Effect of acupuncture at “Neiguan” (PC6) on behavioral deficits and neuroinflammation in rats with autism

To investigate the ameliorative effect of acupuncture at “Neiguan” (PC6) on behavioral deficits, hippocampal neuronal damage, and neuroinflammation in young rats with valproic acid (VPA)-induced autism spectrum disorder (ASD).

Pregnant SD rats were randomly divided into a control group and a model group. The ASD model was established by a single intraperitoneal injection of VPA (600 mg/kg) on gestational day 12.5. After birth, the offspring young rats were then randomly divided into control (CON), ASD model (model), sham-acupuncture (ASD+sham-Acu), acupuncture (ASD+Acu), solvent (DMSO) control (ASD+DMSO), and positive drug bumetanide (ASD+Bu) groups (n=9 in each group). Manual acupuncture (uniform reinforcing-reducing manipulation) was applied to bilateral PC6 of newborn rats for 30 min, once daily, 6 d a week, for 15 d (from the 7^th day to the 21^st day after birth). After acupuncture intervention, behavioral tests including the Morris water maze test, three-chamber social test, and self-grooming test were conducted. The hippocampal tissue was collected for observing changes of neuronal morphology and dendritic spine density by using hematoxylin-eosin and Golgi staining. The expression levels of synapse-related proteins postsynaptic density protein 95 (PSD95), glutamate decarboxylase 67 (GAD67), vesicular GABA transporter (VGAT) and pyroptosis-related proteins NOD-like receptor protein 3 (NLRP3), gasdermin domain-N-terminal (GSDMD-NT) and cleaved Caspase-1 in the hippocampal tissue were detected by Western blot. The levels of hippocampal interleukin (IL)-1β, IL-18 and activity of LDH were measured by enzyme-linked immunosorbent assay. The level of hippocampal reactive oxygen species (ROS) were detected by using commercial kits. The activity of hippocampal Iba1-positive cell (microglia) was assessed by immunofluorescence staining, and the cell apoptosis was detected by TdT-mediated dUTP nick-end labeling staining.

Compared with the CON group, the model group showed a significant increase in the escape latency of water maze test, self-grooming times, LDH activity, ROS level, IL-1β and IL-18 contents, expression of PSD95, NLRP3, ratios of GSDMD-NT/GSDMD and cleaved Caspase-1/pro-Caspase-1, number of Iba1-positive cells and the apoptosis rate (P<0.01, P<0.001), and a considerable decrease in the body mass, number of platform crossings and time spent in the target quadrant, social index and social preference, number of dendritic spine, and the expressions of GAD67 and VGAT proteins (P<0.05, P< 0.01, P<0.001). In comparison with the model group, both the increased and decreased levels of all the indexes mentioned above were reversed in the ASD+Acu and ASD+Bu groups (P<0.05, P<0.01, P<0.001), and the effects of ASD+Acu were evidently superior to those of ASD+Bu in up-regulating the expressions of GAD67 and VGAT proteins (P<0.05), but inferior to those of ASD+Bu in shortening the escape latency, reducing ROS level, and down-regulating the protein expressions of PSD95 and NLRP3, ratio of cleaved Caspase-1/pro-Caspase-1, and apoptosis rate (P< 0.05, P<0.01, P<0.001). The effects of acupuncture were evidently better than those of the sham acupuncture in increasing the body mass and number of platform crossings, shortening the escape latency, prolonging the time spent in the target quadrant, increasing the social index and social preference, reducing the self-grooming times, elevating the number of dendritic spine, and the expressions of GAD67 and VGAT proteins (P<0.05, P<0.01, P<0.001), and in down-regulating the LDH activity and ROS levels, IL-1β and IL-18 contents, and the expressions of PSD95 and NLRP3, ratios of GSDMD-NT/GSDMD and cleaved Caspase-1/pro-Caspase-1, Iba1-positive cell number, and the apoptosis rate (P<0.05, P<0.01, P<0.001). Similarly, in comparison with the ASD+DMSO group, the ASD+Bu group showed a significant improvement in the aforementioned behavioral and molecular biological indexes (P<0.05, P<0.01, P<0.001).

Acupuncture at PC6 can improve social deficits, repetitive stereotyped behavior and cognitive impairment in VPA-induced ASD-like young rats, which may be related to its functions in alleviating hippocampal neuronal damage, regulating the balance of synaptic proteins, and inhibiting oxidative stress and NLRP3 inflammasome-mediated neuroinflammation.