Mechanism of Kongsheng Zhenzhong Dan In Autism-Related Pathways: A Study Based On Network Pharmacology, Molecular Docking, And Experimental Validation.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder, and its pathogenesis is closely associated with chronic neuroinflammation. As a classic traditional Chinese medicine (TCM) formula, Kongsheng Zhenzhong Dan (KSZZD) is used for neurodevelopmental and memory disorders because of its reported neuroprotective effects, but its specific molecular mechanism in ASD remains unclear. This study integrates network pharmacology, molecular docking analysis, and MD simulation to systematically elucidate the core active components and candidate therapeutic targets of KSZZD for ASD at both the systemic and atomic levels. Subsequently, key binding residues are identified by alanine flexible scanning, and the biological effects are assessed using a valproic acid (VPA)-induced ASD rat model. Network pharmacology analysis revealed that quercetin, a key component in KSZZD, may act partly by regulating the TNF-α-mediated inflammatory signaling pathway. Molecular docking results showed that the binding energies of the core components of KSZZD to their targets were all lower than -5.0 kcal/mol, among which quercetin exhibited the strongest binding affinity to TNF (-8.52 kcal/mol). MD simulation suggested that quercetin could maintain stable binding to the active pocket of TNF-α, and alanine scanning further identified GLU104 and GLN102 as key amino acid residues for maintaining the structural stability of the complex. In vivo experiments demonstrated that quercetin intervention significantly improved locomotor and exploratory behaviors in VPA-induced ASD rats, and effectively reduced TNF-α expression levels in serum and brain tissue. These findings suggest that quercetin may contribute to the predicted effects of KSZZD, at least in part, through inhibition of the TNF-α signaling pathway.