(T1530-03-21) Study on the Immunomodulatory Effect of Major Plasma Components of Fufang Shengdi Granules in Imiquimod-Induced Psoriasis Mice

Purpose: This study aims to explore the pharmacokinetics of Fufang Shengdi granules and the immunomodulatory effects of FFSD with elucidation of the immunosuppressive mechanism of its major plasma components.

Methods: The efficacy of FFSD was initially assessed in imiquimod (IMQ) -induced psoriatic mice through psoriasis area, severity index scoring and histopathological evaluation. the major components of FFSD in plasma were identified in psoriasis model mice using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. Pharmacokinetic analysis was performed in IMQ-induced psoriatic mice following administration of Fufang Shengdi Granules at clinical equivalent dosage. Plasma samples were subsequently processed and analyzed using LC-QQQ-MS/MS. The proliferative effects of plasma components on whole spleen cells were evaluated using the CCK-8 assay. Flow cytometry was utilized to evaluate T cell activation, while morphological alterations in spleen cells were examined using the high-content imaging system. The concentrations of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in the supernatant were determined by enzyme-linked immunosorbent assay (ELISA). Component-target prediction and proteomic analysis were performed to identify effector proteins and involved immune cell types. The secretion of inflammatory cytokines, including TNF-α and IL-1β, from LPS-stimulated RAW264.7 cells was quantified by ELISA. Nuclear translocation of NF-κB was assessed using imaging flow cytometry, and transcriptional levels were measured by quantitative PCR (qPCR).

Results: The efficacy of FFSD on psoriasis mice has been confirmed. Loganin, secoxyloganin, and geniposidic acid were the major plasma components of FFSD, with maximum plasma concentrations (Cmax) of 848 nM, 67 nM, and 644 nM, respectively. These compounds, individually or in combination, inhibited the proliferation of whole spleen cells as well as the activation of CD4⁺ and CD8⁺ T cells, accompanied by reduced levels of IL-2 and IFN-γ. Proteomic and target prediction analyses indicated that these immunomodulatory effects may be mediated through the Annexin A protein family and modulation of innate immune responses, with macrophages identified as key target cells. Furthermore, these components significantly decreased TNF-α and IL-1β secretion in LPS-stimulated RAW264.7 cells and suppressed NF-κB nuclear translocation. Quantitative PCR data also confirmed upregulation of Annexin A family gene transcription.

Conclusion: The three major components of FFSD demonstrated significant immunosuppressive effects on whole spleen cells at their respective in vivo Cmax concentrations, and exhibited anti-inflammatory activity by inhibiting NF-κB nuclear translocation in macrophages and upregulating the transcription of Annexin A family proteins.


Figure 1 Graphical abstract. The efficacy evaluation of Fufang Shengdi granules (FFSD) on IMQ-induced psoriasis mice, the pharmacokinetics of FFSD, the bioinformatics analysis of FFSD, and the immune regulatory effect in vitro of the major plasma components of FFSD.


Figure 2 Chemical structure and representative extracted ion chromatographs (MRM) of loganin (LOG), secoxyloganin (SLOG), and geniposidic acid (GPA). (A) Chemical structure of geniposidic acid and representative extracted ion chromatographs (MRM) of geniposidic acid in blank plasma, sample plasma, and spiked plasma. (B) Chemical structure of secoxyloganin and representative extracted ion chromatographs (MRM) of secoxyloganin in blank plasma, sample plasma, and spiked plasma. (C) Chemical structure of loganin and representative extracted ion chromatographs (MRM) of loganin in blank plasma, sample plasma, and spiked plasma. (D) Mean plasma concentrations vs. time profiles of loganin (LOG), secoxyloganin (SLOG), and geniposidic acid (GPA) after oral administration of FFSD decoction to mice.


Figure 3 The immunosuppressive effect of the FFSD components based on plasma concentration. (A) The imaging of whole spleen cell proliferation induced by ConA. (B) The proliferation ratio induced by ConA. (C) The proliferation ratio induced by LPS. (D) The dot plot of T-cell subsets. (E) Statistical analysis of the CD4+ cells proportion. (F) Statistical analysis of the CD8+ cells proportion. (G) The histogram of CD25 MFI in CD4+ cells. (H) The histogram of CD25 MFI in CD8+ cells. (I) Statistical analysis of CD25 MFI in CD4+ cells. (J) Statistical analysis of CD25 MFI in CD8+ cells. (K) The levels of IL-2 in supernatant. (L) The levels of IFN-γ in supernatant. The data are expressed as the mean±SD. Comparison between Control and Naive group is represented by #p < 0.05, ##p < 0.01, ###p < 0.001, and comparison between the ingredient-treated and Control group is represented by *p < 0.05, **p < 0.01, ***p < 0.001).