IL-22对酒精性肝纤维化小鼠回肠紧密连接蛋白及Nrf2/HO-1抗氧化通路的影响

Effect of IL-22 on ileal tight junction protein and nrf2/HO-1 antioxidant pathway in mice with alcoholic liver fibrosis

目的探讨白介素22(IL-22)对酒精性肝纤维化小鼠回肠紧密连接蛋白Occludin、ZO-1及核因子相关因子2(nuclear factor-E2-related factor 2,Nrf2)/血红素氧合酶1(heme oxygenase-1,HO-1)抗氧化通路的影响。方法60只清洁级健康雄性C57/6j小鼠采用慢性乙醇喂养加腹腔注射CCL 4建造ALF复合模型,该实验分为正常组(N组)、对照组(C组)、模型组(M组)、IL-22组、IL-22+ML385(Nrf2抑制剂)组,每组各12只。免疫组织化学检测回肠紧密连接蛋白Occludin、ZO-1和Nrf2及其下游基因血红素氧合酶1(heme oxygenase-1,HO-1)、NAD(P)H醌氧化还原酶1[NAD(P)Hquinone oxidoreductase 1,NQO1]的表达水平,免疫荧光检测回肠黏膜上皮Occludin、ZO-1的表达,观察肝脏及回肠病理学变化和肝纤维化程度。结果与C组相比,M组回肠组织Occludin和ZO-1阳性平均吸光度(A)值降低(0.292±0.015、0.237±0.036比0.236±0.025、0.158±0.025,P<0.05),荧光强度明显减弱;Nrf2、NQO1、HO-1阳性平均A值升高(0.141±0.011、0.196±0.019、0.166±0.033比0.172±0.042、0.223±0.011、0.215±0.044,P<0.05);肝组织胶原纤维沉积明显增加(1.723±0.415比19.759±1.319,P<0.05)。与M组相比,IL-22组回肠组织Occludin、ZO-1、Nrf2、NQO1和HO-1阳性蛋白平均A值升高(0.322±0.020、0.289±0.024、0.217±0.012、0.321±0.018、0.273±0.030,P<0.05);Occludin、ZO-1荧光强度明显增强;肝组织胶原纤维沉积减少(10.851±0.951,P>0.05)。与IL-22组相比,IL-22+ML385组Occludin、ZO-1、Nrf2、NQO1和HO-1阳性蛋白平均A值降低(0.248±0.011、0.175±0.030、0.179±0.013、0.229±0.053、0.218±0.030,P<0.05);Occludin、ZO-1荧光强度减弱;肝组织胶原纤维沉积减少(19.264±0.932,P<0.05),ML385可抑制Nrf2/HO-1通路,明显减弱IL-22对ALF小鼠回肠组织损伤的保护作用。上述结果中N组与C组均无显著差异(P>0.05)。结论ALF小鼠回肠紧密连接蛋白表达下降所致的回肠屏障功能障碍可能与氧化应激有关,而IL-22可上调回肠Nrf2/HO-1抗氧化通路,增加回肠上皮细胞间紧密连接蛋白表达,改善回肠屏障功能,进而阻断ALF的发生发展。

Objective To investigate the effects of interleukin 22(IL-22)on tight junction proteins Occludin and ZO-1 in the ileum of mice with alcohol liver fibrosis,as well as its impact on nuclear factor-E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)antioxidant pathway.Methods Sixty clean-grade healthy male C57/6j mice were used to construct a composite model of ALF by feeding with chronic ethanol plus intraperitoneal injection of CCL4.The mice were divided into a normal group(Group N),a control group(Group C),a model group(Group M),an IL-22 group,and an IL-22+ML385(Nrf2 inhibitor)group,with 12 mice in each group.Immunohistochemistry was used to detect the expression levels of ileal tight junction proteins Occludin,ZO-1 and Nrf2,as well as their downstream genes heme oxygenase-1(HO-1)and NAD(P)H quinone oxidoreductase 1[NQO1].Immunofluorescence was used to detect the expression level of Occludin and ZO-1 in the ileal mucosal epithelium.Researchers observed the pathological changes of liver and ileum,as well as the degree of hepatic fibrosis.Results Compared with group C,the mean optical density values of ileal tissue for Occludin and ZO-1 were lower in group M(0.292±0.015,0.237±0.036 vs.0.236±0.025,0.158±0.025,P<0.05),and the fluorescence intensity was significantly weakened.The mean optical density values of Nrf2,NQO1,and HO-1 were higher(0.141±0.011、0.196±0.019、0.166±0.033 vs.0.172±0.042、0.223±0.011、0.215±0.044,P<0.05).The collagen fiber deposition in liver tissue significantly increased(1.723±0.415 vs.19.759±1.319,P<0.05).Compared with the M group,the mean optical density values of Occludin,ZO-1,Nrf2,NQO1 and HO-1 in ileal tissues were higher in the IL-22 group(0.322±0.020,0.289±0.024,0.217±0.012,0.321±0.018,0.273±0.030,P<0.05).The fluorescence intensity of Occludin and ZO-1 was significantly increased.There was a reduction in collagen fiber deposition in liver tissue(10.851±0.951,P>0.05).Compared with the IL-22 group,the mean optical density values of Occludin,ZO-1,Nrf2,NQO1 and HO-1 were lower in the IL-22+ML385 group(0.248±0.011,0.175±0.030,0.179±0.013,0.229±0.053,0.218±0.030,P<0.05).The fluorescence intensity of Occludin and ZO-1 was reduced,and there was a decrease in collagen fiber deposition in liver tissue(19.264±0.932,P<0.05).ML385 inhibited the Nrf2/HO-1 pathway,resulting in a notable reduction in the protective effect of IL-22 on ileal tissue injury in ALF mice.There was no significant difference between group N and group C in any of the above results(P>0.05).Conclusion The dysfunction of ileal barrier,caused by a decrease in the expression of ileal tight junction proteins in ALF mice,may be influenced by oxidative stress.On the other hand,IL-22 enhanced the Nrf2/HO-1 antioxidant pathway in the ileun,leading to increased expression of tight junction proteins in the ileal epithelial cells and improvement in the barrier function of the ileum.As a result,the progression of ALF was inhibited.