SIRT5-mediated HOXA5 desuccinylation inhibits ferroptosis to alleviate sepsis induced-lung injury

Abstract

Acute lung injury (ALI) is a common and severe complication of sepsis with a high mortality rate. Ferroptosis, an iron-dependent form of cell death, contributes to lung injury. Homeobox A5 (HOXA5) is involved in the regulation of septic acute kidney damage; however, its function on ferroptosis in septic ALI remains unclear. An in vitro model of septic lung injury was established in the pulmonary epithelial cell line (MLE-12) via lipopolysaccharide (LPS) stimulation. Cell viability, ferrous iron (Fe²⁺) level, and cellular lipid reactive oxygen species (ROS) were determined with Cell Counting Kit-8 assay, iron assay kit, and BODIPY™ 665/676 molecular probe, respectively. HOXA5, ferroptosis suppressor protein 1 (FSP1), sirtuin 5 (SIRT5), and glutathione peroxidase 4 (GPX4) expressions were measured using western blotting and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR. Chromatin immunoprecipitation and luciferase reporter assays were performed to validate HOXA5 binding to the FSP1/GPX4 promoter, and regulation of SIRT5 on HOXA5 desuccinylation was confirmed through co-immunoprecipitation. LPS stimulation induced ferroptosis (reduced cell viability, elevated Fe²⁺ and lipid ROS levels, and decreased GPX4 levels) and downregulated FSP1 and HOXA5 protein levels. HOXA5 overexpression neutralized LPS-induced ferroptosis. Moreover, LPS exposure inhibited HOXA5 binding to the FSP1 promoter, which was counteracted via HOXA5 overexpression. Furthermore, SIRT5 overexpression suppressed LPS-induced ferroptosis. In LPS-challenged MLE-12 cells, SIRT5-mediated HOXA5 desuccinylation was reduced. HOXA5 depletion neutralized the suppressive role of SIRT5 overexpression in LPS-induced ferroptosis. SIRT5-mediated HOXA5 desuccinylation inhibited LPS-induced ferroptosis by upregulating FSP1, which may offer a prospective therapeutic strategy for septic lung injury.

Keywords: HOXA5; SIRT5; desuccinylation; ferroptosis; sepsis‐induced lung injury.

FIGURE 1

HOXA5 overexpression suppresses ferroptosis in LPS‐induced septic lung injury in vitro. (A–D) MLE‐12 cells were subjected to LPS stimulation, followed by treatment with Fer‐1. CCK‐8 detected cell viability (A). An iron assay kit measured Fe²⁺ levels (B). Lipid ROS determined with BODIPY™ 665/676 dye (C). Western blotting detected HOXA5 and FSP1 protein levels (D). (E) HOXA5 overexpression vector (OE‐HOXA5) were transfected into MLE‐12 cells, and western blotting was employed to test HOXA5 protein level. (F–I) Transfected cells were stimulated by LPS. Western blotting analyzed HOXA5 and FSP1 protein levels (F). CCK‐8 kit determined cell viability (G). Fe²⁺ level was measured using an iron assay kit (H). Lipid ROS determined with BODIPY™ 665/676 dye (I). *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. Data are shown as mean ± SD. CCK‐8, Cell Counting Kit‐8; FSP1, ferroptosis suppressor protein 1; HOXA5, homeobox A5; LPS, lipopolysaccharide; MLE‐12, mouse lung epithelial cells; ROS, reactive oxygen species.

FIGURE 2

HOXA5 binds to the FSP1 promoter region to inhibit ferroptosis in LPS‐induced septic lung injury in vitro. (A) HOXA5 binding sites, including BS1 (−1501/−1494, 5′‐cagaaatg‐3′) and BS2 (−1537/−1530, 5′‐catgaatt‐3′), in the FSP1 gene promoter predicted with JASPAR database. (B) ChIP analysis validated HOXA5 binding to the promoter of FSP1 in MLE‐12 cells. (C) ChIP assay determined HOXA5 binding to FSP1 promoter in MLE‐12 cells with HOXA5 overexpression. (D) MLE‐12 cells were transfected with OE‐HOXA5, and dual‐luciferase activity was determined through dual‐luciferase assay. (E) MLE‐12 cells transfected with or without OE‐HOXA5 were subjected to LPS stimulation. ChIP assay was performed for measuring HOXA5 binding to FSP1 promoter. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. Data are shown as mean ± SD. BS1, binding site 1; BS2, binding site 2; ChIP, chromatin immunoprecipitation; FSP1, ferroptosis suppressor protein 1; HOXA5, homeobox A5; LPS, lipopolysaccharide; MLE‐12, mouse lung epithelial cells.

FIGURE 3

SIRT5 overexpression suppresses ferroptosis in LPS‐induced septic lung damage in vitro. (A, B) MLE‐12 cells were transfected with SIRT5 overexpression vector (OE‐SIRT5). RT‐qPCR (A) and western blotting (B) analyzed SIRT5 and HOXA5 expressions at mRNA and protein levels. (C–F) MLE‐12 cells transfected with OE‐SIRT5 were exposed to LPS. (C) Western blotting measured the protein expressions of SIRT5, HOXA5, and FSP1. (D) CCK‐8 tested cell viability. (E) Fe²⁺ level was analyzed with an iron assay kit. (F) BODIPY™ 665/676 dye was used to detect lipid ROS level. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. Data are shown as mean ± SD. CCK‐8, Cell Counting Kit‐8; FSP1, ferroptosis suppressor protein 1; HOXA5, homeobox A5; LPS, lipopolysaccharide; MLE‐12, mouse lung epithelial cells; ROS, reactive oxygen species; SIRT5, sirtuin 5.

FIGURE 4

SIRT5‐mediated HOXA5 desuccinylation is decreased in LPS‐induced septic lung injury in vitro. (A) MLE‐12 cells were treated with succinyl‐CoA (0, 0.5, or 1 mM) or with 1 mM succinyl‐CoA and LPS. Co‐IP measured succinylation of HOXA5. (B) Flag‐tagged SIRT5 or Flag‐tagged HOXA5 was transfected into HEK293T cells, and then Co‐IP was performed to assess the interaction of SIRT5 and HOXA5. (C) In MLE‐12 cells, the combination of SIRT5 and HOXA5 was detected using Co‐IP. (D) HEK293T cells transfected with Flag‐labeled HOXA5 were treated with NAM (5 mM). HOXA5 succinylation was assessed via Co‐IP. (E) MLE‐12 cells were incubated with NAM (5 mM) for 0, 2, or 4 h. Co‐IP was then performed to evaluate HOXA5 succinylation. (F) HEK293T cells were transfected with Flag‐labeled HOXA5, HA‐labeled WT‐SIRT5, or its mutant SIRT5‐H158Y. HOXA5 succinylation was determined using Co‐IP assay. (G) MLE‐12 cells transfected with OE‐SIRT5 were subjected to LPS stimulation. Co‐IP analysis of HOXA5 succinylation. Co‐IP, co‐immunoprecipitation; HOXA5, homeobox A5; LPS, lipopolysaccharide; MLE‐12, mouse lung epithelial cells; NAM, nicotinamide; SIRT5, sirtuin 5.

FIGURE 5

SIRT5 inhibits ferroptosis via HOXA5 in LPS‐induced septic lung damage cell model. (A) MLE‐12 cells were transfected with OE‐SIRT5 or/and sh‐HOXA5. Western blotting tested SIRT5, HOXA5, and FSP1 protein expressions. (B–E) Transfected cells were subjected to LPS stimulation. (B) Western blotting determined SIRT5, HOXA5, and FSP1 protein expressions. (C) CCK‐8 detected cell viability. (D) Fe²⁺ level was measured using an iron assay kit. (E) Lipid ROS was assessed with BODIPY™ 665/676 dye. *p < 0.05, **p < 0.01, ***p < 0.001. n = 3. Data are shown as mean ± SD. CCK‐8, Cell Counting Kit‐8; FSP1, ferroptosis suppressor protein 1; HOXA5, homeobox A5; LPS, lipopolysaccharide; MLE‐12, mouse lung epithelial cells; ROS, reactive oxygen species; SIRT5, sirtuin 5.