六味地黄丸促进线粒体自噬调控孤独症大鼠神经炎症及行为障碍的机制OA

Objective:To observe the effect of Liuwei Dihuangwan on behavioral impairments in the rat model of autism spectrum disorder(ASD)and explore the mechanism of action.Methods:Twelve SD pregnant rats were intraperitoneally injected with valproic acid(VPA)(10 rats)or normal saline(2 rats),and male offspring were selected to establish the model of ASD and the control rats.Rats were randomly assigned into model,low-dose(0.75 g·kg-1)and high-dose(1.5 g·kg-1)Liuwei Dihuangwan,vitamin D(positive drug,3.7×10-5 g·kg-1),and blank groups.Each group was administrated with the corresponding concentration of drugs or the same volume of normal saline by gavage for 2 weeks.After the intervention,the three-chamber social test was conducted to evaluate social interaction and social preference.The open field test was carried out to observe spontaneous behavior and anxiety state.Hematoxylin-eosin staining(HE)was used to observe the pathological changes of the prefrontal tissue.Transmission electron microscopy was employed to observe the ultrastructure of mitochondria in prefrontal neurons.Immunofluorescence was used to detect the expression of ionized calcium-binding adapter molecule-1(Iba-1)in the prefrontal tissue.Enzyme-linked immunosorbent assay(ELISA)was adopted to measure the levels of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6).Western blot was employed to assess the expression differences of phosphorylated adenosine monophosphate-activated protein kinase(p-AMPK),adenosine monophosphate-activated protein kinase(AMPK),phosphorylated Unc-51-like autophagy-activating kinase 1(p-ULK1),Unc-51-like autophagy-activating kinase 1(ULK1),and FUN14 domain-containing protein 1(FUNDC1).Results:Compared with the blank group,the model group spent less time sniffing stranger 1 and stranger 2 in the three-chamber social test(P<0.01)and showed reductions in the total distance traveled,average speed,distance traveled in the central area,and time spent in the central area in the open field test(P<0.01).In addition,the model group showed extensive apoptosis of neurons,with shrunken nuclei and red-stained cytoplasm,and extensive necrosis of neurons in the prefrontal tissue,mitochondrial swelling,decreased matrix density,disrupted cristae,and autophagic lysosomes in neurons,increases in the rate of Iba-1 positive cells in the prefrontal area(P<0.01)and the levels of TNF-α and IL-6(P<0.01),and down-regulation in the expression of p-AMPK/AMPK,p-ULK1/ULK1,and FUNDC1(P<0.01).Compared with the model group,low-dose and high-dose Liuwei Dihuangwan and the vitamin D prolonged the time spent sniffing stranger 1 and stranger 2 in the three-chamber social test(P<0.05,P<0.01),increased the total distance traveled,average speed,distance traveled in the central area,and time spent in the central area in the open field test(P<0.05,P<0.01),restored the morphology of neurons in the prefrontal tissue,decreased the number of apoptotic cells,alleviated the swelling of mitochondria in neurons,increased the matrix density,mitigated the fragmentation and disorder of cristae,and increased the number of autophagosomes.Moreover,the drugs decreased the rate of Iba-1 positive cells in the prefrontal area(P<0.01),lowered the levels of TNF-α and IL-6(P<0.01),and up-regulated the expression of p-AMPK/AMPK,p-ULK1/ULK1,and FUNDC1(P<0.01).Conclusion:Liuwei Dihuangwan ameliorate autism-like behaviors and reduce neuronal apoptosis and neuroinflammatory damage in the rat model of ASD by promoting mitophagy mediated by the AMPK/ULK1/FUNDC1 pathway.