Quercetin from Kongsheng Zhenzhong Dan improves ASD-like behaviors by inhibiting TNF-α in rats

Autism spectrum disorder (ASD) is a complex neurodevelopmental condition characterized by social communication deficits and repetitive behaviors. Its pathogenesis is closely linked to chronic neuroinflammation. Current pharmacological treatments for ASD are limited and often target symptoms rather than underlying mechanisms. Kongsheng Zhenzhong Dan (KSZZD), a traditional Chinese medicine (TCM) formula, has reported neuroprotective effects for neurodevelopmental and memory disorders, but its specific molecular mechanisms in ASD, particularly regarding its active components and their targets, have remained largely uncharacterized.

This study employed an integrated approach combining network pharmacology, molecular docking, and molecular dynamics (MD) simulation to systematically identify core active components and candidate therapeutic targets of KSZZD for ASD. Key binding residues were identified using alanine flexible scanning. Subsequently, the biological effects of the identified core component were experimentally validated using a valproic acid (VPA)-induced ASD rat model. The intervention involved quercetin, with primary endpoints assessing locomotor and exploratory behaviors, alongside TNF-α expression levels in serum and brain tissue. The study aimed to elucidate how KSZZD's components modulate ASD-related pathways.

Network pharmacology analysis initially suggested that quercetin, a key component of KSZZD, might exert its effects partly by regulating the TNF-α-mediated inflammatory signaling pathway. Molecular docking results further supported this, showing that core components of KSZZD exhibited binding energies to their targets all lower than -5.0 kcal/mol. Notably, quercetin demonstrated the strongest binding affinity to TNF-α with a binding energy of -8.52 kcal/mol. MD simulation confirmed that quercetin could maintain stable binding within the active pocket of TNF-α. Alanine scanning identified GLU104 and GLN102 as critical amino acid residues for the structural stability of the quercetin-TNF-α complex. > In vivo experiments in VPA-induced ASD rats demonstrated that quercetin intervention significantly improved both locomotor and exploratory behaviors, and effectively reduced TNF-α expression levels in both serum and brain tissue. These findings collectively suggest a direct role for quercetin in modulating TNF-α signaling.