MFG-E8减轻梗阻性黄疸大鼠肠道细胞凋亡和黏膜通透性损害

Milk fat globule-EGF factor Ⅷ attenuates apoptosis and permeability damage of the intestinal mucosa in rats with obstructive jaundice

摘要:

目的 观察梗阻性黄疸大鼠血浆乳脂球上皮生长因子-8（milk fat globule-EGF factor Ⅷ，MFG-E8）水平，小肠组织MFG-E8蛋白表达情况；明确补充MFG-E8能否减轻梗阻性黄疸大鼠肠道细胞凋亡和肠黏膜通透性损害。方法 大鼠梗阻性黄疸模型通过手术结扎胆总管（common bile duct ligation，BDL）建立，32只雄性成年SD大鼠随机分为假手术组（Sham组）、梗阻性黄疸组（BDL组）、MFG-E8治疗组（MFG-E8组）、治疗对照组（Vehicle组）。MFG-E8组和Vehicle组于BDL后第1、3、5天腹腔内注射rmMFG-E8（20 μg/kg）或等体积的生理盐水；建模后第7天收集血及组织标本，Western blot检测血浆MFG-E8水平和肠道MFG-E8蛋白表达；原位末端转移酶标记（TdT-mediated dUTP Nick-End Labeling，TUNEL）法染色观察肠道细胞凋亡情况，Western blot检测肠道Cleaved Caspase-3水平；异硫氰酸荧光素标记葡聚糖（fluorescein isothiocyanate-dextran，FITC-D）法评估肠黏膜通透性，ELISA检测血浆肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）水平。结果 与Sham组（5.675±0.638，1.24±0.157）比较，BDL组血浆MFG-E8水平（3.087±0.272）明显降低（P=0.001），小肠MFG-E8蛋白表达水平（0.484±0.090）明显减少（P=0.000）；与Vehicle组（22.333±5.085，0.887±0.132）比较，MFG-E8组小肠细胞凋亡比例（14.467±3.722）以及Cleaved Caspase-3蛋白表达（0.582±0.145）明显下降（P=0.048，P=0.024）；Vehicle组FITC-D水平（4.515±0.601）以及血浆TNF-α水平（233.84±21.21）较Sham组（1.519±0.346，78.24±13.75）均明显升高（P=0.000，P=0.000），MFG-E8组FITC-D水平（2.826±0.588）较Vehicle组明显降低（P=0.000），同时，血浆TNF-α水平（109.90±24.06）较Vehicle组下降了53%（P=0.000）。结论 补充MFG-E8明显减轻了梗阻性黄疸大鼠小肠细胞凋亡和肠黏膜通透性损害，以及全身炎症反应。

Abstract:

Objective To investigate the level of milk fat globule-EGF factor Ⅷ（MFG-E8） in plasma and the expression of MFG-E8 protein in the small intestine in rats with obstructive jaundice，and to determine whether supplementation of MFG-E8 can reduce apoptosis and permeability damage of the intestinal mucosa in rats with obstructive jaundice.Methods The rat model of obstructive jaundice was established by common bile duct ligation（BDL）. Thirty-two male adult SD rats were randomly divided into sham operation group（sham group），obstructive jaundice group（BDL group），MFG-E8 treatment group（MFG-E8 group），and treatment control group（vehicle group）. The MFG-E8 group and vehicle group were intraperitoneally injected with MFG-E8（20 μg/kg ） or an equal volume of normal saline on the 1st，3rd，and 5th days after BDL. Blood and tissue samples were collected on the 7th day after modeling. Western blot was used to determine plasma MFG-E8 levels，intestinal MFG-E8 protein expression，and intestinal cleaved caspase-3 levels. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was used to observe the apoptosis of intestinal cells. Fluorescein isothiocyanate-dextran（FITC-D） was used to assess intestinal mucosal permeability. Enzyme-linked immunosorbent assay was used to determine plasma tumor necrosis factor alpha（TNF-α） levels.Results Compared with the sham group，the BDL group had significantly decreased plasma MFG-E8 level and MFG-E8 protein expression level in the small intestine（5.675±0.638 vs. 3.087±0.272，P=0.001；1.24±0.157 vs. 0.484±0.090，P=0.000）. Compared with the vehicle group，the MFG-E8 group had significantly decreased apoptosis ratio of small intestinal cells and expression of cleaved caspase-3 protein（22.333±5.085 vs. 14.467±3.722，P=0.048； 0.887±0.132 vs. 0.582±0.145，P=0.024）. The FITC-D and plasma TNF-α levels in the vehicle group were significantly higher than those in the sham group（4.515±0.601 vs. 1.519±0.346，P=0.000；233.84±21.21 vs. 78.24±13.75，P=0.000）. The FITC-D level in the MFG-E8 group（2.826±0.588） was significantly lower than that in the vehicle group（P=0.000）. Compared with the vehicle group，the plasma level of TNF-α（109.90±24.06） in the MFG-E8 group was significantly decreased by 53%（P=0.000）.Conclusion MFG-E8 supplementation significantly attenuates apoptosis and permeability damage of the intestinal mucosa，as well as systemic inflammatory responses in rats with obstructive jaundice.