Network pharmacology, molecular docking, and experimental validation to explore the potential mechanism of Long Mu Qing Xin mixture for the treatment of attention deficit hyperactivity disorder

**Background:** Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a global prevalence of about 5.29%. Dopamine (DA) defects are considered the core pathogenesis. First-line treatment methylphenidate (MPH) has limitations including potential for addiction and adverse effects. Long Mu Qing Xin Mixture (LMQXM) is an empirical Chinese medicine formula that showed clinical efficacy in a prior small randomized trial (86.67% efficacy vs 50% control, p < 0.05), but its mechanisms were unknown.

**Methods:** Network pharmacology screened LMQXM active ingredients using TCMSP (OB ≥ 30%, BBB ≥ −0.3, DL ≥ 0.18) and SwissADME/SwissTargetPrediction. ADHD-related targets were obtained from DrugBank, GeneCards, TTD, and DisGeNET databases. A protein-protein interaction (PPI) network was constructed using STRING and Cytoscape, with 46 core targets identified (degree > 15, BC > 0.0040, CC > 0.4683). GO and KEGG enrichment analyses were performed via Metascape. Molecular docking of 8 active ingredients with 9 targets used AutoDock 4.2.6. For animal experiments, 4-week-old male SHRs (n = 30) were divided into model, MPH (4.22 mg/kg), LMQXM low-dose (5.28 ml/kg), medium-dose (10.56 ml/kg), and high-dose (21.12 ml/kg) groups; WKY rats (n = 6) served as controls. Drugs were administered by gavage twice daily for 4 weeks. Behavioral tests included open field test (OFT) at 0 and 4 weeks and Morris Water Maze (MWM) at 8–9 weeks of age. DA levels were measured by LC-MS, cAMP by ELISA, and DRD1, DRD2, cAMP, PKA expression by immunohistochemistry and qPCR.

**Key Results:** Network pharmacology identified 177 active ingredients and 355 targets from LMQXM, with 121 intersection targets with ADHD. Core targets included DRD1, DRD2, SLC6A3, SLC6A4, AKT1, IL-6, and TNF. KEGG enrichment highlighted dopaminergic synapse and cAMP signaling pathways. Molecular docking showed strong binding (e.g., beta-sitosterol-TNF: −20.13 kJ/mol; stigmasterol-DRD2: −24.02 kJ/mol; rhynchophylline-SLC6A4: −18.95 kJ/mol). In animal experiments, SHRs showed significantly higher total moving distance and average speed vs WKY rats (p < 0.0001). After 4 weeks, MPH, LMQXM-MD, and LMQXM-HD significantly reduced these measures vs SHR (p < 0.01). In MWM, from day 3, MPH and all LMQXM groups had significantly shorter escape latency vs SHR (p < 0.01). Probe trials showed MPH and LMQXM-MD had significantly more annulus visits (p < 0.05) and time in target quadrant (p < 0.05) vs SHR. DA and cAMP levels in PFC and striatum were significantly lower in SHRs vs WKY (p < 0.01). MPH and LMQXM-MD significantly increased DA and cAMP in both regions (p < 0.05). Immunohistochemistry showed lower DRD1, cAMP, and PKA positive cell expression in SHRs vs WKY; MPH and LMQXM-MD significantly reversed cAMP and PKA MOD (p < 0.05). DRD2 showed no significant differences between groups. qPCR confirmed significantly lower PKA mRNA in SHR PFC and striatum vs WKY (p < 0.05), which was increased by MPH, LMQXM-MD, and LMQXM-HD (p < 0.05). DRD1 mRNA was significantly increased in PFC by MPH and LMQXM-MD (p < 0.05) and in striatum by LMQXM-MD (p < 0.05).

**Clinical Implications:** This study provides the first evidence that LMQXM improves ADHD-like behaviors in SHRs through activation of the DRD1/cAMP/PKA signaling pathway, increasing DA levels in the PFC and striatum. The medium dose (10.56 ml/kg) was most effective, outperforming low and high doses. These findings support LMQXM as a potential multi-target, multi-component therapy for ADHD that addresses DA deficiency without the addiction risks associated with stimulant medications. The study also validates the utility of network pharmacology and molecular docking for investigating traditional Chinese medicine mechanisms. Limitations include small sample size, lack of SD rat controls, and no direct investigation of DAT. Future research should expand sample sizes, include additional controls, and explore neuroinflammatory mechanisms.