Sinomenine restrains the proliferation and hyperactivation of B lymphocytes partly by inhibiting interferon...

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.

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.

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.

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.

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.