| Objectives:To investigate the effect of the different dose of rhEPO (recombinant human erythropoietin) on the expression of Bel-2(B-cell lymphoma-2) and caspase-3(cysteine protease-3) in intestinal tissue of neonate rats with NEC (necrotizing enterocolitis).To explore the protective effects and the mechanism of rhEPO in inhibiting intestinal injury of neonatal Sprague-Dewley rat with NEC.Methods:60neonate Sprague-Dewley (SD) rats at the age of48hours,weight5-10g, were randomly selected, both male and female unlimited, and divided into5groups. In control group (n=12), rats stayed with their mothers and were fed normally. In model group (n=12), rats were seperared from their mother,and were fed with rat breast milk replacer4times a day via an orogastric tube and stressed under hypoxia and cold exposure, twice a day of3consecutive days, In the Intervention groups, rats were dealt with hypoxia and cold stress same as rats in model group, at the same time rats were treated with rhEPO mixed in the milk. According to different doses of rhEPO (0.1U/ml、1U/ml and10U/ml), rats were divided into Intervention group1、 group2and group3. The expression of Bcl-2and caspase-3were measured by immunohistochemical test (two step method) and the value of positive expression were analyzed by IOD (integral optical density) image analysis system. SPSS11.5statistical analysis software was used and a=0.05-. P<0.05was statistically significant.Results:1. Manifestation:the newborn rats in control group had normal diet every day without bloating and their growth and development were good; In model group abdominal distention, decreased activity, unresponsiveness occurred in rats24hours after stress exposure and pale skin, decreased skin temperature and respiratory rhythm change appeared in severe cases. The symptoms appeared later and lighter in three intervention groups.2. Changes of body weight:The body weight of each group is as follows(g):control group:1.62+/-0.70, model group:-0.49+/-0.25, intervention group1:-0.23+/-0.25, intervention group2:-0.27+/-0.16, and intervention group3:-0.28+/-0.35. The NEC model group compared to the control group, and the body weight of the model group is negative growth, and there was statistically significant (a’=0.005, P<0.005); The three intervention groups was respectively compared to tihe NEC model group, and the depth of the decline in body weight difference was marked decreased, and there was statistically significant (a’=0.005, P<0.005); Among the three intervention groups, No significant difference of changes of body weight existed between the intervention group1and2(a’=0.005, P>0.005).However,Compared respectively to intervention group3,intervention group1and intervention group2,reductions of body weight were statistically significant (a’=0.005, P≤0.005).3. The survival rate of newborn rats and:The survival rates were as followings respectively:control group:100%(12/12), model group:83.3%(10/12), intervention group1:83.3%(10/12), intervention group2:91.7%(11/12), intervention group3:91.7%(11/12).4. The grades of intestinal Injury and the incidence of NEC:the intestinal tissue pathology score of model group was remarkable higher than control group, and the difference was statistically significant (a’=0.005, P<0.005); Compared with model group, intervention group1, no statistically significant difference existed(a’=0.005, P>0.005).However,Compared respectively to model group, intervention group2and intervention group3, the intestinal injury of rats was significantly different (a’=0.005, P<0.005);no significant difference existed among three intervention groups (a’=0.005, P>0.005). The NEC incidences of newborn rat was as followings respectively:control group:0%, model group:60%, intervention group1:30%, intervention group2:18.2%, intervention group3:9.1%. We found a significant inverse correlation between the dose of EPO and the incidence of NEC in rats (rs=1, P<0.01).5. The IOD of Bel-2and active caspase3protein in rats’gut:There were no significant differences of the IOD of Bel-2protein in gut between the control group and model group (a’=0.005, P>0.005); Compared to model group, the IOD of Bel-2protein in rats’gut of three intervention groups was markedly increased, and there was significant difference (a’=0.005, P<0.005); Among the three intervention groups, There were no significant differences between intervention group1and intervention group2(a’=0.005, P>0.005). Nevertheless, Compared respectively to intervention group3, intervention group1and intervention group2, The IOD of Bel-2protein in rats’gut of intervention group3was significantly increased, and there was statistical significance (a’=0.005, P<0.005). The IOD of active caspase-3protein in rats’gut of model group was more than control group, and the difference was statistically significant (a’=0.005, P<0.005); Compared respectively to model group, intervention group1, group2and group3, the IOD of active caspase-3protein in rats’gut of Three groups was all significantly reduced, and the differences were statistically significant (a’=0.005, P<0.005); Three intervention groups were compared to each other, the IOD of active caspase-3protein in rats’gut of intervention group3was Significantly lower than intervention group1, and there was Statistical differences (a’=0.005, P<0.005). But Compared respectively to intervention group2, intervention group1and group3, there were no statistically significant (a’=0.005, P>0.005).6. The relationship of the expression of Bel-2in intestinal tract of neonatal rats with the dose of rhEPO:There was positive correlation between the expression of Bel-2in intestinal tract of neonatal rats and the dose of rhEPO (rs=0.887, P=0.000).7. The relationship of active caspase3protein in rat gut with dose of rhEPO:There was negative relationship between the IOD of active caspase-3protein in rat gut with the dose of rhEPO (rs=0.872, P=0.000).8. The correlation of IOD of activated caspase3protein in rat gut with damage in intestinal tissue:There was positive correlation between the IOD of activated caspase3protein in rat gut and the pathological damage in intestinal tissue (rs=0.768, P=0.000).Conclusions:1. Artificial feeding, anoxic and cold stimulus could induce intestinal tissue injury in the neonatal SD rats and the pathological changes were close to Necrotizing enterocolitis in humans.2. The results showed that the apoptosis of intestinal cell were involved in the pathogenesis of NEC, and the apoptosis of intestinal cells was positively correlated with the level of the NEC.3. Oral rhEPO could reduce the symptoms, decrease the expression of intestinal active caspase3protein, and up-regulate the expression of Bel-2, and then inhibit the intestinal cell apoptosis and alleviate the pathological damage of rat gut. Finally, it could also reduce the incidence of NEC.4. In this experiment, the optimal dose of rhEPO was10U/ml. |
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