Hua-Feng-Dan Alleviates LPS-induced Neuroinflammation by Inhibiting the TLR4/Myd88/NF-κB Pathway: Integrating Network Pharmacology and Experimental Validation

Abstract

aims: Our study aims to elucidate the molecular mechanism by which HFD mitigates neuroinflammation through combining network pharmacology and experimental validation. background: Neuroinflammation is the pathological basis of many neurological diseases, including neurodegenerative diseases and stroke. Hua-Feng-Dan (HFD) is a well-established traditional Chinese medicine used for the treatment of stroke and various other brain-related ailments. objective: Our study aims to elucidate the molecular mechanism by which HFD mitigates neuroinflammation through combining network pharmacology and experimental validation. method: Active compounds and their related targets were extracted from the TCMSP database and SymMap database. The neuroinflammation-related targets were obtained from the GeneCards database. Protein-protein interaction (PPI) network was constructed using the common target of HFD and neuroinflammation. MCODE plug-in was used to find the hub module genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to dissect the hub module genes. The lipopolysaccharide (LPS)-induced BV2 microglial neuroinflammation model was utilized to assess the therapeutic effects of HFD on neuroinflammation. Western blotting analysis was performed to examine the core target proteins in the TLR4/MyD88/NF-κB signaling pathway, potentially implicated in HFD's therapeutic effects on neuroinflammation. Hoechest 33342 staining and JC-1 staining were employed to evaluate the effect of inflammatory factors secreted by activated microglia on neuronal apoptosis. result: Through network pharmacology, 73 active compounds were identified, with quercetin, beta-sitosterol, luteolin, and (-)-Epigallocatechin-3-Gallate recognized as important compounds. Meanwhile, 115 common targets of HFD and neuroinflammation were identified and 61 targets were selected as the hub targets utilizing the MCODE algorithm. The results of in vitro experiments demonstrated that HFD significantly inhibited microglial-mediated neuronal inflammation induced by LPS. Integrating the predictions from network pharmacology with the results of in vitro experiments, we discerned that the mechanism of HFD in mitigating neuroinflammation is closely related to TLR4/MyD88/NF-κB pathway. Furthermore, HFD demonstrates a capacity to shield neurons from apoptosis by curbing the secretion of pro-inflammatory factors subsequent to microglial activation. conclusion: Our findings demonstrated that HFD had an inhibitory effect on LPS-induced neuroinflammation in microglia and elucidated its underlying mechanism. These findings will provide theoretical underpinning for using HFD in clinical treatment of neurodegenerative diseases related to neuroinflammation. other: No