Sinomenine restrains the proliferation and hyperactivation of B lymphocytes partly by inhibiting interferon regulatory factor 5

Ethnopharmacological relevance: B lymphocytes are central drivers of pathogenic immune responses in autoimmune diseases and represent a promising target for therapeutic intervention. Sinomenine (SIN), a natural alkaloid derived from Sinomenium acutum, exhibits broad immunomodulatory properties and has long been clinically used for treating autoimmune diseases, however, its direct effects on B cells and underlying mechanisms are largely unexplored.

Aim of the study: To investigate the effects of SIN on the proliferation and hyperactivation of B lymphocytes both in vitro and in vivo, and elucidate its therapeutic mechanisms in autoimmune pathologies.

Materials and methods: Firstly, SIN was examined for its ability against lipopolysaccharides (LPS) or CpG oligodeoxynucleotide (CpG) induced B cell proliferation and hyperactivity. Then, the effects of SIN regulating B cell subsets and antibody response were studied in pristane-induced lupus and keyhole limpet hemocyanin (KLH)-immunized murine models. Furthermore, transcriptomics, small molecule-protein interaction assay (SPIA) and a series of validation experiments (including Western blotting, immunofluorescence, cellular thermal shift assay (CETSA), surface plasmon resonance (SPR), and flow cytometry) were performed to identify the key mechanisms involved.

Results: For the first time, our research revealed the significant inhibitory efficacy of SIN on B cell proliferation induced by LPS or CpG. Moreover, SIN significantly reduced CD69 expression, and attenuated IL-6 and IL-10 hypersecretion. Notably, SIN exerted a better inhibitory effect against CpG-mediated activation compared to LPS-induced responses. In the early immune response of pristane-induced lupus model, both SIN and prednisone significantly decreased the spleen coefficient and the proportion of CD19⁺CD69⁺ B cell subsets. In the KLH immunization model, SIN alleviated splenomegaly, lowered serum levels of IgG and IgM, and reduced the cell counts of B220+ cells and proportion of plasma cells (B220^lowCD19-CD138+). Integration of transcriptomic analyses and in vitro verification revealed SIN's regulatory effects on cell cycle, the PI3K-AKT signaling pathway, along with its marked suppression of NF-κB protein activation following both LPS and CpG stimulation. In response to CpG stimulation, SIN also significantly decreased the protein levels of p-STAT3 and STAT3. Further research by SPIA identified the downregulation of interferon regulatory factor 5 (IRF5) by SIN. This effect was then corroborated by Western blot, immunofluorescence, molecular docking, CETSA, and SPR analyses, confirming SIN as a novel modulator of IRF5. Moreover, pharmacological inhibition of IRF5 by IRF5-IN-1 markedly significantly attenuated the proliferation and hyperactivation of B cells following LPS or CpG challenge, implying the critical role of IRF5 in B cells.

Conclusions: Collectively, our findings suggest that SIN restrains the proliferation and hyperactivation of B lymphocytes via inhibiting IRF5 and modulating the PI3K-Akt, STAT3, and NFκB signaling cascades. These results uncover a previously unrecognized mechanism of B cell regulation and support a potential therapeutic approach for autoimmune diseases involving pathogenic B cells by SIN.