Mn₃O₄ nanozymes as potential therapeutic agents for autism spectrum disorder: insights from behavioral and molecular studies

<i>Background</i>: Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder of uncertain etiology. Current studies suggest that ASD progression is closely linked to an imbalance between oxidative stress and antioxidant capacity, marked by elevated levels of reactive oxygen species (ROS) and reduced concentrations of antioxidant molecules such as superoxide dismutase (SOD) and glutathione (GSH). Although the human body does possess endogenous ROS-scavenging enzymes, their sensitivity to environmental conditions and the difficulties of large-scale production limit their practical application. Consequently, substantial efforts have been dedicated in recent years to developing artificial enzymes with ROS-scavenging activity. Among these, ROS-scavenging nanozymes have been widely used due to their enhanced stability and multifunctionality. Notably, only a few manganese-containing nanozymes have been reported to exhibit effective reactive oxygen species (ROS) scavenging activity thus far. <i>Methods</i>: In this study, we utilized Mn₃O₄ nanozymes (Mn₃O₄ NZs) exhibiting superoxide dismutase, catalase, and hydroxyl radical-scavenging activities. We assessed brain injury, as well as the antioxidative and anti-inflammatory effects of Mn₃O₄ NZs through behavioral tests, Nissl staining, immunofluorescence assays, and a laser speckle imaging system. Furthermore, we explored the underlying mechanisms of Mn₃O₄ NZs by employing ELISA kits, oxidative stress detection kits, and immunofluorescence analysis. <i>Results</i>: The results demonstrated that Mn₃O₄ NZs increase cerebral blood flow and effectively ameliorate ischemic and hypoxic conditions in BTBR mice. Moreover, they improve social deficits, repetitive stereotyped behaviors, cognitive impairment, and neuronal morphological damage. Further <i>in vitro</i> experiments confirmed that Mn₃O₄ NZs exert neuroprotective effects in BTBR mice by mitigating oxidative stress and inflammation. <i>Conclusion</i>: These findings indicate that Mn₃O₄ NZs exhibit excellent antioxidant and anti-inflammatory effects <i>in vitro</i> and effectively enhance cerebral blood flow, ameliorate behavioral deficits, and alleviate neuronal damage in BTBR mice <i>in vivo</i>. Collectively, our results suggest that Mn₃O₄ NZs exert neuroprotective effects in the hippocampus of BTBR mice by reducing oxidative stress, mitigating neuroinflammation, and rescuing neuronal injury. Consequently, they hold promise as a potential nanomaterial for the treatment of autism.