一氧化氮合酶在新生鼠肠损伤中的作用

The role of nitric oxide synthase in a neonatal rat model of intestinal injury.

目的 建立新生鼠肠损伤模型 ,探讨不同类型一氧化氮合酶在其发生发展中的作用。方法 新生鼠随机分为对照组 8只 ,实验组每一时相点各 8只。将EcoliO5 5 :B5脂多糖 5mg/kg注入新生鼠胃内 ,分别于注入后 3h、6h、12h、2 4h处死动物 ,分离胃肠道。取部分回肠末端组织固定 ,包埋切片 ,常规HE染色 ,光镜下观察其组织学改变 ,进行损伤评分。其余回肠组织用于测定原生型一氧化氮合酶 (constitutivenitricoxidesynthase ,cNOS)和诱生型一氧化氮合酶 (induciblenitricoxidesynthase,iNOS)活性 ,并分别与组织学评分进行相关性分析。结果 实验组 3h、6h、12h、2 4h损伤评分分别为 (1.5 4± 0 .87)、(1.79± 0 .75 )、(1.92± 0 .4 3)、(2 .2 9± 0 .6 0 ) ,均明显高于对照组 (0 .0 8± 0 .15 ) (P <0 .0 5 ) ;6h、2 4hcNOS活性分别为 (34.2 8±7.34)、(2 2 .96± 2 .93)nmol/gprot/min ,显著低于对照组 (47.5 9± 14 .5 5 )nmol/ gprot/min (P <0 .0 5 )。 6hiNOS活性 (6 .73±2 .4 0 )nmol/ gprot/min ,显著低于对照组 (10 .2 7± 3.36 )nmol/ gprot/min(P <0 .0 5 )。实验组 2 4h内回肠组织cNOS活性与其平均损伤程度呈显著负相关 (P <0 .0 0 1) ;iNOS活性与损伤程度无显著相关性 (P >0 .0 5 )。

Objective: To establish a model in neonatal rats of iintestinal injury and to examine the role of nitric oxide synthase. Methods: The newborn rats were randomized into two groups. A 1.9F catheter was inserted into its stomach of each rat, and 5 mg/kg E coli O55:B5 endotoxin (LPS) was instilled (n=32, 8 per time point). All pups were decapitated respectively at 3 h, 6 h, 12 h, and 24 h after the treatment. The segment of distal ileum was harvested, fixed and embedded in paraffin, sliced, and stained with hematoxylin and eosin for histological injury scoring. The rest of the ileum was snap frozen in liquid nitrogen for measurements of constitutive nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) activity with chemical colorimetry. The correlation between the score of histological changes and the activity of cNOS and iNOS was determined. Results: Significant increases in injury scores at 3 h, 6 h, 12 h, 24 h [respectively, (1.54±0.87), (1.79±0.75), (1.92±0.43) and (2.29±0.60)] were observed than the control (0.08±0.15) (P<0.05). cNOS levels at 6h, 24h in experimental subgroups were respectively (34.28±7.34), (22.96±2.93) nmol/gprot/min. They were significantly less than the control (47.59±14.55) nmol/gprot/min (P<0.05).The activity of iNOS in 6h subgroup (6.73±2.40) nmol/gprot/min was significantly less than the control (10.27±3.36) nmol/gprot/min (P<0.05). There seemed inverse correlation between the injury severity and cNOS activity within 24 h (P<0.05). There was no significant correlation between iNOS activity and median intestinal injury score (P>0.05). Conclusion: cNOS and iNOS play different roles in development of intestinai injury. Much caution should be exerted concerning potential clinical use of NOS inhibitors to treat neonatal nectotizing enterocolitis.