In this study, serum levels of occludin, which is a paracellular TJ protein, and zonulin, which functions as the regulator of the paracellular TJ proteins, were compared between children with ASD and control children of similar age, sex, and socioeconomic level. According to our findings, the serum occludin levels in children with ASD were lower than in the control group, but there was no significant difference between the groups in terms of serum zonulin levels. When the relationship of both markers to CARS scores was evaluated, it was found that the CARS scores increased as the occludin levels decreased, but no significant relationship was found between zonulin and CARS scores.
Occludin, is part of the epithelial and endothelial junction complexes, and recent data suggest that occludin regulates barrier functions of structures such as the gut and BBB . In a review of recent literature, no study investigating the relationship between occludin and ASD was found. When we looked at the studies on occludin, in a study that aimed to identify the biomarkers to watch the active period in MS, serum levels of occludin and zonulin were found to increase during the active period of the disease, and it was emphasized that these findings supported the leaky gut hypothesis . A study by Mankertz et al. showed that proinflammatory cytokines [tumor necrosis factor-alpha (TNF-α), interferon gamma (IFN-γ)] down regulated occludin expression, and the authors suggested that increased proinflammatory cytokines in inflammatory diseases might be an important mechanism in the increased paracellular TJ permeability by mediating the reduction of occludin expression . In two other studies, in which gut barrier function was investigated in inflammatory diseases of the intestines (Crohn's and ulcerative colitis), it was stated that occludin expression decreased during the active period of the disease, unlike other intracellular TJ proteins, and this might play a role in increased paracellular permeability [12, 13]. In addition to these studies, in vivo and in vitro studies have shown that proinflammatory cytokines also disrupt TJs in the BBB [34, 35]. The results of all these studies appear to correlate with the fact that inflammatory processes disrupt TJ proteins, specifically reducing occludin expression. Moreover, in recent years, emphasis has been increasing on the role of inflammatory processes in ASD etiopathogenesis . In fact, a recent meta-analysis concluded that the concentration of pro-inflammatory cytokines [IFN-γ, interleukin-1 (IL-1), IL-6, and TNF-α] in individuals diagnosed with ASD was higher than that of controls . Given these findings, although simultaneous proinflammatory cytokines were not examined in our study, it may be suggested that the low levels of occludin in children with ASD in our study may be the result of increased inflammation. However, this assumption needs to be supported by looking at the simultaneous levels of proinflammatory cytokine and occludin. In addition, the results of our study should be investigated in more detail with gut biopsy samples, in vivo and in vitro studies in a larger sample.
Another biomarker, zonulin has been suggested for use as a serologic marker for the evaluation of epithelial barrier integrity. Zonulin is produced by enterocytes under the influence of environmental stimuli , and mediates increased intestinal permeability through TJs [14, 15]. The results of previous clinical studies on zonulin levels in ASD are inconsistent. There are studies reporting that serum zonulin levels are higher in children with ASD compared with typically developing children [28, 30], as well as studies that reported that they were not different . In a recent study, in vitro IFN-, IL-17A or zonulin exposure was found to increase the small intestine epithelial barrier and BBB permeability by changing the localization of zonula occludens, claudine-5, and occludin, and it was stated that these findings might help to explain the pathogenesis of neuro-inflammatory diseases associated with disorders in the gut-brain axis . Given the studies reporting that intestinal permeability plays a role in ASD, it may be considered that zonulin could contribute to the development of ASD through this mechanism. However, there was no statistically significant difference between the groups in terms of zonulin levels. This findings suggest that the rates of construction and destruction of zonulin were similar to that of between the ASD and control group.
Finally, since zonulin is a regulator of the TJ proteins, we assumed that the levels of occludin and zonulin would correlate positively with each other. However, no correlation was found between occludin and zonulin levels in our study. Based on our findings, we can infer that the difference in occludin levels was independent of zonulin. We believe that occludin's involvement in both the gut barrier and the BBB may mediate our understanding of the pathogenesis of ASD associated with the gut-brain axis. In addition, clarification of the structure represented by occludin subtypes through genetic research will facilitate the interpretation of these results.
Despite the inclusion of more participants with ASD than in previous studies, our study is still limited. The main limitation of the study is it's cross-sectional rather than longitudinal design. This does not allow us to find any cause-effect for our results. The absence of an examination of simultaneous proinflammatory cytokines, the absence of objective tests of intestinal permeability (lactulose/mannitol ratio tests), and the absence of an examination of intestinal biopsy and epithelial barriers were among the other limitations of our study. Further studies with larger sample sizes are required to reach stronger conclusions. Despite all these limitations, our findings suggest that occludin may play a role in influencing the gut-brain axis in children with ASD.
The serum levels of occludin and zonulin were detected in this study. Our findings have shown that there are alterations of the occludin levels in children with ASD. Further studies are needed to investigate the role of occludin in influencing the gut-brain axis in children with ASD. Previous studies suggest that biological abnormalities may be associated with ASD and these abnormalities may be important in diagnosis, follow-up, and/or treatment as potential biomarkers. The investigation of the mechanism underlying the different levels of occludin between ASD and controls may be of importance in clarifying the etiopathogenesis of ASD, as well as its follow-up and treatment.