| Objective Establish neonatal rat model of necrotizing enterocolitis(NEC) to study the dynamic change of nitric oxide(NO), nitric oxide synthase(NOS) and intestinal cells apoptosis, as well as analyse the correlation with them and the scoring of intestinal injury. These result would provide the experimental evidence for clarifying the pathogenesis of NEC and searching for the effective curative drugs.Methods 40 neonatal Sprague-Dawley rats(48 hours olds,weight:5～10g) were randomly divided into the model group(n=32) and the control group(n=8). Rats in the model group were made into NEC models as follows: separating from mother rats and feeding with rat milk substitute, hypoxiaing for 90 seconds and then exposuring at 4℃condition for 10 minutes, twice a day during 3 consecutive days. After the first hypoxia and cold stimulation, The rats in the model group were sacrificed at these different time points: 24 hours later(M24, n=8), 48 hours later(M48, n=8), 72 hours later(M72, n=8), and 96 hours later(24 hours after the last stimulation, M96, n=8). At the end of the experiment, the rats in the control group were sacrificed. Obtained the intestines of both groups for electron microscopic observation and detected the apoptosis rate of intestinal cells(flow cytometer). Then determined the content of NO and activity of NOS in intestinal tissues by means of chemical chromatometry. The ileocecum proximal intestine of all the rats in both groups was obtained for pathological examination and evaluating the score of intestinal injury. SPSS 11.0 software for Windows was used in all statistical tests.α=0.05 was considered significant.Results [1]After the beginning of modeling, the rats in the model group successively suffered from diarrhoea, abdominal distension, languish, reducing activities, growing slowly; Transmission electron microscopy showed a large number of apoptotic cells in the intestinal mucosa, and some formatted apoptotic bodies. [2]The score of intestinal injury and the apoptosis rate of intestinal cells in the control group were 0.08±0.15 and 4.8±2.9. The score of intestinal injury in the M24, M48, M72, M96 group were 1.38±0.42, 1.46±0.69, 1.58±0.30, 3.33±0.59 and the apoptosis rate of intestinal cells were 12.8±6.3, 14.9±5.5, 17.7±5.5, 27.6±9.9, respectively. The intestinal injury extent was significant positive correlated with the apoptosis rates of intestinal cells ( rapoptosis rate=0.853, p<0.01 ). [3]The content of NO (μmol/g prot) in the intestinal tissues of the control group, M24, M48, M72 and M96 were 0.94±0.44, 2.07±0.38, 2.88±0.32, 3.09±0.40 and 3.98±1.15, the total NOS(tNOS, U/mgprot) in the intestinal tissues were 1.49±0.25, 2.21±0.42, 2.77±0.58, 2.95±0.32 and 3.80±1.08, the inducible NOS (iNOS, U/mgprot) in the intestinal tissues were 0.55±0.23, 1.25±0.27, 1.94±0.46, 2.06±0.18 and 2.86±1.07, respectively. The contents of NO, tNOS, iNOS were significant positive correlated with the average degree of the corresponding intestinal injury(r were 0.865, 0.743, 0.807, respectively. P<0.05).Conclusions Feeding with rat milk substitute and repeated suffered from hypoxia with cold stimulation, the apoptosis rate of intestinal cells were significant increased; the content of NO and the activity of tNOS, especially the activity of iNOS in the intestinal tissues were significant enhanced; with the prolonging of time, the increased of apoptotic intestinal cells,the up-regulated expression of iNOS and the increased content of NO in the intestinal tissue were further aggravated. Maybe the apoptosis of intestinal cells was the pathological basis that contribute to the futher damage of intestine in a neonatal rat of necrotizing enterocolitis (NEC). The abnormal apoptosis of intestinal cell can increase the permeability of the intestinal epithelium, activate the bacterial translocation and the inflammatory cascades that harm for organism. NO play an key role in the injury of NEC intestinal barrier. Continued over-expression of NO and its metabolites will mediated injury of intestinal mucosal and the dysfunction of intestinal barrier, thereby triggering NEC.
|
PDF Full Text Request