角质细胞生长因子-2对右旋葡聚糖硫酸钠诱导的小鼠结肠炎和肠道黏膜屏障的保护作用

Protective role of keratinocyte growth factor 2 on dextran sodium sulfate induced murine colitis and intestinal mucosal barrier

目的探究角质细胞生长因子-2(keratinocyte growth factor 2,KGF-2)对右旋葡聚糖硫酸钠(dextran sodium sulfate,DSS)诱导的小鼠结肠炎和肠道黏膜屏障的保护作用及其作用机制。方法 36只C57BL/c雄性小鼠随机分为正常对照组、DSS模型组、中剂量KGF-2(5mg/kg)组和高剂量KGF-2(10mg/kg)组。除正常对照组外小鼠饮用水中均添加3.5%DSS,KGF-2组同时腹腔注射相应剂量的KGF-2。检测小鼠体重、疾病活动指数(disease activity index,DAI)、结肠长度缩短、结肠组织病理评分评价KGF-2对结肠炎的保护作用,多功能酶标仪检测血清异硫氰酸荧光素葡聚糖(fluorescein isothiocyanate-dextran,FITC-D)肠道渗透率,免疫组化和Western blot检测结肠组织ZO-1和occludin蛋白的表达,ELISA分析结肠组织匀浆的TNF-α、IL-6、IL-10、TGF-1β、IFN-γ、IL-1β浓度,以探讨KGF-2对肠道屏障功能保护作用机制。结果与DSS模型组相比,高剂量KGF-2组结肠DAI显著下降(P=0.021 1),体重减轻明显改善(P=0.017 6),HE评分显著降低(12.17±1.222vs.7.000±0.632 5,P=0.001 1),FITC-D渗透率显著降低(168.5±27.01 vs.14.62±1.812,P=0.004 7),结肠较长(4.956±0.2583 vs.6.289±0.2157,P=0.001 1);ZO-1(0.158 6±0.010 51 vs.0.387 9±0.028 64,P<0.000 1)和occludin(0.300 5±0.026 56 vs.0.445 0±0.056 62,P=0.043 4)蛋白表达水平显著升高;肠道组织TNF-α表达显著降低(68.93±3.379 vs.40.41±1.576,P<0.000 1),而IL-10(304.0±21.47 vs.521.2±49.40,P=0.002 4)和IL-6(3755±309.8 vs.5 640±418.0,P=0.004 7)的表达显著升高,疗效呈剂量依赖性。结论 KGF-2能够减轻DSS导致的肠道黏膜损伤,修复肠道黏膜屏障。KGF-2对肠道黏膜屏障的保护作用可能与增加ZO-1和occludin等紧密连接蛋白的表达、抑制TNF-α及促进IL-6和IL-10分泌有关。

Objective To determine the protective role of keratinocyte growth factor 2（KGF-2）on dextran sodium sulfate（DSS）-induced murine colitis and investigate the mechanism of its effect on intestinal mucosal barrier.Methods A total of 36 male C57BL/c mice were divided into 4groups：normal control group,DSS model group,model＋KGF-2（5mg/kg）group and model＋KGF-2（10mg/kg）group.Groups except the control were added 3.5% DSS in drinking water.Disease activity index（DAI）,weight change,colon length loss and histological score were detected to evaluate the protective effect of KGF-2on colitis.Serum fluorescein isothiocyanate-dextran（FITC-D）permeability were assayed by multiscan spectrum.In order to explore the protective role of KGF-2on murine intestinal mucosal barrier,ZO-1and occludin protein concentration were detected by immunohistochemical staining and Western blot.Meanwhile,cytokines including TNF-α,IL-6,IL-10,TGF-1β,IFN-γand IL-1βin colonic tissue were detected by ELISA. Results Compared with DSS-induced colitis model group,10mg/kg KGF-2significantly reduced DAI（P=0.021 1）,weight loss（P=0.017 6）,colon length loss（4.956±0.2583 vs.6.289±0.215 7,P=0.001 1）,histological score（12.17±1.222 vs.7.000±0.632 5,P=0.001 1）,and FITC-D permeability（168.5±27.01 vs.14.62±1.812,P=0.004 7）and reversed the downregulation of tight junction（TJ）proteins（ZO-1：0.158 6±0.010 51 vs.0.387 9±0.028 64,P〈0.000 1;occluding：0.300 5±0.026 56 vs.0.445 0±0.056 62,P=0.043 4）.Significant decrease of TNF-α（68.93±3.379 vs.40.41±1.576,P〈0.000 1）and increase of IL-6（3 755±309.8 vs.5 640±418.0,P=0.004 7）and IL-10（304.0±21.47 vs.521.2±49.40,P=0.002 4）levels were noted in the 10mg/kg KGF-2treated mice.The effect of the KGF-2was dose-dependent.Conclusions KGF-2could ameliorate DSS-induced colitis and it may be associated with the decrease of the damage of mucosal barrier structure and funcntion by preventing ZO-1and occludin from downregulating.The protective effect of KGF-2on intestinal barrier function may also be exerted by inhibition of TNF-αand stimulation of IL-6and IL-10 secretion.