Fig. 3.

Disruption of myeloid-specific TXNIP promotes CYLD and activates the NRF2 pathway in IR-stressed liver.

The WT, TXNIP^FL/FL, and TXNIP^M-KO mice were subjected to 90 min of partial liver warm ischaemia, followed by 6 h of reperfusion. (A) Western-assisted analysis and relative density ratio of CYLD, NOX4, and NRF2 in IR-stressed livers. (B) Western-assisted analysis and relative density ratio of CYLD and NRF2 in the TXNIP^FL/FL and TXNIP^M-KO livers after IR stress. (C) Quantitative RT-PCR analysis of NQO1, GCLC, and GCLM mRNA levels in ischaemic livers (n = 6 samples/group). (D) Immunofluorescence staining of CYLD and CD68 in ischaemic livers (n = 6 mice/group). Scale bars, 100 μm. (E) The Kupffer cells were isolated from ischaemic livers, and then these cells were cultured for 2 h at 37 °C. Western-assisted analysis and relative density ratio of CYLD and nuclear NRF2 in Kupffer cells from the TXNIP^FL/FL and TXNIP^M-KO livers after IR stress. (F) The Kupffer cells (2 × 10⁵/each group) were isolated from ischaemic livers and then cultured for 2 h. Detection of ROS production by Carboxy-H2DFFDA in Kupffer cells from the TXNIP^FL/FL and TXNIP^M-KO livers after IR stress. Quantification of ROS-producing Kupffer cells (green) (n = 6 mice/group). Scale bars, 200 μm. All Western blots represent 4 experiments, and the data represent the mean ± SD. Statistical analysis was performed using the Permutation t test. ∗∗p <0.01, ∗∗∗p <0.005. CYLD, cyclindromatosis; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, glutamate-cysteine ligase regulatory subunit; H2DFFDA, 2',7'-dihydrofluorescein diacetate; IR, ischaemia/reperfusion; NOX4, NADPH oxidase 4; NQO1, NAD(P)H quinone dehydrogenase 1; NRF2, nuclear factor (erythroid-derived 2)-like 2; ROS, reactive oxygen species; TXNIP, thioredoxin-interacting protein; TXNIP^FL/FL, floxed TXNIP; TXNIP^M-KO, myeloid-specific TXNIP knockout; WT, wild type.