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Ulinastatin attenuates LPS-induced inflammation in mouse macrophage RAW264.7 cells by inhibiting the JNK/NF-κB signaling pathway and activating the PI3K/Akt/Nrf2 pathway

Authors: Si-tong LI1, Qi DAI1, Shu-xian ZHANG1, Ya-jun LIU1, Qiu-qiong YU2, Fei TAN2, Shu-hong LU1, Quan WANG1, Jian-wen CHEN1, He-qing HUANG1, Pei-qing LIU1, Min LI1
1 School of Pharmaceutical Sciences, National and Local United Engineering Lab of Druggability and New Drugs Evaluation, Sun Yat-Sen University, Guangzhou 510006, China
2 Techpool Bio-Pharma Co, Ltd, Guangzhou 510520, China
Corresponding to: Pei-qing LIU: liupq@mail.sysu.edu.cn, Min LI: limin65@mail.sysu.edu.cn,
DOI: 10.1038/aps.2017.143
Received: 9 August 2017
Accepted: 18 October 2017
Advance online: 11 January 2018

Abstract

Ulinastatin (UTI) is a broad-spectrum serine protease inhibitor isolated and purified from human urine with strong anti-inflammatory and cytoprotective actions, which is widely used for the treatment of various diseases, such as pancreatitis and sepsis. Although the therapeutic effects of UTI are reported to be associated with a variety of mechanisms, the signaling pathways mediating the anti-inflammatory action of UTI remain to be elucidated. In the present study we carried out a systematic study on the anti-inflammatory and anti-oxidative mechanisms of UTI and their relationships in LPS-treated RAW264.7 cells. Pretreatment with UTI (1000 and 5000 U/mL) dose-dependently decreased the mRNA levels of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, iNOS) and upregulated anti-inflammatory cytokines (IL-10 and TGF-β1) in LPS-treated RAW264.7 cells. UTI pretreatment significantly inhibited the nuclear translocation of NF-κB by preventing the degradation of IκB-α. UTI pretreatment only markedly inhibited the phosphorylation of JNK at Thr183, but it did not affect the phosphorylation of JNK at Tyr185, ERK-1/2 and p38 MAPK; JNK was found to function upstream of the IκB-α/NF-κB signaling pathway. Furthermore, UTI pretreatment significantly suppressed LPS-induced ROS production by activating PI3K/Akt pathways and the nuclear translocation of Nrf2 via promotion of p62-associated Keap1 degradation. However, JNK was not involved in mediating the anti-oxidative stress effects of UTI. In summary, this study shows that UTI exerts both anti-inflammatory and anti-oxidative effects by targeting the JNK/NF-κB and PI3K/Akt/Nrf2 pathways.
Keywords: ulinastatin; inflammation; LPS; cytokines; JNK; NF-κB; ROS; PI3K/Akt; Nrf2; RAW264.7 cells

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