ObjectiveTo investigate the effects of governor vessel pushing manipulation on behavioral outcomes in valproic acid (VPA)-induced autism spectrum disorder (ASD) rats and explore its underlying mechanisms using prefrontal RNA sequencing (RNA-Seq).MethodsNine Sprague-Dawley pregnant rats at gestational day 12.5 were divided into two groups, six received intraperitoneal VPA injection (600 mg/kg) for modeling, and three received saline. Male offspring at postnatal day 21 were evaluated using the three-chamber social test and open field test to validate the ASD model. VPA-induced male offspring were randomly assigned to the model group (n=5) or tuina group (n=5), while saline offspring formed the blank group (n=5). The blank group and model group received no intervention, while the tuina group underwent governor vessel pushing manipulation stimulation along the governor vessel using a custom device, twice a day for 14 days, totaling 28 times. Post-intervention, behavioral assessments included social index (SI) and social preference index (SPI) in the three-chamber test, total distance traveled and central zone time in the open field test, marble-burying test for stereotyped behaviors, and Nissl staining for prefrontal cortical neuron survival. RNA-Seq identified differentially expressed genes (DEGs) in the prefrontal cortex, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Real time fluorogenic quantitative PCR(RT-qPCR) validated DEGs, and Western blotting analyzed proteins in enriched pathways.ResultsPre-intervention, both model and tuina groups showed reduced SI, SPI, total distance, and central zone time compared to the blank group (P<0.05), confirming successful modeling. Post-intervention, the model group exhibited lower SI, SPI, total distance, central zone time, increased marble-burying (P<0.05), and fewer Nissl bodies(P<0.01) versus the blank group. Compared to the model group, the tuina group displayed improved SI, SPI, total distance, central zone time (P<0.05), reduced marble-burying (P<0.05), and increased Nissl bodies(P<0.01). RNA-Seq revealed 213 prefrontal DEGs (181 upregulated, 32 downregulated) in the tuina group. GO analysis highlighted cellular components, while KEGG identified 181 pathways, with 67 significantly enriched (P<0.05), notably the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. RT-qPCR confirmed decreased collagen type I alpha 2 (Col1α2), transforming growth factor-α (TGF-α), epidermal growth factor receptor 3(ErbB3), and serum/glucocorticoid regulated kinase 2(Sgk2)(P<0.05), and increased hepatic growth factor(Hgf)(P<0.01) in the model group, reversed by governor vessel pushing manipulation. Western blotting showed reduced prefrontal NRG1, ErbB3, nNOS, PI3K, AKT, p-nNOS, p-PI3K, and p-AKT in the model group (P<0.05), which were upregulated by tuina.ConclusionGovernor vessel pushing manipulation ameliorates social deficits, anxiety, stereotyped behaviors, and neuronal loss in ASD rats, potentially via activation of the PI3K/AKT signaling pathway.

tuina;governor vessel;autism spectrum disorder;behavioral outcomes;transcriptome sequencing