Yang Ye, Wang Yang, Wang Cuifang, Wu Shuodong, Yao Dianbo
Department of General Surgery, Shengjing Hospital, China Medical University, Shenyang, China.
Department of Pathology, Shenyang DI'AN Medical Testing Company Limited, Shenyang, Liaoning, China.
Clin Res Hepatol Gastroenterol. 2023 Jan;47(1):102062. doi: 10.1016/j.clinre.2022.102062. Epub 2022 Dec 5.
Hepatolithiasis is prevalent in Southeast Asian regions, and the role of endogenous β-glucuronidase (β-GD) in the formation of hepatolithiasis is being gradually recognised. Revealing the regulation mechanism of the expression of endogenous β-GD will provide new therapeutic strategies for intervening in the formation of hepatolithiasis.
Liver specimens from patients with hepatolithiasis were examined by immunohistochemistry to assess the expression of macrophage markers including CD68, CD80, and CD206, as well as that of TNF-α and endogenous β-GD, compared with that in normal liver samples. HiBEpiC cells were co-cultured directly or indirectly with induced M2 macrophages or directly stimulated with TNF-α, and the expression of the endogenous β-GD was examined. A PKC inhibitor, chelerythrine, and an NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), were used to elucidate the possible regulation mechanism.
The expression of macrophage markers including CD68 and CD206, as well as that of TNF-α and endogenous β-GD significantly increased in liver specimens from patients with hepatolithiasis compared with that in normal liver samples. The expression of CD68, CD206 and TNF-α was positively correlated with that of endogenous β-GD. When HiBEpiC cells were co-cultured directly or indirectly with M2 macrophages, following stimulation with lipopolysaccharide (LPS), the expression of endogenous β-GD was significantly higher in the indirect co-culture group than that in the direct co-culture group, or in HiBEpiC cells or M2 macrophages cultured alone. Further experiments revealed that following stimulation with LPS, TNF-α secretion increased in both the indirect and direct co-culture groups compared with that in HiBEpiC cells cultured alone. TNF-α increased the expression of endogenous β-GD in HiBEpiC cells, in a dose- and time-dependent manner. In addition, TNF-α significantly increased the expression levels of p-P65 and proliferating cell nuclear antigen (PCNA), and PDTC effectively inhibited the TNF-α-induced expression of PCNA and β-GD.
Infiltration of macrophages, especially M2 macrophages, may be involved in the hepatolithiasis formation. LPS activates the macrophages, inducing the secretion of TNF-α, which can further increase the expression of endogenous β-GD in the epithelial cells of the bile duct, possibly via the NF-κB/PCNA signalling cascade.
肝内胆管结石在东南亚地区较为普遍，内源性β-葡萄糖醛酸酶（β-GD）在肝内胆管结石形成中的作用正逐渐被认识。揭示内源性β-GD表达的调控机制将为干预肝内胆管结石的形成提供新的治疗策略。
采用免疫组织化学方法检测肝内胆管结石患者肝脏标本中巨噬细胞标志物CD68、CD80和CD206以及TNF-α和内源性β-GD的表达，并与正常肝脏标本进行比较。将人肝内胆管上皮细胞（HiBEpiC）与诱导的M2巨噬细胞直接或间接共培养，或用TNF-α直接刺激，检测内源性β-GD的表达。使用蛋白激酶C（PKC）抑制剂白屈菜红碱和核因子κB（NF-κB）抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）来阐明可能的调控机制。
与正常肝脏标本相比，肝内胆管结石患者肝脏标本中巨噬细胞标志物CD68和CD206以及TNF-α和内源性β-GD的表达显著增加。CD68、CD206和TNF-α的表达与内源性β-GD的表达呈正相关。当HiBEpiC细胞与M2巨噬细胞直接或间接共培养，经脂多糖（LPS）刺激后，间接共培养组内源性β-GD的表达显著高于直接共培养组，或单独培养的HiBEpiC细胞或M2巨噬细胞。进一步实验表明，经LPS刺激后，间接和直接共培养组的TNF-α分泌均高于单独培养的HiBEpiC细胞。TNF-α以剂量和时间依赖性方式增加HiBEpiC细胞内源性β-GD的表达。此外，TNF-α显著增加p-P65和增殖细胞核抗原（PCNA）的表达水平，而PDTC有效抑制TNF-α诱导的PCNA和β-GD的表达。
巨噬细胞尤其是M2巨噬细胞的浸润可能参与肝内胆管结石的形成。LPS激活巨噬细胞，诱导TNF-α分泌，TNF-α可能通过NF-κB/PCNA信号级联反应进一步增加胆管上皮细胞内源性β-GD的表达。
