An Integrated Approach Combining Chemical Profiling, Network Pharmacology, and In Vitro Validation to Uncover the Multitarget Mechanisms of Naokang II Decoction against Vascular Dementia

Vascular dementia (VaD) is a debilitating cognitive disorder with limited pharmacological treatments. Naokang II decoction (NKII) is a traditional Chinese medicine (TCM) formula for VaD with significant efficacy, but its mechanisms remain unclear. This study integrated chemical profiling, network pharmacology, single-nucleus RNA Sequencing (snRNA-seq) analysis, molecular docking, and in vitro validation to elucidate NKII's mechanisms against VaD. LC-MS/MS identified 412 components in NKII, of which 45 compounds were screened as active constituents. Their 1081 predicted targets overlapped with 1592 VaD-related dysregulated genes, yielding 73 common targets. Network analysis identified 10 core targets, including NLRP3, CASP1, and TLR2. snRNA-seq data localized these core targets predominantly to microglia. Molecular docking revealed that the coumarin Herniarin, a component newly identified in NKII, exhibited the strongest binding affinity to NLRP3. In vitro experiments on BV2 and HMC3 microglial cells showed that Herniarin inhibited cell proliferation and migration, induced Apoptosis, arrested the cell cycle at the G2/M phase, and downregulated TLR2, NLRP3, ASC, and CASP1 at both mRNA and protein levels, exerting antineuroinflammatory effects by inhibiting the microglial NLRP3 inflammasome pathway. This study established a multidimensional research paradigm and identified Herniarin as a novel NLRP3 Inhibitor with therapeutic potential for VaD and Other neuroinflammatory diseases.