Yihan Liu, Hao Yu, Jingbin Zhang, Xin Li, Yunu Ma, Chunmei Guang, Wenjun Mu, Huan Chen, Hongwei Hou
These findings indicate that the DR condition was associated with remodeling of LPS-challenged BV2 microglial-like states, attenuation of inflammatory programs, and increased neurotrophic outputs relative to LPS, without significantly reducing CCK-8-assessed metabolic activity. This study provides a single-cell characterization of distinct treatment-associated responses to (R)-nicotine, diosmetin, their combined administration, and galantamine.
BACKGROUND: Microglial neuroinflammation contributes to the progression of neurodegenerative diseases, yet it remains challenging to attenuate inflammatory responses while preserving cellular function. The effects of (R)-nicotine, diosmetin, their combined administration, and galantamine on heterogeneous BV2 transcriptional states have not been compared at single-cell resolution.
METHODS: LPS-stimulated BV2 microglial-like cells were treated with (R)-nicotine, diosmetin, their combination (DR), or galantamine. Single-cell RNA sequencing was performed with three biological replicates per group and integrated with RNA velocity and SCENIC regulon inference to characterize treatment-associated state redistribution, inferred local transcriptional directionality and regulon-activity patterns. Functional validation included CCK-8 metabolic activity assays, multiplex cytokine ELISA, BDNF/GDNF quantification, qPCR, and high-content immunofluorescence analysis of iNOS and Arg1 at single-cell resolution.
RESULTS: LPS decreased the relative abundance of the Itgae+/Plk4+ cluster while increasing the Nmur1+/Limk2+ cluster and inflammatory effector programs. DR treatment suppressed pro-inflammatory cytokine release without significantly reducing CCK-8-assessed metabolic activity, increased the Itgae+/Plk4+ cluster proportion, and increased BDNF/GDNF relative to LPS. RNA velocity and SCENIC analyses suggested that DR and galantamine showed distinct transcriptional and regulatory patterns: DR attenuated Batf-associated inflammatory regulons and was associated with increased Atf3-linked stress-response activity, whereas galantamine preferentially engaged DNA repair and genome-maintenance programs.
CONCLUSION: These findings indicate that the DR condition was associated with remodeling of LPS-challenged BV2 microglial-like states, attenuation of inflammatory programs, and increased neurotrophic outputs relative to LPS, without significantly reducing CCK-8-assessed metabolic activity. This study provides a single-cell characterization of distinct treatment-associated responses to (R)-nicotine, diosmetin, their combined administration, and galantamine.