Chen Liuying, Fan Huiqian, Chu Huikuan, Du Fan, Chen Yixiong, Hu Lilin, Li Zhonglin, Wang Weijun, Hou Xiaohua, Yang Ling
Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Department of Gastroenterology, General Hospital of the Yangtze River Shipping, Wuhan, China.
J Cell Biochem. 2022 Nov;123(11):1857-1872. doi: 10.1002/jcb.30321. Epub 2022 Aug 29.
Cholangiocyte death accompanied by the progression of primary biliary cholangitis (PBC) has not yet been thoroughly investigated. Thus, we are aimed to explore the role of HSP90 and a potential treatment strategy in cholangiocyte necroptosis. First, we detected the expression of HSP90 and necroptotic markers in liver tissues from patients and mice with PBC by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR). Then, the HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), was administered by intraperitoneal injection to evaluate its therapeutic effect for PBC by IHC, real-time PCR, and western blotting. Human intrahepatic bile duct epithelial cells (HIBECs) were induced to necroptosis by toxic bile acid and lipopolysaccharide (LPS) treatment, and evaluated via Cell Counting Kit-8 and flow cytometry assays. Additionally, 17-DMAG, cycloheximide, and a proteasome inhibitor were used to evaluate the role of HSP90 in cholangiocyte necroptosis. We found that the expression of HSP90 was elevated in the cholangiocytes of patients and mice with PBC, along with higher expressions of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), and phosphorylated-MLKL (p-MLKL). Proinflammatory cytokines and antibody levels of the E2 subunit of pyruvate dehydrogenase complex decreased after treatment with 17-DMAG in PBC mice. Meanwhile, RIPK1, RIPK3, phosphorylated-RIPK3, MLKL, and p-MLKL protein expressions decreased with 17-DMAG treatment. In vitro, 17-DMAG and necrostatin-1 prevented glycochenodeoxycholic acid and LPS-induced necroptosis of HIBECs. Immunoprecipitation and high-performance liquid chromatography-mass spectrometry analysis showed that RIPK1 combined with HSP90. Additionally, the 17-DMAG treatment reduced the RIPK1 half-life. Overall, 17-DMAG might be a potential therapeutic agent for PBC via cholangiocyte necroptosis prevention by accelerating RIPK1 degradation.
原发性胆汁性胆管炎（PBC）进展过程中伴随的胆管细胞死亡尚未得到充分研究。因此，我们旨在探讨热休克蛋白90（HSP90）的作用以及胆管细胞坏死性凋亡的潜在治疗策略。首先，我们通过免疫组织化学（IHC）和实时聚合酶链反应（PCR）检测了PBC患者和小鼠肝脏组织中HSP90和坏死性凋亡标志物的表达。然后，通过腹腔注射给予HSP90抑制剂17-二甲基氨基乙基氨基-17-去甲氧基格尔德霉素（17-DMAG），通过IHC、实时PCR和蛋白质印迹法评估其对PBC的治疗效果。通过有毒胆汁酸和脂多糖（LPS）处理诱导人肝内胆管上皮细胞（HIBECs）发生坏死性凋亡，并通过细胞计数试剂盒-8和流式细胞术检测进行评估。此外，使用17-DMAG、放线菌酮和蛋白酶体抑制剂来评估HSP90在胆管细胞坏死性凋亡中的作用。我们发现，PBC患者和小鼠的胆管细胞中HSP90的表达升高，同时受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK1）、RIPK3、混合谱系激酶结构域样蛋白（MLKL）和磷酸化MLKL（p-MLKL）的表达也更高。PBC小鼠经17-DMAG治疗后，促炎细胞因子和丙酮酸脱氢酶复合体E2亚基的抗体水平降低。同时，17-DMAG处理后RIPK1、RIPK3、磷酸化RIPK3、MLKL和p-MLKL蛋白表达降低。在体外，17-DMAG和坏死抑制因子-1可防止甘氨鹅去氧胆酸和LPS诱导的HIBECs坏死性凋亡。免疫沉淀和高效液相色谱-质谱分析表明，RIPK1与HSP90结合。此外，17-DMAG处理缩短了RIPK1的半衰期。总体而言，17-DMAG可能是一种潜在的PBC治疗药物，通过加速RIPK1降解来预防胆管细胞坏死性凋亡。
