School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, Anhui, China; Biopharmaceutical Research Institute, Anhui Medical University, Hefei, 230032, Anhui, China.
Department of Gastroenterology, The First Affiliated Hospital of Anhui Medical University, Hefei, 230022, Anhui, China.
Free Radic Biol Med. 2021 Jan;162:283-297. doi: 10.1016/j.freeradbiomed.2020.10.028. Epub 2020 Oct 28.
Rifampicin (RFP) has been known to be potentially hepatotoxic and often used as an inducer of cholestatic hepatic injury. Here we found that mesencephalic astrocyte-derived neurotrophic factor (MANF), an endoplasmic reticulum (ER) stress inducible protein, is a protector in RFP-induced liver injury. In cholestatic hepatic injury mice induced by RFP, the liver/body ratio and the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) were significantly increased. Meanwhile, the protein and mRNA levels of MANF were remarkably elevated in the liver injury mice. In hepatocyte-specific MANF knockout (HKO) mice, an extra increase in the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels was detected after treatment with RFP. In addition, recombinant human MANF (rhMANF) treatment efficiently reduced the liver/body ratio and serum ALT, AST, ALP, TBA, TBIL, and DBIL levels in RFP-induced liver injury mice. Furthermore, we found there is an increase in the number of the apoptotic cells, detected by TUNEL staining in the liver tissues of HKO mice. Meanwhile, the protein levels of C/EBP-homologous protein (CHOP), Ki67, and the proliferating cell nuclear antigen (PCNA), as well as the mRNA level of Ki67 were elevated after treated with RFP, and these parameters were increased more significantly in HKO mice than that in wild type (WT) controls in RFP-induced liver injury. The rhMANF treatment can rescue the cell apoptosis and reduce the protein and mRNA levels of CHOP, Ki67, and PCNA elevated by MANF deletion and RFP. In HKO mice, immunoglobulin heavy chain binding protein (BIP) and activating transcription factor 4 (ATF4) were predominantly increased after treatment with RFP, which were reduced by rhMANF treatment. Therefore, we conclude that hepatocyte-derived MANF is protective for RFP-induced cholestatic hepatic injury via inhibiting ATF4-CHOP signal activation and subsequent cell apoptosis.
利福平（RFP）已知具有潜在的肝毒性，常被用作胆汁淤积性肝损伤的诱导剂。在这里，我们发现中脑星形胶质细胞衍生的神经营养因子（MANF），一种内质网（ER）应激诱导蛋白，是 RFP 诱导的肝损伤的保护剂。在 RFP 诱导的胆汁淤积性肝损伤小鼠中，肝/体重比和血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、碱性磷酸酶（ALP）、总胆汁酸（TBA）、总胆红素（TBIL）和直接胆红素（DBIL）水平显著升高。同时，肝损伤小鼠中 MANF 的蛋白和 mRNA 水平显著升高。在肝细胞特异性 MANF 敲除（HKO）小鼠中，RFP 处理后检测到肝/体重比和血清 ALT、AST、ALP、TBA、TBIL 和 DBIL 水平额外增加。此外，重组人 MANF（rhMANF）治疗可有效降低 RFP 诱导的肝损伤小鼠的肝/体重比和血清 ALT、AST、ALP、TBA、TBIL 和 DBIL 水平。此外，我们发现 TUNEL 染色检测到 HKO 小鼠肝组织中凋亡细胞数量增加。同时，RFP 处理后，HKO 小鼠中 C/EBP 同源蛋白（CHOP）、Ki67 和增殖细胞核抗原（PCNA）的蛋白水平以及 Ki67 的 mRNA 水平升高，并且这些参数在 HKO 小鼠中比 WT 对照中更显著增加在 RFP 诱导的肝损伤中。rhMANF 治疗可挽救细胞凋亡，并降低 MANF 缺失和 RFP 引起的 CHOP、Ki67 和 PCNA 蛋白和 mRNA 水平升高。在 HKO 小鼠中，RFP 处理后免疫球蛋白重链结合蛋白（BIP）和激活转录因子 4（ATF4）明显增加，rhMANF 处理后减少。因此，我们得出结论，肝细胞源性 MANF 通过抑制 ATF4-CHOP 信号激活和随后的细胞凋亡对 RFP 诱导的胆汁淤积性肝损伤具有保护作用。
