| Objectives:1. To observe the pathological changes of lung tissue of neonatal rats after hyperoxia exposure according to the rat model of hyperoxia-induced lung injury Ozdemir[1]established.2. To study the expression of the ROS、p-JNK、apoptosis in neonatal rats’ lung tissue after hyperoxia exposure and the role of ROS 、 p-JNK 、 apoptosis in hyperoxia-induced lung injury..3. To study the effect of N- acetylcysteine on expression of the ROS、p-JNK、apoptosis in neonatal rats’ lung tissue after hyperoxia exposure, and to explore its possible mechanism to provide experimental evidence for becoming drug of early of treatment of neonatal hyperoxia-induced lung injury.Methods:1. Experimental grouping: Totally 72 SD rats of three days old were randomly divided into 3 groups(n=24 in each group): air and normal saline(air control group),hyperoxia and normal saline group(hyperoxia exposure group), NAC and normal saline group(NAC intervention group).2. Model establishment and drug intervention: The hyperoxia exposure group and NAC intervention group were placed in high oxygen container which contains100% oxygen. The air control group was placed in the air in the same room. The NAC intervention group was injected of NAC(150mg/kg) through abdominal cavity from the first day to the fourteenth day. The air control group and the hyperoxia exposure group were injected at the same volume of normal saline through abdominal cavity at the same time point.3. Sample collection and measure target: Eight neonatal rats were decapitated and removed lung tissue from each group at 3 day, 7day, and 14 day of the experiment.The right lung tissue was placed in a small amount of PBS, and then frozen in liquidnitrogen to detect the concentration of ROS by Elisa. The left lung tissue was first placed in the neutral formaldehyde and then paraffin-embedded for HE staining,immunohistochemistry and TUNEL. The pathological changes of lung tissue were observed by HE staining. The expression of p-JNK protein was detected by immunohistochemistry. The Integrated option density of p-JNK protein was calculated then. The apoptosis of lung tissue cells was detected by TUNEL. The apoptotic index was calculated then.Results:1. Pathological changes of lung tissue: Under the light microscope, lung structure was normal in the air control group. The lung tissue showed alveolar congestion edema, less inflammatory cells in the hyperoxia exposure group of 3d.The lung tissue showed widened alveolar septum, serious congestion edema, more inflammatory cell, and disorder structure in the hyperoxia exposure group of 7d. The lung showed decreased congestion edema, inflammation and disorder structure in the hyperoxia exposure group of 14 d. The lung showed decreased congestion edema,inflammation and in the NAC intervention group compared to the hyperoxia oxygen exposure group at each time point, and there was no obvious disorder of lung tissue structure.2. The change of concentration of ROS in lung: The concentration of ROS was higher in the hyperoxia exposure group and NAC intervention group than that in the air control group at each time point(P<0.01). The concentration of ROS was lower in NAC intervention group than that in hyperoxia exposure group at each time point(P<0.01). The concentration of ROS was not significant difference between the time point in the air control group(P>0.05). The concentration of ROS began to increase at 3d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group.There was significant difference between each two time points(P<0.01). The concentration of ROS began to increase at 3d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group.There was significant difference between each two time points(P<0.01).3. The change of expression and distribution of p-JNK protein in lung: Theexpression of p-JNK protein was higher in the hyperoxia exposure group and NAC intervention group than that in the air control group at each time point(P<0.01),and the positive cells were found in alveolar epithelial cells, endothelial cells,macrophages and inflammatory cells. The expression of p-JNK protein was lower in NAC intervention group at each time point than that in hyperoxia exposure group at each time point(P<0.01).The expression of p-JNK protein was not significant difference between the time point in the air control group(P>0.05). The expression of p-JNK protein began to increase at 3d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group.There was significant difference between each two time points(P<0.01). The expression of p-JNK protein began to increase at 3d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group.There was significant difference between each two time points(P<0.01).4. The changes of lung tissue cell apoptosis and distribution: The apoptosis cells increased in the hyperoxia exposure group and NAC intervention group than that in the air control group, and the positive cells were found in alveolar epithelial cells,endothelial cells, macrophages and inflammatory cells. The apoptosis index was higher in the hyperoxia exposure group and NAC intervention group than that in the air control group at each time point(P<0.01). The apoptosis index was lower in NAC intervention group at each time point than that in hyperoxia exposure group at each time point(P<0.01). The apoptosis index was not significant difference between the time point in the air control group(P>0.05). The apoptosis index began to increase at3 d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group. There was significant difference between each two time points(P<0.01). T The apoptosis index began to increase at 3d, reached the peak at 7d, decreased at 14 d in the hyperoxia exposure group.There was significant difference between each two time points(P<0.01).5. Correlation analysis between ROS, p-JNK protein and AI: The ROS, p-JNK and AI were positively correlated in hyperoxia exposure group(P<0.01).Conclusion:1.we have successfully established the model of hyperoxia-induced lung injuryin neonatal rats. The pathological change of lung tissue is alveolar edema,inflammation, enlarged alveolar septum, disorder structure.2. The expression of ROS, p-JNK protein and apoptosis increased in lung tissue of neonatal rats of after hyperoxia exposure, prompted that the rise of ROS-JNKapoptosis pathway may be one of the reasons of by hyperoxia-induced lung injury of neonatal rats3. The ROS, p-JNK and AI were positively correlated in hyperoxia exposure group, prompted that the three factors play a synergistic role in hyperoxia-induced lung injury of neonatal rats.4. NAC can inhibit the production of ROS, reduce the expression of p-JNK protein and apoptosis, which play a protective role in hyperoxia- induced lung injury in neonatal rats. |
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