| AimSevere acute pancreatitis is one of the life-threatening diseases. Previous literature reported the mortality rate of ANP was ranging from 10%-25%. Forty to seventy percent of the patients with pancreatic necrosis developed pancreatic infection, which was considered as the most determinative factor associated with mortality. Small bowel ileus associated with ANP has been validated to participate in the process of infection of pancreatic necrosis and systemic inflammatory response syndrome since it is closely associated with small bowel bacterial overgrowth and the following bacterial translocation. However, the underlying mechanism of the ileus during ANP remains unclear. Gastrointestinal motility disorders in ANP may have multiple causes. They could derive from deficiencies in neurons, smooth muscle cells, and/or interstitial cells of Cajal (ICC). In this study, we adopt the model of L-ornithine-induced pancreatitis for the study of intestinal motility. We assessed the role of ICC, myenteric neurons and the associated mechanism in the pathogenesis of ileus during experimentally induced acute pancreatitis.MethodsANP was induced by intraperitoneal injections of 30% L-ornithine at a dose of 3 g/kg at hourly intervals. We explored the alterations of ICC and enteric nerves 24h,48h,72h after induction of ANP and evaluated the effect of octreotide on the intestinal motility by the methods of fuctional, morphological and molecular biological study. Fuctional study included in vivo small intestine electrical motility(migrating motor complexes, MMCs and slow waves) and in vitro smooth muscle contractions with the organ bath technique. Morphological study was designed to detect the impairment of pancrease, ICC, and the PGP9.5, nNOS, CHAT and SSTR2 immunoreactive enteric neurons. Molecular biological study aimed to explore the protein expression of ICC, nNOS and CHAT under the circumstance of ANP.Results(1) The pancreas from all ANP groups (24h,48h and 72h) exhibited signs of ANP. L-ornithine-induced necrotizing pancreatitis manifested a series of disturbed intestinal motility. (2) The disturbed MMC cycle length(P=0.052), decreased dominant frequency (P = 0.005) and dominant power (P=0.034) of slow waves in vivo, decreased amplitude of spontaneous contractions (P= 0.004) in small intestinal smooth muscle, declined contractile response to ACh (P= 0.05) in vitro were observed in our study. We also found the difference in reactivity to TTX (control, P= 0.045; ANP, P= 0.197) and L-NNA (P =0.042) between ANP and control group. Furthermore, the morphological studies demonstrated the damage of ICC (ANP group vs control, P= 0.000), myenteric neurons (ANP group vs control, P= 0.001) and nNOS immunoreactive neurons (ANP group vs control, P= 0.000). A substantial loss of expression of nNOS protein in muscular layer was also observed in our study (ANP group vs control, P= 0.032).(3) Compared with the basal MMC cycle length, the tendency of alteration of ANP+ octreotide group was significantly different from that of ANP+saline group. In the ANP +saline group, a significant increase in MMC cycles length was observed at 48 h (P= 0.002) or 72 h (P= 0.006) after ANP induction. In contrast, in the ANP+octreotide group, a significant increase was only observed at 24 h after ANP induction (P= 0.002), but not at 48 h (P= 0.14) nor at 72 h time point (P= 0.14). Compared with the ANP+saline group, the ratios of MMC cycle length in 48 h and 72 h to the basal were significantly lower in the ANP+octreotide group (48 h, P= 0.005; 72 h, P= 0.004). Although ANP+saline group showed rhythmic slow waves at all the 3 measured time points, the DF of slow waves was lower than the basal measurement (24 h, P= 0.046; 48 h P= 0.017; 72 h, P= 0.004). Compared with the basal DF of slow waves, the alteration tendency of ANP+octreotide group after ANP induction was different from that of ANP+saline group. The DF in the ANP+octreotide group did not decrease significantly at any time point. The ratios of DF in 48 h or 72 h to the basal were significantly higher in the ANP+octreotide group than the ANP+saline group (48 h, P= 0.021; 72 h, P= 0.001). In the saline and octreotide groups of control rats, dense network of c-Kit-positive cells were observed throughout the deep muscular plexus (DMP) of small intestine. In contrast, in the ANP+saline rats, c-Kit-positive cells were disrupted or their number dropped. Compared with the ANP+ saline groups, the integrated optical density (IOD) of ICC-DMP per field in ANP+ octreotide groups showed a significant lower decrease at 48 h and 72 h (48 h, P= 0.028; 72 h, P= 0.01). Drastic difference in protein expression of c-Kit between ANP+saline group and ANP+octreotide group was observed at 72 h (P= 0.01; n= 8), but not at 24 h or 48 h. (4) The amplitude of the spontaneous contractions significantly decreased in ANP+ saline group at 24 h and 48 h time points, compared with the control+saline group (24 h, P= 0.004; 48 h, P= 0.011). For the three groups, control,24 h and 48 h ANP group, the administration of octreotide appeared to protect spontaneous contractions, compared with the administration of saline (24 h, P= 0.000; 48 h, P= 0.008). Contractions to ACh and L-NNA were also impaired in the ileal muscle from ANP+saline rats, compared with those generated by specimens obtained from control+saline rats. Octreotide also appeared to be a protective factor for those responses after 24h ANP induction, compared with the saline group (ACh, P= 0.002; L-NNA, P= 0.053). The CHAT-positive neurons in all groups of ANP+saline rats significantly decreased (24 h, P= 0.000; 48 h, P= 0.000; 72 h, P= 0.005), while at the same time point, the CHAT-positive neurons in ANP+octreotide group appeared more than those in ANP+saline group (24 h, P= 0.034; 48 h, P= 0.024). The similar alterations happened to the number of nNOS-positive neurons in all groups of ANP+saline rats, compared with the control+saline group (24 h, P= 0.000; 48 h, P= 0.000; 72 h, P= 0.000). And at the same time point, octreotide appeared to protect the nNOS-positive neurons, compared with the saline group (24 h, P= 0.003; 48 h, P= 0.046). At 48 h time point, CHAT protein expression in octreotide groups was obviously higher than that of the saline rats (P= 0.032). However, at 72 h time point after ANP induction the nNOS protein expression appeared higher in ANP+octreotide group than that of the ANP+saline group (P= 0.057)ConclusionsOur results suggested that small bowel ileus exsited under the circumstance of ANP, the pathogenesis of which may involve deficiencies and alterations in ICC and myenteric neurons (nNOS and CHAT nerves). Octreotide administrated during the process of ANP was considered to protect the impairment of intestinal motility by pathways of muscle and enteric neuronal origin. That was, the protection to the ICC, nNOS and CHAT nerves from octreotide, at least in part, ameliorated the small bowel ileus associated with ANP. |
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