Nilotinib protects the murine liver from ischemia/reperfusion injury

Abstract

Background & aims: The mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK), and p38, mediate liver ischemia/reperfusion (I/R) injury via cell death and inflammatory cytokine expression, respectively. Nilotinib is an orally available receptor tyrosine kinase inhibitor used for chronic myelogenous leukemia that also has in vitro activity against JNK and p38. In this study, we examine its therapeutic potential against hepatic I/R injury.

Methods: The effects of nilotinib on liver I/R injury were tested using a murine model of warm, segmental liver I/R. Serum ALT was measured and livers were analyzed by histology, RT-PCR, Western blot, and flow cytometry. The in vitro effects of nilotinib on hepatocyte and non-parenchymal cell (NPC) MAPK activation and cytokine production were also tested.

Results: Mice receiving nilotinib had markedly lower serum ALT levels and less histologic injury and apoptosis following liver I/R. Nilotinib did not inhibit its known receptor tyrosine kinases. Nilotinib lowered intrahepatic expression of IL-1β, IL-6, MCP-1, and MIP-2 and systemic levels of IL-6, MCP-1, and TNF. Nilotinib reduced NPC activation of p38 MAPK signaling and decreased the recruitment of inflammatory monocytes and their production of TNF. Nilotinib attenuated JNK phosphorylation and hepatocellular apoptosis. In vitro, nilotinib demonstrated direct inhibition of JNK activation in isolated hepatocytes cultured under hypoxic conditions, and blocked activation of p38 MAPK and cytokine production by stimulated NPCs.

Conclusions: Nilotinib lowers both liver JNK activation and NPC p38 MAPK activation and may be useful for ameliorating liver I/R injury in humans.

Figure 1. Nilotinib protects against liver I/R injury

WT mice were treated with vehicle or nilotinib prior to undergoing sham or liver I/R. (A) Serum ALT at serial time points. (B) Representative H&E staining (40× magnification). (C) Histologic injury score based on Suzuki criteria. (D) Ischemic whole liver protein from the indicated time points was assessed for receptor tyrosine kinase expression and phosphorylation. Each lane represents 1 animal. (E) WT mice were treated with vehicle or imatinib prior to undergoing sham or liver I/R. Serum was analyzed for ALT at the indicated time points. Data are representative of 2 experiments with similar results. n = 2–3 mice/group (sham) and 7–8 mice/group per time point (I/R). S – sham. P – phospho. *p < 0.05.

Figure 2. Nilotinib blocks p38 MAPK activation, decreases cytokine production, and modulates leukocyte recruitment to the liver following I/R

WT mice were treated with vehicle or nilotinib prior to undergoing sham or I/R. (A) Ischemic whole liver was analyzed by RT-PCR for cytokine expression. Values are normalized to sham animals. (B) 12h serum cytokines were measured by cytometric bead array. (C) NPCs were isolated as described from sham or ischemic lobes at 1h and 3h and analyzed by western blot for activation of the p38 MAPK pathway. (D) Intrahepatic immune infiltrate in sham animals or ischemic lobes 12h following I/R as determined by flow cytometry. (E) Representative TNF staining on inflammatory monocytes from ischemic lobes at 12h. Data are representative of 2 experiments with similar results. n = 2–3 mice/group (sham) and 7–8 mice/group per time point (I/R). S – sham. P – phospho. *p < 0.05.

Figure 3. Nilotinib attenuates JNK signaling and hepatocellular apoptosis after hepatic I/R

WT mice were treated with vehicle or nilotinib prior to undergoing sham or I/R. (A) Western blot of ischemic whole liver protein for total and phospho-JNK, p38 MAPK, and p44/42 (ERK1/2). Each lane represents 1 animal. (B) Representative immunostaining for phospho-JNK on liver sections (40× magnification). (C) Western blot of ischemic whole liver protein for total and phospho-MKK4. (D) Ischemic whole liver mRNA transcript level for Gadd45β as measured by RT-PCR. Values are normalized to sham animals. (E) Representative TUNEL staining of liver sections from sham livers or ischemic lobes (40× magnification). (F) Western blot of ischemic whole liver nuclear protein extract for total and cleaved PARP. Each lane represents 1 animal. (G) mRNA transcript levels for Bcl-2 and Bax from whole liver as determined by RT-PCR. Values are normalized to sham animals. Data are representative of 2 experiments with similar results. n = 2–3 mice/group (sham) and 7–8 mice/group per time point (I/R). S – sham. P – phospho. *p < 0.05.

Figure 4. Nilotinib mediates direct effects against hepatocyte JNK and NPC p38 MAPK activation in vitro

(A) WT hepatocytes were isolated and pre-treated with vehicle or nilotinib, followed by overnight culture under normoxic or hypoxic conditions. Cell lysates were isolated and analyzed by western blot for phospho-JNK. (B and C) WT NPCs were treated with vehicle or nilotinib prior to stimulation with CpG. (B) Supernatant cytokines were measured by cytokine bead array after overnight culture. (C) Western blot for activation of p38 MAPK after 2h of stimulation with CpG. Data are representative of 2 experiments with similar results. Veh – vehicle. P – phospho. *p < 0.05.