Mechanism Of An Shen Ding Zhi Ling In Regulating Neuroinflammation Of Attention Deficit Hyperactivity Disorder

Objective:To observe the effect of An Shen Ding Zhi Ling(ASDZL)on the behavior of young spontaneously hypertensive rats(SHRs),and to explore the neuroinflammatory mechanism of ADHD and the intervention effect of ASDZL from inflammatory factors,immune cells,immune function,blood-brain barrier and inflammatory pathways.And to explore the modern medical connotation of "Syndrome of hyperactivity of heart and liver fire" of ADHD.Methods:(1)The constituents of ASDZL were analyzed by ultra performance liquid chromatography-mass spectrometry.(2)Three-week-old SHRs were divided into model group,atomoxetine(ATX)group and ASDZL group.Wistar Kyoto(WKY)rats were set as normal control group.(3)The effects of ASDZL on spontaneous and impulsive behavior,and spatial learning and memory ability of SHR rats were evaluated by open field test(OFT),elevated plus maze(EPM)and Morris water maze(MWM)test.(4)Flow cytometry was used to detect the expression of dendritic cells,macrophages and T lymphocytes in the spleen of rats to evaluate the immune function of SHRs and the effect of ASDZL on them.(5)ELISA was used to detect the regulatory effects of ASDZL on IL-1?,IL-4,IL-6,IL-10,TNF-? and MCP-1 in serum,prefrontal cortex(PFC)and hippocampus of SHRs.(6)Immunofluorescence,immunohistochemistry and toluidine blue staining were used to observe the expression of microglia,astrocytes and mast cells in the PFC and hippocampus of SHRs and the effect of ASDZL on them.(7)Transmission electron microscopy was used to observe the tight junction structure of vascular endothelial cells in PFC.Western blot was used to evaluate the regulatory effects of ASDZL on tight junction proteins ZO-1 and occludin in PFC and hippocampus of SHRs.(8)Western blot was used to detect the effect of ASDZL on key proteins and their phosphorylation expression in the MAPK and NF-?B signaling pathways of SHRs.Results:(1)Ten components of ASDZL were determined by comparison with the standards under positive and negative ion modes:Catalpol,Acteoside,Isorhynchophylline,Baicalin,Rhynchophylline,Phyllyrin,Quercetin,Saikosaponin A,Curcumin and Emodin.(2)ASDZL significantly reduce hyperactivity and impulsive behavior of SHR,and improved the ability of spatial learning and memory.(3)ASDZL showed inhibitory effect on the proliferation of macrophages and dendritic cells in SHRs,and improved the ratio of CD4+/CD8+ratio.(4)ASDZL inhibited the release of pro-inflammatory factors IL-1?,IL-4,IL-6,TNF-? and MCP-1 in serum,PFC and hippocampus of SHRs,and increased the level of anti-inflammatory factor IL-10.(5)ASDZL showed inhibitory effect on the activation of microglia,astrocytes and mast cells in prefrontal cortex,hippocampus of SHR rats.(6)ASDZL improved the expression of tight junction proteins ZO-1 and occludin in PFC and hippocampus of SHRs,and protected the structural integrity of blood-brain barrier.(7)ASDZL inhibited phosphorylation of key factors in MAPK and NF-?B signaling pathways.Conclusion:This study preliminarily explored the neuroinflammatory mechanism of ADHD,and found that ASDZL achieved the purpose of anti-inflammation by inhibiting the activation of immune cells,improving the imbalance of immune function,repairing the damage of blood-brain barrier,and inhibiting the phosphorylation of MAPK and NF-?B signaling pathways,so as to improve the behavioral characteristics of ADHD in SHRs and play a therapeutic role in ADHD.Chronic neuroinflammation may be the modern medical connotation of "Syndrome of hyperactivity of heart and liver fire" in ADHD.