[Mechanism of irisin in alleviating sepsis-associated acute kidney injury by inhibiting ferroptosis via the Nrf2/HO-1 signaling pathway].

内容摘要

To investigate the ameliorative effect of irisin on sepsis-associated acute kidney injury (SA-AKI) by inhibiting ferroptosis through the regulation of the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. Sixty C57BL/6 mice were divided into four groups using a random number table method: control group, lipopolysaccharide (LPS) group, LPS+irisin group, and LPS+irisin+ML385 (Nrf2 inhibitor) group, with 15 mice in each group. The SA-AKI model was established by intraperitoneal injection of LPS at a dose of 10 mg/kg, while the control group received an equal volume of normal saline. In the LPS+irisin group, irisin (1 &#x3bc;g/kg) was intravenously injected 30 minutes prior to LPS administration. In the LPS+irisin+ML385 group, ML385 (30 mg/kg) was intraperitoneally injected 60 minutes before LPS injection, followed by irisin injection 30 minutes before LPS. All mice were anesthetized and sacrificed 24 hours after modeling. Blood samples were collected from the eyeballs for serum creatinine (SCr) and blood urea nitrogen (BUN) measurement using an automatic biochemical analyzer. Renal tissues were harvested for the following assessments: the levels of interleukin-6 (IL-6) and tumor necrosis factor-&#x3b1; (TNF-&#x3b1;) were measured by enzyme linked immunosorbent assay (ELISA); histopathological changes were observed under light microscope after hematoxylin-eosin (HE) staining; mitochondrial morphology was observed under transmission electron microscope after uranium acetate staining; protein expressions of glutathione peroxidase 4 (GPX4), acyl-CoA synthetase long-chain family member 4 (ACSL4), Nrf2, and HO-1 were detected by Western blotting; the contents of Fe2+, malondialdehyde (MDA), reduced glutathione (GSH), and relative fluorescence intensity of reactive oxygen species (ROS) were also measured in renal tissues. 1) The levels of SCr, BUN, IL-6, and TNF-&#x3b1; in the LPS, LPS+irisin, and LPS+irisin+ML385 groups were higher than those in the control group (all P<0.05). Compared with the LPS group, the LPS+irisin group showed decreased levels of SCr, BUN, IL-6, and TNF-&#x3b1; [SCr (&#x3bc;mol/L): 31.70&#xb1;3.94 vs. 65.75&#xb1;7.02, BUN (mmol/L): 11.03&#xb1;2.25 vs. 29.23&#xb1;2.58, IL-6 (ng/L): 239.96&#xb1;14.93 vs. 1 080.40&#xb1;20.06, TNF-&#x3b1; (ng/L): 105.85&#xb1;14.92 vs. 299.12&#xb1;19.94, all P<0.05]. However, the levels of these indicators in the LPS+irisin+ML385 group were higher than those in the LPS+irisin group (all P<0.05). 2) Light microscopy revealed that compared with the control group, the LPS, LPS+irisin, and LPS+irisin+ML385 groups exhibited varying degrees of renal tubular dilation, tubular cell shedding, cellular vacuolization, and intratubular cast formation, with the most severe damage observed in the LPS group and the mildest damage in the LPS+irisin group. 3) Transmission electron microscopy showed that compared with the control group, the other three groups displayed varying degrees of mitochondrial enlargement, rounding and swelling, sparse and dissolved matrix, and reduced cristae, with the most pronounced changes in the LPS group, while mitochondrial morphology was only mildly altered in the LPS+irisin group. 4) Compared with the control group, the protein expression of GPX4 was decreased, while the protein expressions of Nrf2, HO-1, and ACSL4 were increased in the LPS, LPS+irisin, and LPS+irisin+ML385 groups (all P<0.05). Compared with the LPS group, the LPS+irisin group exhibited increased expression of GPX4, Nrf2, and HO-1, and decreased expression of ACSL4 (GPX4/GAPDH: 0.68&#xb1;0.07 vs. 0.49&#xb1;0.03, Nrf2/Lamin B: 1.03&#xb1;0.04 vs. 0.82&#xb1;0.06, HO-1/GAPDH: 0.93&#xb1;0.02 vs. 0.66&#xb1;0.04, ACSL4/GAPDH: 0.69&#xb1;0.02 vs. 0.93&#xb1;0.07, all P<0.05). Compared with the LPS+irisin group, the LPS+irisin+ML385 group showed decreased expressions of GPX4, Nrf2, and HO-1, and increased expression of ACSL4 (all P<0.05). 5) Compared with the control group, the levels of MDA, ROS, and Fe2+; were increased, while the level of GSH was decreased in the LPS, LPS+irisin, and LPS+irisin+ML385 groups (all P<0.05). Compared with the LPS group, the LPS+irisin group exhibited decreased levels of MDA, ROS, and Fe2+, and increased level of GSH [MDA (nmol/mg): 9.35&#xb1;2.07 vs. 15.65&#xb1;4.03, ROS (relative fluorescence intensity): 584.26&#xb1;74.48 vs. 1 655.68&#xb1;405.71, Fe2+ (nmol/mg): 9.24&#xb1;3.82 vs. 26.99&#xb1;7.95, GSH (nmol/mg): 91.50&#xb1;7.99 vs. 43.13&#xb1;11.85, all P<0.05]. Compared with the LPS+irisin group, the LPS+irisin+ML385 group showed increased levels of MDA, ROS, and Fe2+, and decreased level of GSH (all P<0.05). Irisin ameliorates LPS-induced SA-AKI in mice by inhibiting ferroptosis through the Nrf2/HO-1 signaling pathway, suggesting that irisin may serve as a potential therapeutic strategy for organ protection in sepsis.