Signal transducer and activator of transcription 6 mediates skeletal muscle cell injury in septic mice by regulating ferroptosis

Abstract

To explore the effect of signal transducer and activator of transcription 6 (STAT6) on ferroptosis in skeletal muscle cells in sepsis model and its potential mechanism. Twenty-four 8-week-old male specific pathogen free Kunming mice were divided into normal control group, sham group, sepsis model group and STAT6 inhibitor pretreatment group according to random number table method with 6 mice in each group. A mouse sepsis model was reproduced by cecal ligation and perforation (CLP). In the sham group, the skin of mice was sutured after exposing the cecum tissue. In the STAT6 inhibitor pretreatment group, 10 mg/kg AS1517499 was injected intraperitoneally 1 hour before model reproduction. The sham group and the model group were intraperitoneally injected with the same volume of normal saline. Mice in the normal control group did not receive any operation or drug intervention. The mice were sacrificed 24 hours after model reproduction, and the muscle tissue of hind limb was obtained under sterile condition. Hematoxylin-eosin (HE) staining was used to observe the histopathology with optical microscope, and mitochondrial morphological changes were observed by transmission electron microscopy after double staining with uranium acetate lead citrate. The ferroptosis marker proteins expressions of chitinase-3-like protein 1 (CHI3L1), cyclooxygenase-2 (COX-2), acyl-CoA synthetase long-chain family member 4 (ACSL4), ferritin heavy chain 1 (FTH1), and glutathione peroxidase 4 (GPx4) were detected by Western blotting. Under the optical microscope, the morphology and structure of skeletal muscle tissues in the normal control and sham groups were normal. In the model group, the structure of skeletal muscle tissues was loose, the muscle fiber became smaller and atrophic, inflammatory cell infiltration and even muscle fiber loss were found. Compared with the model group, the structure of skeletal muscle tissues was tight and skeletal muscle atrophy was improved in the STAT6 inhibitor pretreatment group. The ultrastructure of skeletal muscle cell in the normal control and sham groups was normal under transmission electron microscope. The ultrastructure characteristics of skeletal muscle in the model group showed that cell membrane was broken and blister, mitochondria became smaller and membrane density increased, the mitochondrial crista decreased or disappeared, the mitochondrial outer membrane was broken, and the nucleus was normal in size but lacked chromatin condensation. Compared with the model group, the STAT6 inhibitor pretreatment group had a significant improvement in the ultrastructure of muscle cells. Compared with the normal control and sham groups, the protein expressions of CHI3L1, COX-2, ACSL4 and FTH1 in the muscle of the model group were significantly increased, while the protein expression of GPx4 was decreased significantly, indicating that the skeletal muscle cells in the mouse sepsis model showed characteristic mitochondrial injury and abnormal expression of ferroptosis markers. Compared with the model group, the protein expressions of CHI3LI, COX-2, ACSL4 and FTH1 in the STAT6 inhibitor pretreatment group were significantly decreased [CHI3L1 protein (CHI3L1/GAPDH): 0.70±0.08 vs. 0.97±0.09, COX-2 protein (COX-2/GAPDH): 0.61±0.03 vs. 0.83±0.03, ACSL4 protein (ACSL4/GAPDH): 0.75±0.04 vs. 1.02±0.16, FTH1 protein (FTH1/GAPDH): 0.49±0.06 vs. 0.76±0.13, all P < 0.05], while the protein expression of GPx4 was significantly increased (GPx4/GAPDH: 1.14±0.29 vs. 0.53±0.03, P < 0.05). Sepsis can induce ferroptosis in skeletal muscle cells of mice. STAT6 may mediate ferroptosis in mouse skeletal muscle cells by regulating the expressions of COX-2, ACSL4, FTH1 and GPx4, thereby inducing skeletal muscle cell injury in sepsis.