HSCR is a congenital disorder of the intestinal nervous system, which is characterized by the absence of muscular and submucosal ganglion cells in the distal colon due to the impeded migrations of the neural crest cells, leading to intestinal motility dysfunction and functional obstruction[8, 9]. HAEC is one of the most serious complications of HSCR. HAEC is associated with a variety of risk factors, including family history, concomitant trisomy 21, Bardet-Biedl syndrome, long-segment HSCR, delayed diagnosis of HSCR, and repeated HAEC. The pathogenesis of HAEC is still unclear. It was found that mechanical obstruction, mucosal immunodeficiency, abnormal mucin, and infection were associated with HAEC. Among them, immune cells and cytokines play important roles in the development of HAEC. The gastrointestinal mucosa contains numerous macrophages. These resident macrophages participate in the regulation of inflammatory responses to bacteria and antigens, protect the mucosa from damage by pathogenic microorganisms, and remove dead cells and debris. Therefore, intestinal macrophages are the first line of defense against microbes in the innate immune system to maintain the intestinal microenvironment and normal physiological functions[4, 11].
In HSCR, pathogenic bacteria have the opportunity to enter the lamina propria through the mucosal barrier of injury, activate resident macrophages, and recruit mononuclear cells in the blood to release high levels of cytokines to activate the adaptive immune response, further inducing tissue damage. Macrophages play an important role in inflammatory responses, and their activation is mainly initiated by intestinal lipopolysaccharide (LPS). LPS activates the MAPK and NF-κB signaling pathway by binding to TLR4, which results in the production of cytokines interleukin-6 (IL-6) and TNF-α. NF-κB is a major transcription factor for the expression of numerous cytokines and inflammatory mediators, which plays a key role in the regulation of the expression of many inflammatory factors and growth factors. Phosphorylation of IκBα is an important marker of NF-κB activation. In our study, we found that the expression levels of p-IκBα and IκBα proteins and IκBα mRNA in the dilated segment were higher than those in the narrow segment, which suggested that the inflammation of HSCR mice mainly occurred in the dilated segment.This result was consistent with HE staining.
Gene mutations, regulatory sequences, microRNAs and environmental factors can cause disorders of the intestinal nervous system. In human HSCR, acetylcholine transferase was elevated in the non-ganglionic intestine, but neuronal nitric oxide synthase was reduced in the non-ganglionic intestine. These neurotransmitter changes were also confirmed in HSCR animal models[14, 15]. Our study suggested that the acetylcholine content in the colon of HSCR mice varied in the different location of colon. However, contrary to the location of inflammation, the acetylcholine content in the colon of HSCR mice decreased in the dilated segment but increased in the narrow segment. Agrawal RK et al. found that hyperplastic parasympathetic fibers were distributed in the intestinal wall of the narrow segment of HSCR children,but the cause of parasympathetic fiber hyperplasia was unclear.Parasympathetic nerve fibers can produce a large amount of acetylcholine, which may explain the significant increase in acetylcholine content in the narrow section; however, in the dilated segment, the expansion of the intestinal wall may reduce the distribution density of parasympathetic nerve fibers per unit area, resulting in a relative decrease in acetylcholine content.
The main receptor for acetylcholine on immune cells is α7nAChR, which is expressed in a variety of immune cells[17, 18, 19]. Studiesalso found that the distributions of intestinal cholinergic nerve fibers were tightly associated with macrophages with positive expression of α7nAChR, which laid a morphological foundation for acetylcholine to inhibit the activation of macrophages through α7nAChR. Acetylcholine was found to alleviate inflammation by inhibiting the expression of CD14, TLR4, icam-1, B7.1 and CD40 in inflammatory cells via binding α7nAChR.The mechanism may be to dampen nuclear translocation of NF-κB, activate the Jak2-stat3 signaling cascade and up-regulate of heme oxygenase-1 and PI3K[21, 22, 23]. In this study, we found the inflammation was more severe in the dilated colon of HSCR mice. The signal pathway proteins Jak2, p-jak2, Stat3, and p-Stat3 and the mRNA expression levels of Jak2 and Stat3 in the narrow colon of HSCR mice were higher than those in the dilated segment. The acetylcholine content in the narrow colon was higher than that in the dilated colon, which may inhibit the occurrence of inflammation by activating the jak2-stat3 signaling pathway through α7nAChR.
Macrophages are the targets in the CAP, which play anti-inflammatory roles mainly by activating α7nAChR on macrophages. After α7nAChR activation, the expressions of TNF-α, IL-1β, IL-6, and high-mobility group box-1 were significantly inhibited. This is the main mechanism of its anti-inflammatory effect. Activation of α7nAChR mainly affects the NF-κB pathway and Jak2-Stat3 pathway. First, it rapidly inhibits the activation of NF-κB inhibitory protein kinase, which prevents IκB from being phosphorylated. Therefore, it maintains the inhibition of NF-κB by IκB and further inhibits the production of proinflammatory factors. Second, activation of α7nAChR phosphorylates Jak2, and subsequently activates the downstream transcription factor Stat3. Moreover, stat3 is a key anti-inflammatory transcription factor, and its anti-inflammatory effect is believed to be related to the anti-inflammatory effect of IL-10 rather than directly inhibit the transcription of pro-inflammatory cytokines. Third, the Jak2-stat3 pathway also interacts with the NF-κB signaling pathway to inhibit the production of inflammatory cytokines. In our study, we found that activation of the two major anti-inflammatory pathways of α7nAChR in the narrow segment of HSCR mice was associated with increased acetylcholine content:firstly, it decreased the expression of p-IκBα and IκBα proteins and IκBα mRNA, thereby inhibiting NF-κB; secondly, it increased the expression levels of p-Jak2, Jak2, p-Stat3 and Stat3 proteins and mRNA of Jak2 and Stat3, thereby inhibiting the occurrence of inflammation.