EGF-ERK1/2-IL-8通路在胆道闭锁患儿Kasai术后胆管炎中的作用机制研究

摘要：目的　明确白细胞介素8（IL-8）在胆道闭锁（BA）患儿中的表达水平及其与肝门空肠吻合术（Kasai术）后胆管炎间的关系，探索调控IL-8的上游信号通路。方法　（1）纳入33例BA患儿（BA组，其中胆管炎组18例，无胆管炎组15例），6例胆道扩张患儿（胆道扩张组）以及36例体检健康婴幼儿（正常对照组）。获取全部BA患儿和健康婴幼儿血清样本，酶联免疫吸附试验（ELISA）检测血清IL-8和表皮生长因子（EGF）表达水平。取6例BA和6例胆道扩张患儿肝组织标本，免疫组化染色检测磷酸化胞外信号调节激酶1/2（p-ERK1/2）活化情况。（2）体外培养人正常肝细胞L-02，给予磷酸盐缓冲液（PBS）和25、50、100 μg/L EGF干预24 h，定量聚合酶链反应（qPCR）检测IL-8水平。细胞系HIBEpic、L-02、LX-2分别给予PBS和100 μg/L EGF干预24 h，qPCR检测IL-8水平。L-02细胞经100 μg/L EGF和（或）5 mmol/L的ERK通路抑制剂KO-947干预后，检测ERK1/2活化情况和IL-8表达水平。结果　（1）BA组血清IL-8水平高于正常对照组，胆管炎组术前血清IL-8水平高于无胆管炎组（P＜0.01）。BA组血清EGF与IL-8水平呈正相关（rs=0.422，P＜0.01）。免疫组化染色显示，BA组肝组织中大量表达p-ERK1/2。（2）PBS和25、50、100 μg/L EGF干预后，L-02细胞IL-8表达呈浓度依赖性增高。3种细胞经100 μg/L EGF处理后，HIBEpic、LX-2细胞IL-8表达水平差异无统计学意义（P＞0.05），L-02细胞IL-8表达升高（P＜0.01）。100 μg/L的EGF还可诱导L-02细胞p-ERK1/2大量表达，添加KO-947干预可逆转上述改变。结论　BA患儿术前血清IL-8表达显著升高，与Kasai术后胆管炎关系密切，EGF可通过ERK1/2信号通路促进肝细胞IL-8表达升高。

Study on the mechanism of EGF-ERK1/2-IL-8 pathway in cholangitis after Kasai procedure in biliary atresia

Abstract: Objective　To investigate the expression level of interleukin-8 (IL-8) in children with biliary atresia (BA) and its relationship with cholangitis after Kasai procedure, and to explore the upstream signaling pathway regulating IL-8. Methods　(1) A total of 33 children with BA (18 cases with cholangitis and 15 cases without cholangitis after Kasai procedure), 6 children with biliary dilatation and 36 healthy children were included in this study. Serum samples were collected from children with BA and healthy infants. The serum expression levels of IL-8 and epidermal growth factor (EGF) were determined by enzyme-linked immunosorbent assay (ELISA). The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by immunohistochemical staining in liver tissue samples from 6 children with BA and 6 children with biliary dilatation. (2) Human normal hepatocytes L-02 were cultured in vitro, and treated with phosphate buffer (PBS) and 25, 50 and 100 μg/L EGF for 24 h. The level of IL-8 was detected by quantitative polymerase chain reaction (qPCR). Cell lines HIBEpic, L-02 and LX-2 were treated with PBS and 100 μg/L EGF for 24 h, and the levels of IL-8 were detected by qPCR. L-02 cells were treated with 100 μg/L EGF and/or 5 mmol/L ERK pathway inhibitor KO-947, and the activation of ERK1/2 and the expression level of IL-8 were detected. Results　(1) The serum level of IL-8 was significantly higher in theBA group than that in the normal control group (P＜0.01), and the serum level of IL-8 was significantly higher in the cholangitis group than that in the non-cholangitis group (P＜0.01). Serum EGF was positively correlated with IL-8 in the BA group (rs=0.422, P＜0.01). Immunohistochemical staining showed that p-ERK1/2 was highly expressed in liver tissues of BA group. (2) The IL-8 expression in L-02 cells increased in a concentration-dependent manner after PBS and 25, 50, 100 μg/L EGF intervention. There were no significant differences in the expression levels of IL-8 in HIBEpic and LX-2 cells after 100 μg/L EGF treatment (P＞0.05), but the expression of L-8 was increased in L-02 cells (P＜0.01). EGF at 100 μg/L also induced the expression of p-ERK1/2 in L-02 cells, and KO-947 could reverse the changes. Conclusion　The serum IL-8 expression is significantly increased in children with BA before surgery, which is closely related to the postoperative cholangitis of Kasai. EGF could promote the expression of IL-8 in hepatocytes through ERK1/2 signaling pathway.