MiR-125b-5p ameliorates ventilator-induced lung injury in rats by suppressing ferroptosis via the regulation of the Keap1/Nrf2/GPX4 signaling pathway

Abstract

Ventilator induced lung injury (VILI) is caused by improper use of mechanical ventilation, and its pathogenesis remains unclear. The aim of this study was to establish animal and cell models of VILI, and to explore the mechanism of miR-125b-5p in alleviating VILI by inhibiting ferroptosis through targeted regulation of Keap1/Nrf2/GPX4 axis. Firstly, ferrostain-1(Fer-1), a ferroptosis inhibitor, was used to confirm that ferroptosis was involved in the progression of VILI. Secondly, overexpression and knockdown of miR-125b-5p were performed to validate its function; Further, mechanistically, miR-125b-5p targets negatively regulated Keap1 to activate Nrf2 and then increased the expression of GPX4, thereby inhibiting the occurrence of ferroptosis. Finally, the rescue experiment shows, overexpression of Keap1 and use of the GPX4 inhibitor RSL3 reversed the miR-125b-5p effect, respectively. Through real-time quantitative polymerase chain reaction (qRT-PCR), western blotting (WB), immunofluorescence (IF), hematoxylin and eosin (H&E), and iron death related factor detection, it was confirmed that, overexpression of miR-125b-5p upregulates ferroptosis inhibitory protein and downregulates ferroptosis promoting protein, leading to alleviation of lung injury. However, overexpression of Keap1 and RSL3 reverses the effect of miR-125b-5p, respectively. Therefore, miR-125b-5p can inhibit ferroptosis and alleviate lung injury in VILI rats by targeting the Keap1/Nrf2/GPX4 axis, miR-125b-5p may be a potential intervention target for VILI.

Keywords: Ferroptosis; GPX4; Keap1; Ventilator-induced lung injury; miR-125b-5p.

Fig. 1

Ferroptosis is involved in the progression of VILI in rats. (A) Observation of the lung tissue with the naked eye. (B) Wet/dry weight ratio of the lung tissue. (C) Total protein content in BALF. (D) Lung tissue HE staining (200 × and 400 ×) based on observation with a light microscope and lung injury score. (E) Protein expression of GPX4, SLC7A11 and ACSL4 were detected by western blotting, with β-actin as the internal control. (F) DAB-enhanced Perls’ Prussian blue staining was used to evaluate the content of lung tissue ferric ions. Detection of biochemical indicators of ferroptosis: (G) GSH, (H) MDA, (I) Tissue iron. (J) Immunofluorescence microscopy analysis of the distribution of fluorescence labeled GPX4 protein. (***P < 0.001. ****P < 0.0001.) VILI, ventilator-induced lung injury; BALF, bronchoalveolar lavage fluid; HE, Hematoxylin and eosin; ACSL4, Acyl-CoA synthetase long-chain family member 4; GPX4, Glutathione peroxidase-4; SLC7A11, Solute carrier family 7 member 11; GSH, Glutathione; MDA, malondialdehyde.

Fig. 2

Ferroptosis is involved in the progression of VILI in rats. (A) Observation of the lung tissue with the naked eye. (B) Wet/dry weight ratio of the lung tissue. (C) Total protein content in BALF. (D) Lung tissue HE staining (200 × and 400 ×) based on observation with a light microscope and lung injury score. (E) Protein expression of GPX4, SLC7A11 and ACSL4 were detected by western blotting, with β-actin as the internal control. (F) DAB-enhanced Perls’ Prussian blue staining was used to evaluate the content of lung tissue ferric ions. Detection of biochemical indicators of ferroptosis: (G) GSH, (H) MDA, (I) Tissue iron. (J) Immunofluorescence microscopy analysis of the distribution of fluorescence labeled GPX4 protein. (*P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001.) VILI, ventilator-induced lung injury; BALF, bronchoalveolar lavage fluid; HE, Hematoxylin and eosin; ACSL4, Acyl-CoA synthetase long-chain family member 4; GPX4, Glutathione peroxidase-4; SLC7A11, Solute carrier family 7 member 11; GSH, Glutathione; MDA, malondialdehyde.

Fig. 3

Overexpression of miR-125b-5p alleviates ferroptosis and lung tissue injury in VILI rats. (A,B) Expression of miR-125b-5p in the lung tissue of rats based on qRT-PCR. (C) Observation of the lung tissue with the naked eye. (D) Wet/dry weight ratio of the lung tissue. (E) Total protein content in BALF. (F) Lung tissue HE staining (200 × and 400 ×) based on observation with a light microscope and lung injury score. (**P < 0.01.***P < 0.001.****P < 0.0001). VILI, ventilator-induced lung injury; BALF, bronchoalveolar lavage fluid; HE, Hematoxylin and eosin; qRT-PCR, real-time quantitative polymerase chain reaction.

Fig. 4

Overexpression of miR-125b-5p alleviates ferroptosis and lung tissue injury in VILI rats. (A) Protein expression of GPX4, SLC7A11 and ACSL4 were detected by western blotting, with β-actin as the internal control. (B) DAB-enhanced Perls’ Prussian blue staining was used to evaluate the content of lung tissue ferric ions. Levels of GSH (C), MDA (D) and total iron (E) in the lung tissues. (F) GPX4 immunofluorescent staining results in lung tissue of miR-125b-5p treated VILI rats. (**P < 0.01.***P < 0.001.****P < 0.0001). VILI, ventilator-induced lung injury; ACSL4, Acyl-CoA synthetase long-chain family member 4; GPX4, Glutathione peroxidase-4; SLC7A11, Solute carrier family 7 member 11; GSH, Glutathione; MDA, malondialdehyde.

Fig. 5

Ferroptosis was present in MS-induced injury cell models. (A) Protein expression of SLC7A11, GPX4 and ACSL4 in ATII cells were detected by western blotting, with β-actin as the internal control. (B) ROS was detected by immunofluorescence. Green: ROS. Magnification: 40x. Content of GSH (C), MDA (D) and cell iron (E) in MS-induced injury cell model. (**P < 0.01. ***P < 0.001. ****P < 0.0001). MS, mechanical stretch; SLC7A11, Solute carrier family 7 member 11; GPX4, Glutathione peroxidase-4; ACSL4, Acyl-CoA synthetase long-chain family member 4; ROS, Reactive oxygen species; GSH, Glutathione; MDA, malondialdehyde.

Fig. 6

Ferroptosis was present in MS-induced injury cell models. (A) Protein expression of SLC7A11, GPX4 and ACSL4 in ATII cells were detected by western blotting, with β-actin as the internal control. (B) ROS was detected by immunofluorescence. Green: ROS. Magnification: 40×. Content of GSH (C), MDA (D) and cell iron (E) in MS-induced injury cell model. (**P < 0.01. ***P < 0.001. ****P < 0.0001). MS, mechanical stretch; SLC7A11, Solute carrier family 7 member 11; GPX4, Glutathione peroxidase-4; ACSL4, Acyl-CoA synthetase long-chain family member 4; ROS, Reactive oxygen species; GSH, Glutathione; MDA, malondialdehyde.

Fig. 7

Overexpression of miR-125b-5p alleviates MS induced injury to ATII cells by inhibiting ferroptosis. (A,B) mRNA levels of miR-125b-5p in MS-induced injury cell was detected by qRT-qPCR. (C) Protein expression levels of SLC7A11, GPX4 and ACSL4 in MS-induced injury cell were measured by western blotting, with β-actin as the internal control. (D) ROS detected by immunofluorescence in MS-induced injury cell model. Green: ROS. Magnification: 40×. Detection of biochemical indicators of GSH (E), MDA (F) and total iron (G) in MS-induced injury cell models. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. MS, mechanical stretch; ATII, Type II alveolar epithelial cell; qRT-PCR, real-time quantitative polymerase chain reaction. SLC7A11, Solute carrier family 7 member 11; GPX4, Glutathione peroxidase-4; ACSL4, Acyl-CoA synthetase long-chain family member 4; ROS, Reactive oxygen species; GSH, Glutathione; MDA, malondialdehyde.

Fig. 8

MiR-125b-5p alleviated ferroptosis via upregulating GPX4 in in MS-induced injury cell models. (A) concentration gradient for the GPX4 inhibitor RSL3 by western blotting. (B) Protein expression levels of SLC7A11, GPX4 and ACSL4 in MS-induced injury cell were measured by Western blotting, with β-actin as the internal control. (C) ROS detected by immunofluorescence in MS-induced injury cell model. Green: ROS. Magnification: 40×. Detection of biochemical indicators of GSH (D), MDA (E) and total iron (F) in MS-induced injury cell models. *P < 0.05. **P < 0.01. ***P < 0.001. ****P < 0.0001. MS, mechanical stretch; SLC7A11, Solute carrier family 7 member 11; GPX4, Glutathione peroxidase-4; ACSL4, Acyl-CoA synthetase long-chain family member 4; ROS, Reactive oxygen species; GSH, Glutathione; MDA, malondialdehyde.

Fig. 9

Keap1 was a target of miR-125b-5p. (A) Prediction of the binding site between miR-125b-5p and Keap1 using the online bioinformatics website. (B) Structure and luciferase result of dual luciferase reporter gene. (*****P < 0.0001).

Fig. 10

Keap1 was a target of miR-125b-5p. (A,D) Level of miR-125b-5p based on qRT-PCR. (B,C,E–J) Protein and mRNA expression of Keap1, with β-actin as the internal control. (**P < 0.01. ***P < 0.001. ****P < 0.0001). qRT-PCR, real-time quantitative polymerase chain reaction.

Fig. 11

MiR-125b-5p inhibits ferroptosis by targeting the Keap1/Nrf2/GPX4 axis, thereby alleviating VILI. (A,B) expression of miR-125b-5p and Keap1 mRNA in the lung tissue of rats in the different groups were determined by qRT-PCR. (C) Protein expression of Keap1 was detected by western blotting, with β-actin as the internal control. (D) Observation of the lung tissue with the naked eye. (E) Wet/dry weight ratio of the lung tissue. (F) Total protein content in BALF. (G) Lung tissue HE staining (200 × and 400 ×) based on observation with a light microscope and lung injury score. (**P < 0.01. ***P < 0.001. ****P < 0.0001). VILI, ventilator-induced lung injury; BALF, bronchoalveolar lavage fluid; HE, Hematoxylin and eosin; qRT-PCR, real-time quantitative polymerase chain reaction.

Fig. 12

MiR-125b-5p inhibits ferroptosis by targeting the Keap1/Nrf2/GPX4 axis, thereby alleviating VILI. (A) Protein expression of Keap1, GPX4, SLC7A11 and ACSL4 were detected by western blotting, with β-actin as the internal control. (B) Protein expression level of Nrf2 was measured by Western blotting, with Histone H3 as the internal control. (C,D) Immunofluorescence microscopy analysis of the distribution of fluorescence labeled Keap1 and GPX4 protein. (E) DAB-enhanced Perls’ Prussian blue staining was used to evaluate the content of lung tissue ferric ions. Detection of biochemical indicators of ferroptosis: (F) GSH, (G) MDA, (H) Tissue iron. (**P < 0.01. ***P < 0.001. ****P < 0.0001). VILI, ventilator-induced lung injury; BALF, bronchoalveolar lavage fluid; HE, Hematoxylin and eosin; Keap1, Kelch-like epichlorohydrin-associated protein 1; GPX4, Glutathione peroxidase-4; SLC7A11, Solute carrier family 7 member 11; ACSL4, Acyl-CoA synthetase long-chain family member 4; GSH, Glutathione; MDA, malondialdehyde.

Fig. 13

MiR-125b-5p inhibits ferroptosis by targeting the Keap1/Nrf2/GPX4 axis, thereby alleviating MS-induced injury cell. (A,B) expression of miR-125b-5p and Keap1 mRNA in the different groups of MS-induced injury cell models were determined by qRT-PCR. (C,D) Protein expression of Keap1, GPX4, SLC7A11 and ACSL4 were detected by western blotting, with β-actin as the internal control. (E) Protein expression level of Nrf2 was measured by western blotting, with Histone H3 as the internal control. (**P < 0.01. ***P < 0.001. ****P < 0.0001). MS, mechanical stretch; qRT-PCR, real-time quantitative polymerase chain reaction; Keap1, Kelch-like epichlorohydrin-associated protein 1; GPX4, Glutathione peroxidase-4; SLC7A11, Solute carrier family 7 member 11; ACSL4, Acyl-CoA synthetase long-chain family member 4; Nrf2, Nuclear factor-erythroid2-related factor 2.

Fig. 14

MiR-125b-5p inhibits ferroptosis by targeting the Keap1/Nrf2/GPX4 axis, thereby alleviating MS-induced injury cell. (A) ROS detected by immunofluorescence in MS-induced injury cell model. Green: ROS. Magnification: 40x. Detection of biochemical indicators of GSH (B), MDA (C) and total iron (D) in MS-induced injury cell models. (**P < 0.01. ****P < 0.0001). MS, mechanical stretch; ROS, Reactive oxygen species; GSH, Glutathione; MDA, malondialdehyde.