肝吸虫(granulin)是肝吸虫体外/分泌产物中的一种多功能生长因子，可促进胆管癌细胞的转移。然而，它对人体肝内胆管上皮细胞（HIBECs）的影响尚不清楚。在这里，我们研究了肝吸虫(granulin)对HIBECs恶性转化及其潜在机制的影响。通过EdU-488纳入实验、集落形成实验、愈合实验、Transwell实验和西方印迹法评估了CsGRN处理后HIBECs的恶性转化表型。用西方印迹、免疫组织化学染色和苏木精-伊红染色检测CsGRN处理小鼠的胆道损伤。在体外和体内分别通过流式细胞术、免疫荧光和免疫组织化学方法分析了巨噬细胞（人单核细胞白血病细胞系（THP-1））的表型。建立了一个共培养系统来探究THP-1和HIBECs在含有CsGRN的培养基中的相互作用。酶联免疫吸附试验和西方印迹法用于检测白细胞介素6（IL-6）的活化、信号转导子和活化因子3（p-STAT3）的磷酸化以及有丝分裂原激活蛋白激酶/细胞外信号调节激酶（MEK/ERK）途径。使用MEK/ERK通路抑制剂PD98059，确定这个通路是否参与CsGRN介导的细胞相互作用以及STAT3磷酸化和HIBECs的恶性转化。CsGRN处理后，在体内和体外观察到了HIBECs的过度增生和异常增殖、肝脏促炎性细胞因子和趋化因子的增分泌以及胆道损伤。相比对照组，CsGRN处理的THP-1细胞和胆管组织中M2型巨噬细胞标记物的表达显著增加。此外，在THP-1-HIBECs共培养组中，HIBECs在经过CsGRN处理后经历了恶性转化。另外，观察到在CsGRN处理的共培养液中高水平表达了IL-6，这激活了STAT3、JAK2、MEK和ERK的磷酸化。然而，使用MEK/ERK通路抑制剂PD98059减少了CsGRN处理的HIBEC中p-STAT3的表达，并进一步抑制了HIBECs的恶性转化。我们的研究结果表明，通过诱导巨噬细胞的极化和激活IL-6/JAK2/STAT3和MEK/ERK通路，CsGRN促进了HIBECs的恶性转化。

Clonorchis sinensis granulin (CsGRN), a component of the excretory/secretory products of this species, is a multifunctional growth factor that can promote the metastasis of cholangiocarcinoma cells. However, the effect of CsGRN on human intrahepatic biliary epithelial cells (HIBECs) is unclear. Here, we investigated the effect of CsGRN on the malignant transformation of HIBECs and its possible underlying mechanism.The malignant transformation phenotypes of HIBECs after CsGRN treatment were estimated by EdU-488 incorporation assay, colony formation assay, wound-healing assay, Transwell assay and western blot. The biliary damage of CsGRN-treated mice was detected by western blot, immunohistochemical staining and hematoxylin and eosin staining. The phenotypes of the macrophages [human monocytic leukemia cell line (THP-1)] were analyzed by flow cytometry, immunofluorescence and immunohistochemistry, both in vitro and in vivo. A co-culture system was developed to explore the interaction between THP-1 and HIBECs in CsGRN-containing medium. Enzyme-linked immunosorbent assay and western blot were used to detected the activation of interleukin 6 (IL-6), phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. An inhibitor of the MEK/ERK pathway, PD98059, was used to determine whether this pathway is involved in CsGRN-mediated cell interactions as well as in STAT3 phosphorylation and malignant transformation of HIBECs.Excessive hyperplasia and abnormal proliferation of HIBECs, enhanced secretion of hepatic pro-inflammatory cytokines and chemokines, as well as biliary damage, were observed in vitro and in vivo after treatment with CsGRN. The expression of the markers of M2 macrophages significantly increased in CsGRN-treated THP-1 cells and biliary duct tissues compared with the controls. Moreover, following treatment with CsGRN, the HIBECs underwent malignant transformation in the THP-1-HIBECs co-culture group. In addition, high expression of IL-6 was observed in the CsGRN-treated co-culture media, which activated the phosphorylation of STAT3, JAK2, MEK and ERK. However, treatment with an inhibitor of the MEK/ERK pathway, PD98059, decreased expression of p-STAT3 in CsGRN-treated HIBECs and further repressed the malignant transformation of HIBECs.Our results demonstrated that, by inducing the M2-type polarization of macrophages and activating the IL-6/JAK2/STAT3 and MEK/ERK pathways in HIBECs, CsGRN promoted the malignant transformation of the latter.© 2023. The Author(s).