Responses to Nei-Xiao-Luo-Li-San in Toledo Diffuse Large B-Cell Lymphoma Cells: Potential Involvement of ATF5 and HDAC-Associated Epigenetic Changes

Meth-ods: NXLLS was chemically characterized to assess its compositional profile and compositional similarity among batches. Its biological effects were evaluated by as-sessing cell viability, migration, invasion, and cell-cycle progression in Toledo DLBCL cells. Molecular responses to NXLLS were investigated through an integrated approach combining transcriptomic analysis, network pharmacology, and molecular assays.

The functional role of ATF5 was further evaluated using lentiviral overexpression and knockdown. Results: Chromatographic profiling provided an exploratory assessment of compositional similarity among NXLLS samples and supported the selection of eight representative compounds using LC–MS annotation and HPLC authentic-standard confirmation. NXLLS reduced cell viability, migration, and invasion in Toledo DLBCL cells within the tested concentration range.

At the molecular level, NXLLS was associ-ated with altered cell-cycle distribution and potential G1-phase accumulation at later time points, reduced VEGF-associated ELISA signal, and modulated stress-response genes. RNA-seq revealed transcriptional changes involving stress-response and HDAC-associated epigenetic pathways. Correspondingly, HDAC expression was re-duced and histone acetylation (Ac-H3K9 and Ac-H3K27) was increased.

ATF5 knock-down recapitulated several cellular and epigenetic changes observed following NXLLS treatment, supporting ATF5 as a potential downstream mediator of the NXLLS re-sponse. Conclusions: This study identified potential molecular targets associated with the response to NXLLS in Toledo DLBCL cells, with ATF5 further investigated as a potential downstream mediator. The findings also suggest an association between the NXLLS response and HDAC-related epigenetic changes, providing a basis for further investigation of their mechanistic relationships.