谷氨酰胺预处理对大鼠肠缺血再灌注损伤的保护作用及其对eNOS-NO通路的影响

The protective effect of glutamine pretreatment on intestinal ischemia-reperfusion injury and eNOS/NO levels in rats

目的探讨谷氨酰胺(Gln)预处理对大鼠肠缺血再灌注损伤(IR)后的保护作用及其对内皮型一氧化氮合酶(e NOS)/一氧化氮(NO)信号通路的影响。方法将30只健康雄性Wistar大鼠随机分为假手术(Sham)组、IR组、Gln组,每组10只,Gln组给予谷氨酰胺1 g/(kg·d),连续灌胃7 d。Sham组和IR组以同等剂量生理盐水灌胃7 d。Sham组仅分离肠系膜上动脉(SMA)而根部不夹闭。IR组和Gln组均用无损伤血管夹夹闭SMA根部,30 min后放松血管夹形成再灌注损伤模型。各组大鼠均于制模后24 h采集腹主动脉血和回肠标本。应用HE染色法观察肠黏膜组织形态学改变,ELISA试剂盒双抗体夹心法测定D-乳酸、内毒素含量,硝酸还原酶法检测血清NO的含量,比色法检测血清e NOS、诱导型一氧化氮合酶(i NOS)的含量,荧光定量PCR(RT-PCR)检测大鼠肠组织e NOS、i NOS m RNA表达水平。结果再灌注后,与Sham组相比,IR组肠黏膜绒毛上皮脱落,固有层崩解,部分绒毛顶端出血,腺体明显受损;Gln组表现为肠黏膜绒毛上皮下间隙扩大,但不明显,绒毛轻度水肿,腺体大致正常,固有层轻度水肿。IR组血清D-乳酸、内毒素、i NOS水平及肠组织i NOS m RNA表达水平均高于Sham组和Gln组(P<0.05)。IR组血清NO、e NOS含量及组织中e NOS的m RNA表达量均低于Sham组和Gln组(P<0.05)。结论谷氨酰胺预处理可以减轻肠缺血再灌注后的组织形态学改变及损伤,其机制可能与抑制i NOS表达,增加e NOS表达,从而增加NO活性有关。

Objective To investigate the protective effect of glutamine（Gln） pretreatment on intestinal ischemia-reperfusion （I/R） injury and endothelial nitric oxide synthase （eNOS）/nitric oxide （NO） signaling pathway in rat model. Methods Thirty male Wistar rats were randomly divided into three groups（n=10 for each group）：sham group, I/R group and Gln group. Animals were pretreated with 1 g/（kg·d）Gln by orogastric route for 7 days in Gln group, and normal saline was given to the other two groups in the same dose. Intestinal I/R was induced by 30 min occlusion of the superior mesenteric artery followed by 24 h of reperfusion. After the operation, the intestinal histopathological changes, the plasma endotoxin level, serum D-lactic acid, eNOS, inducible NOS（iNOS）activity and NO levels were detected by ultraviolet spectrophotometer. The mRNA expressions of myocardial eNOS and iNOS were detected by real-time fluorescence quantitative PCR （RT-PCR）. Results After reperfusion, in IR group, extensive epithelial sloughing and mucosal ulceration of villous tips were observed, whereas these findings did not occur in Gln group and sham group. Compared with IR group, the serum NO, eNOS levels and eNOS mRNA expression of intestinal tissue were elevated in Gln group （P〈0.01）, but the plasma endotoxin level, serum D-lactic acid, serum iNOS and intestinal iNOS mRNA expression decreased in IR group（P〈0.05）. Conclusion Glutamine pretreatment has protective effects on intestinal ischemia-reperfusion injury in vivo. The mechanism may be related to the inhibition of iNOS expression and the increased expression of eNOS, thereby increasing NO activity.