NETs promote a ferroptosis-prone phenotype, activate NF-κB signaling, and induce profibrotic protein expression in H9c2 cells. (A) Representative immunofluorescence images showing EdU proliferating nuclei and DAPI-stained total nuclei under indicated treatments. (B) Quantitative analysis of EdU+ cell ratio (*P < 0.05, **P < 0.01). (C–F) NETs induce ferroptotic responses: (C) Increased intracellular Fe²+ (*P < 0.05, **P < 0.01), (D) decreased MDA (*P < 0.05, **P < 0.01), (E) increased SOD activity (*P < 0.05, **P < 0.01), and (F) elevated GSH (*P < 0.05, **P < 0.01). Dysregulation is rescued by DNase I pretreatment, Fer-1, JSH-23, or combined inhibitors. (G) Representative Western blot analysis of fibrotic markers in H9c2 cells under indicated treatments. (H) Quantitative analysis of α-SMA protein expression in H9c2 cells (**P < 0.01). (I) Quantitative analysis of Collagen II protein expression in H9c2 cells (**P < 0.01). (J) Quantitative analysis of FN protein expression in H9c2 cells (**P < 0.01). (K) Quantitative analysis of MMP3 protein expression in H9c2 cells (**P < 0.01). (L) Western blot analysis of ferroptosis markers ACSL4, FTH1, and GPX4 in H9c2 cells under indicated conditions. (M) Quantitative analysis of ACSL4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (N) Quantitative analysis of FTH1 protein expression in H9c2 cells (**P < 0.01). (O) Quantitative analysis of GPX4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (P) Western blot analysis of NF-κB pathway activation in H9c2 cells under indicated conditions. (Q) Quantitative analysis of the p-NF-κB/NF-κB ratio densitometry derived from blots (*P < 0.05, **P < 0.01). less

NETs promote a ferroptosis-prone phenotype, activate NF-κB signaling, and induce profibrotic protein expression in H9c2 cells. (A) Representative immunofluorescence images showing EdU proliferating nuclei and DAPI-stained total nuclei under indicated treatments. (B) Quantitative analysis of EdU+ cell ratio (*P < 0.05, **P < 0.01). (C–F) NETs induce ferroptotic responses: (C) Increased intracellular Fe²+ (*P < 0.05, **P < 0.01), (D) decreased MDA (*P < 0.05, **P < 0.01), (E) increased SOD activity (*P < 0.05, **P < 0.01), and (F) elevated GSH (*P < 0.05, **P < 0.01). Dysregulation is rescued by DNase I pretreatment, Fer-1, JSH-23, or combined inhibitors. (G) Representative Western blot analysis of fibrotic markers in H9c2 cells under indicated treatments. (H) Quantitative analysis of α-SMA protein expression in H9c2 cells (**P < 0.01). (I) Quantitative analysis of Collagen II protein expression in H9c2 cells (**P < 0.01). (J) Quantitative analysis of FN protein expression in H9c2 cells (**P < 0.01). (K) Quantitative analysis of MMP3 protein expression in H9c2 cells (**P < 0.01). (L) Western blot analysis of ferroptosis markers ACSL4, FTH1, and GPX4 in H9c2 cells under indicated conditions. (M) Quantitative analysis of ACSL4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (N) Quantitative analysis of FTH1 protein expression in H9c2 cells (**P < 0.01). (O) Quantitative analysis of GPX4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (P) Western blot analysis of NF-κB pathway activation in H9c2 cells under indicated conditions. (Q) Quantitative analysis of the p-NF-κB/NF-κB ratio densitometry derived from blots (*P < 0.05, **P < 0.01). less

NETs promote a ferroptosis-prone phenotype, activate NF-κB signaling, and induce profibrotic protein expression in H9c2 cells. (A) Representative immunofluorescence images showing EdU proliferating nuclei and DAPI-stained total nuclei under indicated treatments. (B) Quantitative analysis of EdU+ cell ratio (*P < 0.05, **P < 0.01). (C–F) NETs induce ferroptotic responses: (C) Increased intracellular Fe²+ (*P < 0.05, **P < 0.01), (D) decreased MDA (*P < 0.05, **P < 0.01), (E) increased SOD activity (*P < 0.05, **P < 0.01), and (F) elevated GSH (*P < 0.05, **P < 0.01). Dysregulation is rescued by DNase I pretreatment, Fer-1, JSH-23, or combined inhibitors. (G) Representative Western blot analysis of fibrotic markers in H9c2 cells under indicated treatments. (H) Quantitative analysis of α-SMA protein expression in H9c2 cells (**P < 0.01). (I) Quantitative analysis of Collagen II protein expression in H9c2 cells (**P < 0.01). (J) Quantitative analysis of FN protein expression in H9c2 cells (**P < 0.01). (K) Quantitative analysis of MMP3 protein expression in H9c2 cells (**P < 0.01). (L) Western blot analysis of ferroptosis markers ACSL4, FTH1, and GPX4 in H9c2 cells under indicated conditions. (M) Quantitative analysis of ACSL4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (N) Quantitative analysis of FTH1 protein expression in H9c2 cells (**P < 0.01). (O) Quantitative analysis of GPX4 protein expression in H9c2 cells (*P < 0.05, **P < 0.01). (P) Western blot analysis of NF-κB pathway activation in H9c2 cells under indicated conditions. (Q) Quantitative analysis of the p-NF-κB/NF-κB ratio densitometry derived from blots (*P < 0.05, **P < 0.01). less

mtDNA participates in NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. After transfection with DNase I, IPEC-J2 cells were infected with PEDV (MOI = 1). IL-1β mRNA expression (a) and secretion in cell supernatants detected by ELISA (b). NLRP3 inflammasome mRNA expression (c) and caspase-1 enzyme activity (d). Western blot detection of NLRP3 inflammasome and downstream protein expression after transfection with DNase I protein (e). Data represent mean ± SD (n = 3). ** p < 0.01. less

mtDNA participates in NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. After transfection with DNase I, IPEC-J2 cells were infected with PEDV (MOI = 1). IL-1β mRNA expression (a) and secretion in cell supernatants detected by ELISA (b). NLRP3 inflammasome mRNA expression (c) and caspase-1 enzyme activity (d). Western blot detection of NLRP3 inflammasome and downstream protein expression after transfection with DNase I protein (e). Data represent mean ± SD (n = 3). ** p < 0.01. less

mtDNA participates in NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. After transfection with DNase I, IPEC-J2 cells were infected with PEDV (MOI = 1). IL-1β mRNA expression (a) and secretion in cell supernatants detected by ELISA (b). NLRP3 inflammasome mRNA expression (c) and caspase-1 enzyme activity (d). Western blot detection of NLRP3 inflammasome and downstream protein expression after transfection with DNase I protein (e). Data represent mean ± SD (n = 3). ** p < 0.01. less

mtDNA participates in NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. After transfection with DNase I, IPEC-J2 cells were infected with PEDV (MOI = 1). IL-1β mRNA expression (a) and secretion in cell supernatants detected by ELISA (b). NLRP3 inflammasome mRNA expression (c) and caspase-1 enzyme activity (d). Western blot detection of NLRP3 inflammasome and downstream protein expression after transfection with DNase I protein (e). Data represent mean ± SD (n = 3). ** p < 0.01. less

mtDNA participates in NLRP3 inflammasome activation in PEDV-infected IPEC-J2 cells. After transfection with DNase I, IPEC-J2 cells were infected with PEDV (MOI = 1). IL-1β mRNA expression (a) and secretion in cell supernatants detected by ELISA (b). NLRP3 inflammasome mRNA expression (c) and caspase-1 enzyme activity (d). Western blot detection of NLRP3 inflammasome and downstream protein expression after transfection with DNase I protein (e). Data represent mean ± SD (n = 3). ** p < 0.01. less

NET degradation inhibited the pyroptosis in the corpus cavernosum tissues of diabetic ED rats. (A) Assessment of NLRP3 expression in the corpus cavernosum tissues of rats using immunohistochemistry (n = 6/group; scale bar = 50 μm). (B) Assessment of the protein expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC in the corpus cavernosum tissues of rats using western blotting (n = 6/group); the uncropped protein blots can be seen in Supplementary file 2. (C) Assessment of plasma IL-1β and IL-18 levels in rats using ELISA (n = 6/group). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were expressed as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, NLRP3 NOD-like receptor family, pyrin domain-containing protein 3, GSDMD gasdermin D, GSDMD-N N-terminal proteolytic fragment of gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, IL interleukin, ELISA enzyme-linked immunosorbent assay. less

NET degradation inhibited the pyroptosis in the corpus cavernosum tissues of diabetic ED rats. (A) Assessment of NLRP3 expression in the corpus cavernosum tissues of rats using immunohistochemistry (n = 6/group; scale bar = 50 μm). (B) Assessment of the protein expression levels of NLRP3, GSDMD, GSDMD-N, pro-caspase-1, cleaved-caspase-1, and ASC in the corpus cavernosum tissues of rats using western blotting (n = 6/group); the uncropped protein blots can be seen in Supplementary file 2. (C) Assessment of plasma IL-1β and IL-18 levels in rats using ELISA (n = 6/group). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were expressed as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, NLRP3 NOD-like receptor family, pyrin domain-containing protein 3, GSDMD gasdermin D, GSDMD-N N-terminal proteolytic fragment of gasdermin D, ASC apoptosis-associated speck-like protein containing a CARD, IL interleukin, ELISA enzyme-linked immunosorbent assay. less

NET degradation alleviated diabetic ED symptoms of rats. (A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group). (B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm). (C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/group). (D) Assessment of erection number and latency in rats (n = 6/group). (E) Assessment of the serum testosterone level using ELISA (n = 6/group). (F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm). (G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were presented as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin–eosin. less

NET degradation alleviated diabetic ED symptoms of rats. (A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group). (B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm). (C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/group). (D) Assessment of erection number and latency in rats (n = 6/group). (E) Assessment of the serum testosterone level using ELISA (n = 6/group). (F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm). (G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were presented as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin–eosin. less

NET degradation alleviated diabetic ED symptoms of rats. (A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group). (B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm). (C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/group). (D) Assessment of erection number and latency in rats (n = 6/group). (E) Assessment of the serum testosterone level using ELISA (n = 6/group). (F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm). (G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were presented as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin–eosin. less

NET degradation alleviated diabetic ED symptoms of rats. (A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group). (B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm). (C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/group). (D) Assessment of erection number and latency in rats (n = 6/group). (E) Assessment of the serum testosterone level using ELISA (n = 6/group). (F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm). (G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were presented as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin–eosin. less

NET degradation alleviated diabetic ED symptoms of rats. (A) Assessment of the levels of plasma H3Cit, NE, and cf-DNA (n = 6/group). (B) Assessment of expression levels of H3Cit and MPO in the corpus cavernosum tissues of rats using immunofluorescence (n = 6/group; scale bar = 50 μm). (C) Blood routine examination of WBC, RBC, Hb, and PLT levels in rats (n = 6/group). (D) Assessment of erection number and latency in rats (n = 6/group). (E) Assessment of the serum testosterone level using ELISA (n = 6/group). (F) Histopathological examination of the corpus cavernosum tissues of rats using HE staining (n = 6/group; scale bar = 50 μm). (G) Histopathological examination of the corpus cavernosum tissues of rats using Masson staining (n = 6/group; scale bar = 50 μm). Rats in diabetic ED + DNase I group were intraperitoneally injected with DNase I (a NET degrading agent) at the dose of 1 mg/kg for 8 successive days. Meanwhile, rats in Control and diabetic ED groups were intraperitoneally injected with the same amount of normal saline. Data were presented as mean ± standard deviation. **p < 0.01. NET neutrophil extracellular trap, ED erectile dysfunction, H3Cit citrullinated histone H3, NE neutrophil elastase, cf-DNA cell-free DNA, MPO myeloperoxidase, WBC white blood cell, RBC red blood cell, Hb hemoglobin, PLT platelet, ELISA enzyme-linked immunosorbent assay, HE hematoxylin–eosin. less