecular docking method. GD model was initiated by administering Ad-TSH289. Subsequently, the mice underwent a four-week regimen that included oral gavage of XHXY at doses of 17 g/kg·d and 34 g/kg·d, along with intraperitoneal injections of Gentiopicroside (GPS). Utilizing the principles of pharmacological chemistry in traditional Chinese medicine, we employed high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (HPLC-QTOF/MS) to discern prescribed prototype composition of XHXY in serum samples from mouse. TMT proteomics research provided evidence of XHXY's putative targets and important pathways in vivo. The binding activity of probable action targets and prototype composition was detected by molecular docking. Finally, Immunohistochemistry (IHC) and TUNEL staining were used to verify the mechanism of XHXY and GPS in anti-GD. XHXY and GPS alleviated GD by ameliorating the pathological changes and reducing thyroxine and TRAb levels. In mouse serum, a total of 31 prototypical XHXY ingredients were detected, and the majority of these components were from monarch and minister medicine. Proteomics study results indicated that the XHXY may mainly regulate targets including FAS-associated death domain protein (FADD), Apolipoprotein C-III, etc. and main pathways are Apoptosis, Cholesterol metabolism, TNF signalling pathway, etc. Strong binding activity of the prototypical active ingredient and GPS towards FADD, Caspase 8, and Caspase 3 was demonstrated by molecular docking. XHXY and its primary component, GPS, elevated the expression of FADD, Caspase 8, and Caspase 3, and enhance apoptosis in thyroid cells, as lastly validated by TUNEL and IHC staining. XHXY exhibits a favorable therapeutic effect in treating GD by promoting apoptosis in thyroid cells through the upregulation of FADD, Caspase 8, and Caspase 3 expression. And GPS is the main efficacy material basis for its therapeutic effect in anti-GD. [Display omitted] • FAS-associated death domain protein (FADD) was identified as the central target for XHXY in GD treatment through TMT-based proteomics, showing strong binding interactions with prototype components of XHXY and Gentiopicroside. • XHXY relieved GD through enhances apoptosis in thyroid cells by upregulating levels of FADD, Caspase 8, Caspase 3. • Gentiopicroside is the main pharmacodynamic material basis for its therapeutic effect in anti-GD. [ABSTRACT FROM AUTHOR]

Huangqin Decoction (HQD), a traditional Chinese medicinal (TCM) formula chronicled in Shang Han Lun , has been used to treat gastrointestinal diseases for nearly 1800 years. To investigate the effects and underlying mechanisms of HQD on ulcerative colitis (UC). The bioactive compounds in HQD were obtained from the traditional Chinese medicine systems pharmacology database. Then, the HQD and UC-related targets were analyzed by establishing HQD-Compounds-Targets (H–C-T) and protein-protein interaction (PPI) networks. Enrichment analysis was used for further study. The candidate targets for the effects of HQD on UC were validated using a dextran sulfate sodium-induced UC mouse experiment. The results showed that 51 key targets were gained by matching 284 HQD-related targets and 837 UC-related targets. Combined with H–C-T and PPI network analyses, the key targets were divided into endothelial growth, inflammation and signal transcription-related targets. Further experimental validation showed that HQD targeted estrogen receptor alpha (ESR1) and endothelial growth factor receptors to relieve endothelial dysfunction, thereby improving intestinal barrier function. The expression of inflammatory cytokines and signal transducers was suppressed by HQD treatment and inflammation was inhibited. HQD may acts on UC via the regulation of targets and pathways related to improving the intestinal mucosal barrier and ameliorating endothelial dysfunction. Additionally, ERS1 may be a new target to explore the mechanisms of UC. Image 1 [ABSTRACT FROM AUTHOR]

Huangqin Decoction (HQD), a traditional Chinese medicine (TCM) formula chronicled in Shang Han Lun , is safe and effective for treatment of ulcerative colitis (UC). To investigate the effect of HQD against dextran sulfate sodium (DSS)-induced UC mice by regulating gut microbiota and metabolites, and further explore the mechanism of fatty acid metabolism on macrophage polarization. Based on 3% dextran sulfate sodium (DSS)-induced UC mice model, clinical symptoms observation (body weight, DAI, and colon length) and histological inspection were used to evaluate the efficacy of HQD and fecal microbiota transplantation (FMT) from HQD-treated mice. The gut microbiota and metabolites were detected by 16S rRNA sequencing and metabolomics analysis. The parameters of fatty acid metabolism, macrophage polarization, and FFAR1/FFAR4-AMPK-PPARα pathway were analyzed by immunofluorescence analysis, western blotting, and real-time PCR. Then, the effects of FFAR1 and FFAR4 on macrophage polarization were examined by agonists based on LPS-induced RAW264.7 cell model. The results showed that FMT, like HQD, ameliorated UC by improving weight loss, restoring colon length, and reducing DAI scores and histopathological scores. Besides, HQD and FMT both enhanced the richness of gut microbiota, and modulated intestinal bacteria and metabolites to achieve a new balance. Untargeted metabolomics analysis revealed that fatty acids, especially long-chain fatty acids (LCFAs), dominated in HQD against DSS-induced UC by regulating the gut microenvironment. Further, FMT and HQD recovered the expression of fatty acid metabolism-related enzymes, and simultaneously activated FFAR1/FFAR4-AMPK-PPARα pathway but suppressed NF-κB pathway. Combined with cell experiment, HQD and FMT promoted macrophage polarization from M1 toward M2, which were well associated with anti-inflammatory cytokines and combined with the activated FFAR4. The mechanism of HQD against UC was related to regulating fatty acid metabolism to mediate M2 macrophage polarization by activating the FFAR4-AMPK-PPARα pathway. Huangqin decoction (HQD) ameliorated dextran sulfate sodium (DSS)-induced ulcerative colitis by regulating fatty acid metabolism to mediate macrophage polarization via activating FFAR4-AMPK-PPARα pathway. [Display omitted] • HQD alleviated DSS-induced colitis by restructuring gut microbiota and improving intestinal long-chain fatty acids. • HQD regulated fatty acid metabolism to mediate M2 macrophage polarization by activating FFAR1/FFAR4-AMPK-PPARα pathway. • FFAR4 was more central than FFAR1 in M2 macrophage polarization and FFAR1/FFAR4-AMPK-PPARα pathway. [ABSTRACT FROM AUTHOR]