Center for Liver Diseases, Nantong Third People's Hospital, Nantong University, 60 Mid-Youth Road, Nantong, 226006, Jiangsu, China.
Division of Gastroenterology and Hepatology, Shanghai Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200001, China.
Dig Dis Sci. 2020 Feb;65(2):470-479. doi: 10.1007/s10620-019-05758-5. Epub 2019 Aug 3.
Primary biliary cholangitis (PBC) is an autoimmune liver disease characterized by the highly selective autoimmune injury of small intrahepatic bile ducts. Studies reported that the cholangiocytes from PBC patients expressed significantly higher levels of both receptor activator of nuclear factor-kappa B (RANK) and its ligand RANKL. However, the accurate role of RANK/RANKL axis in PBC remains unclear.
Forty patients with PBC were enrolled according to the inclusion criteria. The biochemical parameters (alkaline phosphatase, ALP; gamma-glutamyltransferase, GGT; alanine aminotransferase, ALT; aspartate transaminase, AST; total bilirubin, TB) were collected at baseline and followed-up after 6 months of treatment with ursodeoxycholic acid (UDCA, 15 mg/kg d). Stages of PBC were diagnosed based on liver biopsy histopathology according to Nakanuma's criteria. RANK expression in hepatic tissues was detected by immunohistochemistry. The cellular immunofluorescence method was used to locate the distribution of RANK in the human intrahepatic biliary epithelial cells (HIBECs) cultured in vitro. HIBECs were treated with RANKL at a concentration of 100 ng/ml or transfected with RANK-overexpressing lentivirus (LV-RANK). CCK-8 assay and cell cycle assay were used to detect the cell proliferation. Real-time PCR was used to detect the expression of IL-6, E-cadherin, VCAM, ICAM-1, TNF-α, and CD80.
RANK expression in liver biopsies from early PBC patients (stage I + stage II) was significantly lower than that from advanced PBC patients (stage III + stage IV) (1.7 ± 0.63 vs. 2.3 ± 0.45 scores, P < 0.05). High-RANK patients seemed to have better response to UDCA than low-RANK patients (88.9% vs. 40.9%, P < 0.05). The baseline biochemical parameters between the two groups were comparable. The decline percentages of ALP and GGT after UDCA treatment were more obvious in high-RANK patients than those in low-RANK patients (53.90% ± 9.82% vs. 23.93% ± 6.24%, P < 0.05; 74.11% ± 7.18% vs. 48.00% ± 8.17%, P < 0.05, respectively). HIBECs proliferation was significantly inhibited after treatment of RANKL or transfection with LV-RANK. Increased expression of IL-6 and E-cadherin was observed in HIBECs treated with RANKL or LV-RANK.
The overall hepatic RANK expression was associated with disease severity and biochemical response in PBC patients. Activation of RANK/RANKL signaling pathway inhibited cholangiocytes proliferation in vitro. Our study suggested that RANK/RANKL pathway might be a potential target of immunotherapy of PBC based on its involvement in the occurrence and development of the disease.
原发性胆汁性胆管炎（PBC）是一种自身免疫性肝病，其特征为小肝内胆管的高度选择性自身免疫损伤。研究报道，PBC 患者的胆管细胞表达的核因子-κB 受体激活剂（RANK）及其配体 RANKL 水平显著升高。然而，RANK/RANKL 轴在 PBC 中的准确作用仍不清楚。
根据纳入标准，共纳入 40 例 PBC 患者。基线时收集生化参数（碱性磷酸酶、γ-谷氨酰转移酶、丙氨酸氨基转移酶、天冬氨酸氨基转移酶、总胆红素），并在使用熊去氧胆酸（UDCA，15mg/kg/d）治疗 6 个月后进行随访。根据 Nakanuma 标准，通过肝活检组织病理学诊断 PBC 分期。采用免疫组织化学法检测肝组织中 RANK 的表达。采用细胞免疫荧光法定位体外培养的人肝内胆管上皮细胞（HIBECs）中 RANK 的分布。用浓度为 100ng/ml 的 RANKL 或转染过表达 RANK 的慢病毒（LV-RANK）处理 HIBECs。CCK-8 检测和细胞周期检测用于检测细胞增殖。实时 PCR 用于检测白细胞介素 6、E-钙黏蛋白、VCAM、ICAM-1、TNF-α 和 CD80 的表达。
早期 PBC 患者（I 期+II 期）肝活检组织中 RANK 的表达明显低于晚期 PBC 患者（III 期+IV 期）（1.7±0.63 与 2.3±0.45 评分，P<0.05）。高 RANK 患者对 UDCA 的反应似乎优于低 RANK 患者（88.9%与 40.9%，P<0.05）。两组患者的基线生化参数相当。与低 RANK 患者相比，高 RANK 患者经 UDCA 治疗后 ALP 和 GGT 的下降百分比更为明显（53.90%±9.82%与 23.93%±6.24%，P<0.05；74.11%±7.18%与 48.00%±8.17%，P<0.05）。RANKL 处理或转染 LV-RANK 后，HIBECs 的增殖明显受到抑制。用 RANKL 或 LV-RANK 处理的 HIBECs 中观察到白细胞介素 6 和 E-钙黏蛋白的表达增加。
总体肝 RANK 表达与 PBC 患者的疾病严重程度和生化反应相关。RANK/RANKL 信号通路的激活抑制了体外胆管细胞的增殖。我们的研究表明，RANK/RANKL 通路可能是基于其在疾病发生和发展中的作用，成为 PBC 免疫治疗的潜在靶点。
