乌司他丁对腹部创伤大鼠肠黏膜屏障及ERK/MAPK通路的影响

Influence of Ulinastatin on Intestinal Mucosal Barrier and ERK/MAPK Access in Rats with Abdominal Trauma

目的:探讨乌司他丁(UST)对腹部创伤大鼠肠黏膜屏障的保护作用及对胞外调节蛋白激酶/酪丝裂原活化蛋白激酶(ERK/MAPK)通路的影响。方法:将60只雄性SD大鼠按照随机数字表法分为UST组、模型组和对照组,每组20只。UST组:造模后立即给予腹腔注射UST(20 000 U·kg^(-1))处理;模型组:造模后立即给予等量0.9%生理盐水腹腔注射处理;对照组:给予等量0.9%生理盐水腹腔注射处理。采用苏木精-伊红(HE)染色法观察肠黏膜形态;采用透射电子显微镜观察肠黏膜超微结构;检测血清二胺氧化酶(DAO)、肠型脂肪酸结合蛋白(I-FABP)、D-乳酸盐(D-lactate)、内毒素(LPS)评估肠黏膜屏障损伤程度;采用荧光定量PCR(qRT-PCR)法检测ERK、P-38MAPKmRNA水平;采用蛋白印迹(WB)法检测ERK、p-38MAPK蛋白水平及磷酸化水平。结果:与对照组比较,腹部创伤模型组大鼠肠黏膜上皮细胞变形且排列疏松,上皮下间隙增大;微绒毛排列稀疏、混乱,胞浆中出现空泡;血清中DAO、I-FABP、D-lactate、LPS含量明显增加,差异有统计学意义(P<0.05);ERK、P-38MAPKmRNA和蛋白水平显著升高(P<0.05);ERK、P-38MAPK磷酸化水平显著增加(P<0.05)。UST作用24 h后,与模型组比较,UST组肠黏膜上皮细胞排列较为规则;肠黏膜绒毛排列紧密、较为规则,胞浆中无空泡出现;血清中DAO、I-FABP、Dlactate、LPS含量显著降低(P<0.05);ERK、P-38MAPKmRNA和蛋白水平明显降低(P<0.05);ERK、P-38MAPK磷酸化水平显著降低(P<0.05)。结论:乌司他丁能有效保护腹部创伤所致肠黏膜屏障损伤,推测其机制是通过抑制ERK和p38-MAPK磷酸化水平,抑制ERK/MAPK信号通路激活,实现这一保护作用。

Objective： To explore the protective effect of ulinastatin （UST） on intestinal mucosal barrier in the rats with abdominal trauma and the effect on the access of extracellular regulated protein kinases/mitogen-activated protein kinase（ERK/MAPK）. Meth- ods： Sixty male Sprague-Dawley rats were randomly divided into UST group, the model group and the control group according to the random number table method with 20 rats in each. The rats in UST group were given intraperitoneal injection of UST （20 000 U ~ kg-1 ） immediately after the modeling. The rats in the model group were injected 0.9% saline immediately after the modeling and the control group was injected O. 9% saline intraperitoneally. The intestinal mucosal morphology was observed by using hematoxylin eosin （HE） staining method, and the ultrastrueture of intestinal mucosa was observed under a transmission electron microscope. The serum levels of DAO, I-FABP, D-lactate and LPS were tested to evaluate the damage extent of intestinal mucosal barrier. The ERK and P- 38MAPKmRNA levels were detected by using a quantitative PCR （qRT-PCR） method. The levels of ERK and p-38MAPK proteins and phosphorylation were tested by using Western blot （WB） method. Results： Compared with those in the control group, intestinal muco- sa epithelial ceils in the abdominal trauma model group deformed and arranged loosely, the subepithelial space increased, the arrange- ment of microvilli was sparse and disordered, and vacuoles appeared in cytoplasm. The serum levels of DAO, I-FABP, D-lactate and LPS increased significantly with statistically significant difference （P 〈0.05 ）. The levels of ERK, P-38MAPKmRNA and protein in- creased significantly （P 〈 0.05 ）, and the levels of ERK and P-38MAPK phosphorylation increased significantly （P 〈 0.05 ）. Com- pared with those in the model group, the intestinal epithelial cells in UST group were more regular after 24-hour UST acting. The villi of the intestinal tract were regular and closely arranged without vacuoles in the cytoplasm. The contents of DAO, I-FABP, D-lactate and LPS in serum decreased significantly （P 〈 0.05 ）. The levels of ERK, P-38MAPKmRNA and protein deereased obviously （P 〈 0.05 ）. The levels of ERK and P-38MAPK phosphorylation decreased significantly （ P 〈 0.05 ）. Conclusion： Ulinastatin can effective- ly protect intestinal mucosal barrier in abdominal trauma, and the mechanism is the inhibition of ERK/MAPK signaling pathway by decreasing ERK and p38-MAPK phosphorylation levels.