In our study, the 32 mice were randomly divided for four groups (n = 8 per group): (1) room-air-expose (sham), (2) hyperoxia-expose with Sal (Sal + Hyperoxia), (3) hyperoxia-exposed (Hyperoxia), (4) hyperoxia-exposed with Y-320 (an inhibitor of IL-17) (Y-320 + Hyperoxia). The mice exposed to normoxia groups were placed in room air with 21% oxygen, and the mice exposed to hyperoxia were placed in over 90% oxygen for 24 h. The continue exposure to over 90% oxygen was achieved in a self-made airtight box which attached to a medical oxygen cylinder, and the O2 level inside was continuously monitored with O2 analyzer, mice had free access to food and water. In the first three days before exposure to the hyperoxia, mice in the Sal + Hyperoxia group or Y-320 + Hyperoxia group were treated with Sal (100 mg/Kg) or Y-320 (2 mg/Kg) once orally every day, while the rest of groups were given equal isotonic saline. Based on the above experiments, eight 8-week-old KM mice were randomly divided into two groups: Sal + Hyperoxia group and Sal + Hyperoxia + IL-17A group. Sal + Hyperoxia + IL-17A group, mice were i.v. injected with 50 ug/kg of recombinant mouse IL-17A (210-17, Pepro Tech, USA). Animal were sacrificed following reperfusion, and lungs were stored at -80 until further experimental analysis.

The animals were randomly assigned to six groups (7 mice in each) as follows: (I) Normal saline (NS) group, (II) Ast-IV 100 mg/kg (Ast) group, (III) PM2.5 group, (IV) Ast-IV 50 mg/kg + PM2.5 (Ast-L) group, (V) Ast 100 mg/kg + PM2.5 (Ast-H) group, and (VI) Ast-IV 100 mg/kg + erastin 20 mg/kg + PM2.5 (Era) group. Based on our previous results, this study adopted anintraperitoneal injection(i.p.) of Ast-IV (dissolved in normal saline containing 0.1% DMSO for preventive treatment. After all the mice were adaptively fed for 5 days, in the NS and PM2.5 groups, mice received the normal saline containing 0.1% DMSO viai.p.once a day for the next three consecutive days. Similar to the NS group, in the Ast, Ast-H, and Era groups, mice received Ast-IV (100 mg/kg) viai.p. Ast-L group received Ast-IV (50 mg/kg) viai.p. To evaluate the effect of Ast-IV on ferroptosis in PM2.5-induced lung injury, we used the ferroptosis agonist erastin to activate ferroptosisin vivo. In the Era group, mice received erastin (20 mg/kg, 10% DMSO + 40% PEG300 + 5%Tween80 + 45% normal saline) 30 min before each preventive treatment of Ast-IV.

The male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8-10 weeks old, weight = 23-25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 ul of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively.

The male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8-10 weeks old, weight = 23-25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 ul of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively.

C57BL/6J male mice (6-8 weeks) were purchased from Slac Lab Animals (Shanghai, China). The basic principle of the CLP method was to find the caecum through anatomy and puncture at the blind end and extrude the contents into the abdominal cavity. Diffuse peritonitis was formed, and systemic infection appeared in mice. Mice in the control group were only treated with laparotomy.

6-week-Babl/c female mice were randomized to the following three groups of seven mice each: vehicle group, LPS group, Astaxanthin plus LPS group. Astaxanthin plus LPS group mice were pretreated with astaxanthin (20 mg/kg) byi.v injectionfor daily for 7 consecutive days. Astaxanthin was dissolved in 2%DMSO (vol/vol), 40% PEG-400 (vol/vol), 2% Tween 80 (vol/vol), and 56% PBS (vol/vol). On the last day, the mice were intraperitoneally injected with 5 mg/kg LPS or normal saline 2 h after the injection of astaxanthin. After 6 h of LPS stimulation, mice were euthanized to collect the BALF, and lung tissue samples. BALF was collected three times through a tracheal cannula with autoclaved normal saline, instilled up to a total volume of 1.8 ml.

Specific pathogen-free (SPF) male C57BL/6 mice (6-8 weeks old, 20-24 g body weight) were purchased from the Experimental Animal Center, Anhui Medical University (Hefei, China). All mice were randomly divided into five groups (8 mice every group): control group, LPS group, PX+LPS group (administered 20 mg/kg PX), Fe+LPS group (administered 15 mg/kg Fe-citrate (III)), and PX+Fe+LPS group (administered 20 mg/kg PX and 15 mg/kg Fe-citrate (III)). Fe-citrate (III) was dissolved in stroke-physiological saline solution (SPSS). PX was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich), and further diluted in SPSS. Intravenous injection of Fe or/and intraperitoneal injection of PX were performed from day 0 to day 2. At 1 h after the final Fe and PX treatment, the mice were anesthetized with 30 mg/kg of pentobarbital sodium (Beijing Chemical Co., China) and then LPS (10 ug/mouse; InvivoGen, San Diego, CA, USA) or SPSS was injected into the trachea.

The animals were randomly assigned to six groups (7 mice in each) as follows: (I) Normal saline (NS) group, (II) Ast-IV 100 mg/kg (Ast) group, (III) PM2.5 group, (IV) Ast-IV 50 mg/kg + PM2.5 (Ast-L) group, (V) Ast 100 mg/kg + PM2.5 (Ast-H) group, and (VI) Ast-IV 100 mg/kg + erastin 20 mg/kg + PM2.5 (Era) group. Based on our previous results, this study adopted anintraperitoneal injection(i.p.) of Ast-IV (dissolved in normal saline containing 0.1% DMSO for preventive treatment. After all the mice were adaptively fed for 5 days, in the NS and PM2.5 groups, mice received the normal saline containing 0.1% DMSO viai.p.once a day for the next three consecutive days. Similar to the NS group, in the Ast, Ast-H, and Era groups, mice received Ast-IV (100 mg/kg) viai.p. Ast-L group received Ast-IV (50 mg/kg) viai.p. To evaluate the effect of Ast-IV on ferroptosis in PM2.5-induced lung injury, we used the ferroptosis agonist erastin to activate ferroptosisin vivo. In the Era group, mice received erastin (20 mg/kg, 10% DMSO + 40% PEG300 + 5%Tween80 + 45% normal saline) 30 min before each preventive treatment of Ast-IV.

C57BL/6J male mice (6-8 weeks) were purchased from Slac Lab Animals (Shanghai, China). The basic principle of the CLP method was to find the caecum through anatomy and puncture at the blind end and extrude the contents into the abdominal cavity. Diffuse peritonitis was formed, and systemic infection appeared in mice. Mice in the control group were only treated with laparotomy.

A total of 30 male C57/B6J mice (8-10 weeks, 21-23 g) were acquired from Chinese Academy of Medical Sciences (Beijing, China). The sepsis-induced ALI model was constructed by administering LPS intratracheally (5 mg/kg) for 12 h as previously reported. The control groups were given an isovolumetric sterile saline. Then, 12 h after LPS installation, the animals were sacrificed by cervical dislocation under deep anesthesia with an intraperitoneal injection of 2% sodium pentobarbital (60 mg/kg).

6-week-Babl/c female mice were randomized to the following three groups of seven mice each: vehicle group, LPS group, Astaxanthin plus LPS group. Astaxanthin plus LPS group mice were pretreated with astaxanthin (20 mg/kg) byi.v injectionfor daily for 7 consecutive days. Astaxanthin was dissolved in 2%DMSO (vol/vol), 40% PEG-400 (vol/vol), 2% Tween 80 (vol/vol), and 56% PBS (vol/vol). On the last day, the mice were intraperitoneally injected with 5 mg/kg LPS or normal saline 2 h after the injection of astaxanthin. After 6 h of LPS stimulation, mice were euthanized to collect the BALF, and lung tissue samples. BALF was collected three times through a tracheal cannula with autoclaved normal saline, instilled up to a total volume of 1.8 ml.

Male C57BL/6 mice (8 weeks, 23-25 g) were obtained from Beijing Hua Fu Kang Biotechnology Co. LTD (Beijing, China). A total of 96 mice were randomly divided into 6 groups (n = 16 per group): Sham group, LPS group, Fer-1 group, Fe group, Sev group, and Fe + Sev group. Mice were given 5 mg/kg LPS intranasally to construct LPS-induced ALI model. To study the role of ferroptosis in ALI, the mice in the Fer-1 or Fe groups were administered with Fer-1 (0.8 mg/kg; Ferrostatin-1, the inhibitor of ferroptosis) or Fe (8 mg/kg; Fe-citrate (III), ferroptosis inducer) via tail vein injection once a day for 3 consecutive days before treatment with LPS, respectively. To study the effect of Sev on ALI mice, the mice in Sev group were treated with LPS for 2 h, and then Sev, delivered by gaseous admixture (oxygen) at a concentration of 3% via a calibrated vaporizer, was administered via an endotracheal tube for 4 h. In order to study the effect of Sev on ferroptosis, the mice in Fe + Sev group were administered with Fe via tail vein injection once a day for 3 consecutive days, and then treated with LPS and Sev. Sham group was given 0.9% NaCl (containing 0.1% DMSO).

8- to 10-week-old male C57BL/6 mice were administered 2.5 ug/mL lipopolysaccharide (Escherichia coliserotype 0111: B4) via the trachea. After LPS administration for 6, 12, 24 and 48 h, the mice were euthanized and exsanguinated by cardiac puncture, and the lung tissue was removed for subsequent analysis.

The sepsis mouse model was established by cecal ligation and puncture (CLP) of male C57BL/6 mouse (6-8 weeks, Guangzhou Animal Medical Center). Firstly, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 ml/10g). The cecum was found by cutting a 1 cm longitudinally in the abdomen of the mice, and ligating the cecum from the end of the cecum to half the length of the ileocecal valve with 5-0 silk suture. Then a 21 G needle was used to puncture the ligature and the midpoint of the cecum once, and finally the wound was sutured. In the sham operation group, only laparotomy was performed, without ligation and perforation of the cecum. All animal-related experiments in this study were approved by hospital ethics committee according to following the Nation Institutes of Health Guide for the Laboratory Animals Care and Use (approval No. Med-Eth-Re [2022] 168).

The sepsis mouse model was established by cecal ligation and puncture (CLP) of male C57BL/6 mouse (6-8 weeks, Guangzhou Animal Medical Center). Firstly, the mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (0.1 ml/10g). The cecum was found by cutting a 1 cm longitudinally in the abdomen of the mice, and ligating the cecum from the end of the cecum to half the length of the ileocecal valve with 5-0 silk suture. Then a 21 G needle was used to puncture the ligature and the midpoint of the cecum once, and finally the wound was sutured. In the sham operation group, only laparotomy was performed, without ligation and perforation of the cecum. All animal-related experiments in this study were approved by hospital ethics committee according to following the Nation Institutes of Health Guide for the Laboratory Animals Care and Use (approval No. Med-Eth-Re [2022] 168).

The animals were randomly assigned to six groups (7 mice in each) as follows: (I) Normal saline (NS) group, (II) Ast-IV 100 mg/kg (Ast) group, (III) PM2.5 group, (IV) Ast-IV 50 mg/kg + PM2.5 (Ast-L) group, (V) Ast 100 mg/kg + PM2.5 (Ast-H) group, and (VI) Ast-IV 100 mg/kg + erastin 20 mg/kg + PM2.5 (Era) group. Based on our previous results, this study adopted anintraperitoneal injection(i.p.) of Ast-IV (dissolved in normal saline containing 0.1% DMSO for preventive treatment. After all the mice were adaptively fed for 5 days, in the NS and PM2.5 groups, mice received the normal saline containing 0.1% DMSO viai.p.once a day for the next three consecutive days. Similar to the NS group, in the Ast, Ast-H, and Era groups, mice received Ast-IV (100 mg/kg) viai.p. Ast-L group received Ast-IV (50 mg/kg) viai.p. To evaluate the effect of Ast-IV on ferroptosis in PM2.5-induced lung injury, we used the ferroptosis agonist erastin to activate ferroptosisin vivo. In the Era group, mice received erastin (20 mg/kg, 10% DMSO + 40% PEG300 + 5%Tween80 + 45% normal saline) 30 min before each preventive treatment of Ast-IV.

The male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8-10 weeks old, weight = 23-25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 ul of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively.

The male C57BL/6 mice were divided randomly into 4 groups (n = 4 per group, 8-10 weeks old, weight = 23-25 g): the control group receiving 0.9% NaCl (containing 0.1% DMSO), the LPS group receiving LPS plus 0.9% NaCl (containing 0.1% DMSO), the Fer-1 group receiving Fer-1 only, and the LPS + Fer-1 group receiving both Fer-1 and LPS. The LPS-induced ALI model was induced by instilling intratracheally 50 ul of LPS solution (0.2 g/L), then Fer-1 (0.8 mg/kg) was administered after LPS challenge via tail vein injection. The Fer-1 was dissolved in DMSO first, and diluted with 0.9% NaCl. The final concentration of Fer-1 and DMSO was 0.2 mg/ml and 0.1% respectively.

For the models of CS and LPS exposure, mice were anesthetized and intratracheally instilled with CS suspensions (3 mg/50 ul) or LPS (1 mg/kg). For the models of CS + Ferr-1/DFO, mice were intraperitoneally injected with Ferr-1 (1.25 umol/kg) or intranasal instilled with DFO (10 mg/kg) for 7 consecutive days after CS instillation. For the models of LPS + Ferr-1/DFO, mice were pretreated with Ferr-1 or DFO for 2 consecutive days and then intratracheally instilled with LPS. Mice were sacrificed 24 h after LPS instillation. For the X-ray exposure model, mice were exposed to ionizing radiation (IR) at 20 Gy, which was delivered at the dose rate of 2 Gy/min and a source skin distance of 51 cm by an X-ray generator (Model X-RAD320iX; Precision X-Ray, Inc., North Branford, CT, USA), and sacrificed 3 days after radiation.

For the models of CS and LPS exposure, mice were anesthetized and intratracheally instilled with CS suspensions (3 mg/50 ul) or LPS (1 mg/kg). For the models of CS + Ferr-1/DFO, mice were intraperitoneally injected with Ferr-1 (1.25 umol/kg) or intranasal instilled with DFO (10 mg/kg) for 7 consecutive days after CS instillation. For the models of LPS + Ferr-1/DFO, mice were pretreated with Ferr-1 or DFO for 2 consecutive days and then intratracheally instilled with LPS. Mice were sacrificed 24 h after LPS instillation. For the X-ray exposure model, mice were exposed to ionizing radiation (IR) at 20 Gy, which was delivered at the dose rate of 2 Gy/min and a source skin distance of 51 cm by an X-ray generator (Model X-RAD320iX; Precision X-Ray, Inc., North Branford, CT, USA), and sacrificed 3 days after radiation.

We received 60 C57BL/6J mice and 48 Nrf2 knockout (Nrf2-/-) mice of the same genetic background from the RIKEN Bio-Resource Centre via the National BioResource Project, MEXT, Japan. Intestinal ischemia was simulated by clamping the superior mesenteric artery following the intraperitoneal administration of 50 mg/kg sodium pentobarbital. Forty-five minutes later, the intestine was allowed to re-perfuse for 3 h.

For the models of CS and LPS exposure, mice were anesthetized and intratracheally instilled with CS suspensions (3 mg/50 ul) or LPS (1 mg/kg). For the models of CS + Ferr-1/DFO, mice were intraperitoneally injected with Ferr-1 (1.25 umol/kg) or intranasal instilled with DFO (10 mg/kg) for 7 consecutive days after CS instillation. For the models of LPS + Ferr-1/DFO, mice were pretreated with Ferr-1 or DFO for 2 consecutive days and then intratracheally instilled with LPS. Mice were sacrificed 24 h after LPS instillation. For the X-ray exposure model, mice were exposed to ionizing radiation (IR) at 20 Gy, which was delivered at the dose rate of 2 Gy/min and a source skin distance of 51 cm by an X-ray generator (Model X-RAD320iX; Precision X-Ray, Inc., North Branford, CT, USA), and sacrificed 3 days after radiation.