The Down-Regulation Of Neuroligin-2 And The Correlative Clinical Significance Of Serum GABA Over-expression In Hirschsprung’s Disease

Background and objective:Hirschsprung’s disease (DH) is a congenital disorder and is characterized by colonic stasis due to the absence of enteric neurons in the distal gut. To date, more and more cause and basic research of HD is in progress, but there is a common understanding that HD is a complex disease influenced by multiple genetic factors,such as RET, SOX 10, EDNRB and so on. Neuroligin2 is expressed in CNS and it play an important role in synapse formation, development, specification and synaptic function cycle. Studies have demonstrated that there is bi-directional communication between the ENS and the CNS. To investigate the expression of Neuroligin2 in the different segments of colon in patients with HD, and to explore the correlation of Neuroligin2 with pathogenesis of HD. It has been confirmed that Neuroligin2 is distributed in GABAergic synapses in CNS and the expression level of GABA could reflect the expression level of Neuroligin2.Clinically, there is no serum diagnosis of HD, and whether the serum GABA could be used as a new diagnostic method of HD. So, in this study, we tried to detect the expression level of serum GABA.Methods We collected 52 surgical excision waste tissues and preoperative routine blood collections as the experimental samples from the surgical excision waste tissue and preoperative routine blood collection from Department of Pediatric Surgery, Qilu Hospital, Shandong University for this study.52 patients with indirect inguinal hernia (ⅡH) were regarded as control group. Immunohistochemical staining, Western blotting, and qRT-PCR were applied to detect the expression of Neuroligin2 in different colon segments.We used Enzyme-linked Immunosorbent Assay (ELISA) method to detect the serum GABA.Results Western blotting revealed that Neuroligin2 protein levels in spastic segments (0.012±0.0011) were less than that in transitional segments and ganglionic segments (0.039±0.0014,0.057±0.0018, respectively; P< 0.05). qRT-PCR revealed that Neuroligin2 mRNA levels were consistent with the trend of protein expression. A large number of Neuroligin2 positive gangliocytes were observed in the ganglionic segments, while none was observed in spastic segments. The level of serum GABA was significantly higher in HD (23.69±2.30)than that in ⅡH(7.65±1.16,P< 0.05).Conclusion Neuroligin2 are down-regulated significantly in spastic colon segments, which may be associated with the pathogenesis of HD. The expression level of serum GABA coincided with neuroligin-2 may be considered as a new diagnostic method of HD.