To investigate the clinical efficacy of Longmu Ningxin Formula (LMNXF) in treating children with attention deficit hyperactivity disorder (ADHD) and to explore its potential mechanism of action through animal experiments and proteomics.

A total of 80 children with ADHD were randomly divided into a western medicine group (n=40) and a Chinese medicine group (n=40). The western medicine group was treated with methylphenidate hydrochloride extended-release tablets (MPH), while the Chinese medicine group received LMNXF. The treatment lasted for 12 weeks. The scores of the Swanson, Nolan, and Pelham-Ⅳ Rating Scale (SNAP-Ⅳ, parent version), the Weiss Functional Impairment Rating Scale (WFIRS, parent version), the Conners' Parent Rating Scale (CPRS), and the Chinese medicine syndrome scores were compared between the two groups before and after treatment. Safety was also evaluated. For the animal experiment, the spontaneously hypertensive rat (SHR), a classic ADHD model, was used. A total of 18 4-week-old male SHRs were randomly divided into a model group, a Chinese medicine group, and a western medicine group. A total of 6 Wistar-Kyoto rats served as the control group. The Chinese medicine group received LMNXF at 38.00 g·kg^-1·d^-1, the western medicine group received MPH at 4.22 mg·kg^-1·d^-1, and the control and model groups were administered an equal volume of saline by gavage. Treatment lasted for 4 weeks. Behavioral tests were conducted to assess the rats' performance. After the experiment, prefrontal cortex tissues were collected for quantitative proteomic analysis, and Western Blot analysis was performed to validate differentially expressed proteins.

After treatment, the scores of the SNAP-Ⅳ, WFIRS, CPRS, and the primary and secondary Chinese medicine syndrome scores in the Chinese medicine group were significantly decreased compared to those before treatment (all P<0.001). The improvements in the WFIRS and CPRS scores and the Chinese medicine syndrome scores in the Chinese medicine group were greater than those in the western medicine group (all P<0.05). No serious adverse reactions related to the study drugs were observed in any child during the treatment period. LMNXF significantly ameliorated hyperactivity/impulsivity and spatial learning/memory deficits in SHRs (all P<0.05). Proteomic analysis of the prefrontal cortex identified 273 differentially expressed proteins between the model group and the Chinese medicine group. These proteins were primarily involved in seven pathways, including the NOD-like receptor signaling pathway, FoxO signaling pathway, and intestinal immune network for IgA production. Western Blot results showed that LMNXF reversed the upregulation of Ripk2, Tab1, and IL-6 in the prefrontal cortex of SHRs.

LMNXF demonstrates favorable efficacy and a high safety profile in treating children with ADHD. Its mechanism of action may involve modulating Ripk2, Tab1, and IL-6 expression in the prefrontal cortex, thereby inhibiting NOD-like receptor signaling pathway-mediated neuroinflammation.