A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

Ziqiao Yuan; Zihang Yuan; Muhammad Hasnat; Haoran Zhang; Peishi Liang; Lixin Sun; Zhenzhou Jiang; Luyong Zhang

doi:10.1016/j.apsb.2020.02.009

A new perspective of triptolide-associated hepatotoxicity: the relevance of NF- κ B and NF- κ B-mediated cellular FLICE-inhibitory protein

Acta Pharm Sin B. 2020 May;10(5):861-877. doi: 10.1016/j.apsb.2020.02.009. Epub 2020 Feb 28.

Authors

Ziqiao Yuan¹, Zihang Yuan¹, Muhammad Hasnat^{1

2}, Haoran Zhang¹, Peishi Liang³, Lixin Sun¹, Zhenzhou Jiang^{1

4}, Luyong Zhang^{1

5

6

7}

Affiliations

¹ Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009, China.
² Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore 54000, Pakistan.
³ College of Traditional Chinese Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, China.
⁴ Key Laboratory of Drug Quality Control and Pharmacovigilance (China Pharmaceutical University), Ministry of Education, Nanjing 21009, China.
⁵ Center for Drug Research and Development, Guangdong Pharmaceutical University, Guangzhou 510006, China.
⁶ Jiangsu Center for Pharmacodynamics Research and Evaluation, China Pharmaceutical University, Nanjing 210009, China.
⁷ State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China.

Abstract

Previously, we proposed a new perspective of triptolide (TP)-associated hepatotoxicity: liver hypersensitivity upon lipopolysaccharide (LPS) stimulation. However, the mechanisms for TP/LPS-induced hepatotoxicity remained elusive. The present study aimed to clarify the role of LPS in TP/LPS-induced hepatotoxicity and the mechanism by which TP induces liver hypersensitivity upon LPS stimulation. TNF-α inhibitor, etanercept, was injected intraperitoneally into mice to investigate whether induction of TNF-α by LPS participated in the liver injury induced by TP/LPS co-treatment. Mice and hepatocytes pretreated with TP were stimulated with recombinant TNF-α to assess the function of TNF-α in TP/LPS co-treatment. Additionally, time-dependent NF-κB activation and NF-κB-mediated pro-survival signals were measured in vivo and in vitro. Finally, overexpression of cellular FLICE-inhibitory protein (FLIP), the most potent NF-κB-mediated pro-survival protein, was measured in vivo and in vitro to assess its function in TP/LPS-induced hepatotoxicity. Etanercept counteracted the toxic reactions induced by TP/LPS. TP-treatment sensitized mice and hepatocytes to TNF-α, revealing the role of TNF-α in TP/LPS-induced hepatotoxicity. Mechanistic studies revealed that TP inhibited NF-κB dependent pro-survival signals, especially FLIP, induced by LPS/TNF-α. Moreover, overexpression of FLIP alleviated TP/LPS-induced hepatotoxicity in vivo and TP/TNF-α-induced apoptosis in vitro. Mice and hepatocytes treated with TP were sensitive to TNF-α, which was released from LPS-stimulated immune cells. These and other results show that the TP-induced inhibition of NF-κB-dependent transcriptional activity and FLIP production are responsible for liver hypersensitivity.

Keywords: CIAPs, cellular inhibitor of apoptosis proteins; Etan, etanercept; FADD, FAS-associated protein with death domain; FLIP; FLIP, cellular FLICE-inhibitory protein; IκB-α, NF-κB inhibitor alpha; LDH, lactate dehydrogenase; LPS; LPS, lipopolysaccharide; MLKL, mixed lineage kinase domain like pseudokinase; MPO, myeloperoxidase; NF-κB; PAS, periodic acid-schiff; RIPK1/3, receptor-interacting protein kinase 1/3; TNF-R1, tumor necrosis factor receptor type 1; TNF-α; TNFAIP3, TNF-α-induced protein 3; TP, triptolide; TRADD, TNF receptor-associated death domain; TRAF2, TNF receptor-associated factor 2; Triptolide; XIAP, X-linked inhibitor of apoptosis protein.