Transcriptomic Analysis Identifies Putative Hematopoietic Co-Expression Networks in Icariin-Mediated Recovery from Cyclophosphamide-Induced Immunosuppression

Abstract

Background: Cyclophosphamide (CTX)-induced immunosuppression involves systemic toxicity, splenic atrophy, and broad transcriptomic disruption. Icariin (ICA) has immunomodulatory activity, but the systems-level features associated with splenic recovery remain incompletely defined. Methods and Results: Male mice were assigned to control, CTX, and CTX plus ICA (20, 40, or 80 mg/kg) groups. Phenotypic measurements were integrated with splenic RNA sequencing, differential expression analysis, targeted gene-set analysis, and weighted gene co-expression network analysis (WGCNA). ICA produced dose-associated improvement in spleen index and body weight trajectory, with the most consistent phenotypic response at 80 mg/kg. Overall transcriptomic separation was supported by permutational multivariate analysis of variance (PERMANOVA) (F = 17.18, R2 = 0.873, p < 0.001). WGCNA identified a recovery-associated turquoise module; Myb was assigned to this module, whereas Gata1 belonged to a distinct royalblue module. Complement and chemokine gene sets provided the strongest targeted enrichment evidence, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed dose-associated changes in selected innate immune transcripts. Marker-based lineage signatures indicated non-uniform recovery, including persistent depression of the B-cell signature. Conclusion: ICA-associated phenotypic recovery coincided with partial, non-uniform remodeling of splenic transcriptional programs. The Myb- and Gata1-associated findings are hypothesis-generating co-expression signals and do not establish transcription-factor binding or causality.