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

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

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、100μg/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100μg/L EGF干预24 h,qPCR检测IL-8水平。L-02细胞经100μg/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、100μg/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100μg/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)。100μg/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。

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)and25,50 and 100μp/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 100μg/L EGF for 24 h,and the levels of IL-8 were detected by qPCR.L-02 cells were treated with 100μg/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 the BA 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(r_(s)=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 25,50,100μg/L EGF intervention.There were no significant differences in the expression levels of IL-8 in HIBEpic and LX-2 cells after100μg/L EGF treatment(P>0.05),but the expression of L-8 was increased in L-02 cells(P<0.01).EGF at 100μg/L also induced the expression of p-ERKl/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.