Modulation of Autistic-Related Factors in Hippocampal Neurons: Role of Oxytocin

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Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with dysregulation of several cellular processes. Accumulating evidence links ASD to the abnormality of cellular growth and programmed cell death (apoptosis). According to many postmortem and animal studies, abnormalities of several apoptotic signaling pathways have been linked to the induction of ASD, such as the ERK and p53 signaling pathways. Besides, the participation of neuroinflammation and oxidative stress in ASD induction and perpetuation has been identified. It has been reported that the levels of ROS and interleukin-1β are abnormally increased in neuronal brain cells in individuals with ASD. Therefore, agents that can improve cellular growth, regulate apoptosis, and reduce oxidative stress and neuroinflammation, like the neuropeptide oxytocin (OXT), may be effective in managing ASD. Objective: Our main goal was to investigate the effects of OXT on autistic-related factors, including cellular growth, oxidative stress, and neuroinflammation, as well as the intracellular signaling pathways involved in these effects. Methodology: We evaluated the effect of OXT on cell growth and death by performing cell counting (hemocytometer), MTT assay, and Bresto blue assay in hippocampal neurons (mHippoE-2). The proliferative effect mechanisms were evaluated using western blotting and MTT assay. In the survival experiments, viability was assessed by MTT assay in cells incubated in the presence or absence of OXT 1000 nM and/or 1000nM OXTA with oxidative stress inducers (H2O2, DMNQ, and CPT) and neuroinflammatory inducer (LPS). The mechanisms of the protective effect were evaluated using western blotting, ELISA. Also, we used the DCFDA kit to evaluate the antioxidant effect of OXT. Moreover, we employed the immunocytochemistry technique to assess the effect of 1000 nM OXT and/or 1000 nM OXTA against the induced morphological alterations. v Results: This study revealed that OXT significantly induced cell growth in hippocampal neurons (mHippoE-2). The OXTA (L-371,257) significantly reduced cell growth. The proliferative effect of OXT is mediated through MEK/ERK signaling pathway. In addition, treatment with 1000 nM OXT significantly reduced the reduction in cell viability induced by oxidative stress inducers (H2O2, CPT, and DMNQ) but not inflammatory inducer (LPS). In addition, OXT significantly reduced ROS generation when the cells were exposed to H2O2 and DMNQ but not CPT. The western blotting technique demonstrated that OXT significantly reduced the protein levels of p53-caspase 3 and increased the levels of Mdm2 induced by H2O2 and DMNQ, but not CPT. Our morphological studies showed that OXT countered the reduction in cellular projection length induced by H2O2, CPT, and DMNQ. Furthermore, OXT significantly reduced the protein levels of PI3K and p-AKT but not the NLRP3-caspase 1 signaling pathway. Conclusion: Our results indicate that OXT has a proliferative effect by activating the ERK signaling pathway. Furthermore, we revealed that the protective effect of OXT was mediated through the modulation of oxidative stress and mitochondrial apoptosis pathway. Moreover, OXT decreased the levels of some inflammatory-mediated proteins. On the other hand, these effects were lacking in the presence of OXTA. These results will contribute to a better understanding of OXT’s potential role in autistic-related factors associated with cell loss, oxidative stress, and neuroinflammation.
Oxytocin, autism, oxidative stress, apoptosis, neuroinflammation, cell proliferation