Mechanism of action of Liuwei Dihuang Pill in treating autism spectrum disorder based on network pharmacology and animal experiments
Objective To explore the potential mechanism of action of Liuwei Dihuang Pill(LWDHP)in treating autism spectrum disorder(ASD)using network pharmacology,molecular docking technology,and animal experiments.Methods The active ingredients and corresponding target gene information of LWDHP were obtained through the TCMSP database,and ASD disease target genes were obtained through the databases of GeneCards,OMIM,and DrugBank;the above target genes were intersected with Venn diagram,and then the STRING database was used to construct the PPI network of the key targets;GO and KEGG enrichment analyses of the key targets was performed using the Metascape database.The Autodock software was used for molecular docking of the core targets and active ingredients of LWDHP.Animal experimental verification:Eight SPF SD pregnant rats were randomized into blank pregnant rat group(n=1)and model pregnant rat group(n=7)which was modeled by intraperitoneal injection of sodium valproate(VPA).Thirty neonatal male rats conforming to the ASD disease model were selected and randomly subdivided into model group,vitamin D group,high-,medium-,and low-dose LWDHP groups,meanwhile,the neonatal rats from blank pregnant rat group were selected as blank group,with six rats in each group.Low-,medium-,and high-dose LWDHP groups were given 0.75,1.5,and 3 g/kg LWDHP suspension,respectively,vitamin D group 1 480 IU/kg vitamin D drops,and model and blank groups an equal amount of normal saline,once a day by gavage,continuously intervened for two weeks.Then,three-box social test and open field test were conducted to evaluate the social behaviors of rats.ELISA was used to determine the levels of glycogen synthase kinase 3β(GSK3β)and β-catenin in the hippocampus of the neonatal rats in each group;Western blot was used to examine the protein expression levels of β-catenin,cellular myelocytomatosis oncogene(c-Myc),and cell cycle protein D1(Cyclin D1)in the hippocampus.Results A total of 74 active ingredients and 208 drug target genes of LWDHP were obtained,as well as 3,903 ASD disease target genes and 127 key targets including core targets of protein kinase B α(Akt1),interleukin-6(IL-6),interleukin-1(IL-1),and tumor necrosis factor(TNF),etc.;GO function enrichment showed that LWDHP mainly acted on cellular responses of organic nitrogen-containing compounds,postsynaptic membranes,and postsynaptic neurotransmitter receptor activity,and others;KEGG pathway enrichment showed that it regulated signaling pathways of TNF,nuclear transcription factor-κB(NF-κB),Wnt,and others;the molecular docking showed that the active ingredients in LWDHP could be structurally stable with the core protein Akt1.Animal experiments showed that in the test of social ability,compared with blank group,the neonatal rats in model group,vitamin D group,and low-,medium-,and high-dose LWDHP groups had shorter contact time with unfamiliar rat I(P＜0.05);after 2 weeks of administration,the contact time with unfamiliar rat I of the neonatal rats in vitamin D group and low-,medium-,and high-dose LWDHP groups was prolonged(P＜0.05).In the test of social novelty,the neonatal rats in model group,vitamin D group,and low-,medium-,and high-dose LWDHP groups had shorter contact time with unfamiliar rat Ⅱ compared with those in blank group(P＜0.05);after 2 weeks of administration,the contact time with unfamiliar rat Ⅱ of the neonatal rats in vitamin D group and low-,medium-,and high-dose LWDHP groups was prolonged(P＜0.05).The open field test showed that after treatment,both the total activity distance and activity distance in the central area of the neonatal rats in low-and medium-dose LWDHP groups increased(P＜0.05),and the total activity distance of the neonatal rats in vitamin D and high-dose LWDHP groups increased(P＜0.05).Compared with model group,vitamin D group and low-,medium-,and high-dose LWDHP groups showed higher GSK-3β level(P＜0.05),lower β-Catenin level(P＜0.05),and reduced protein expressions of β-Catenin and c-Myc(P＜0.05);the protein expression of Cyclin D1 in low-dose LWDHP group decreased(P＜0.05).Conclusion LWDHP can effectively regulate the Wnt signaling pathway,and improve the social behaviors and reduce anxiety of rats with VPA-induced ASD.
万方数据
维普
