Extracellular histone H3 facilitates ferroptosis in sepsis through ROS/JNK pathway

Abstract

Introduction: Previous evidence realized the critical role of histone in disease control. The anti-inflammatory function of estradiol (E2) in sepsis has been documented. We here intended to unveil the role of extracellular histone H3 in sepsis regarding cell ferroptosis and the role of E2 in a such mechanism.

Methods: Clinical sample, cecal ligation and puncture (CLP)-induced animal models and lipopolysaccharides (LPS)-induced cell models were prepared for testing relative expression of extracellular histone H3 and E2 as well as analyzing the role of extracellular histone H3 and E2 in sepsis concerning cell viability, reactive oxygen species (ROS), and ferroptosis.

Results: Under sepsis, we found increased ferroptosis and extracellular histone H3 content, but reduced E2 concentration. Extracellular histone H3 facilitated ferroptosis of human umbilical vein endothelial cells (HUVECs) induced by LPS through activating the ROS/c-Jun N-terminal kinase (JNK) pathway. Moreover, E2 antagonized the effect of extracellular histone H3 on LPS-induced HUVEC ferroptosis and sepsis injury in CLP-induced animal models.

Conclusion: We highlighted that extracellular histone H3 facilitated lipopolysaccharides-induced HUVEC ferroptosis via activating ROS/JNK pathway, and such an effect could be antagonized by E2.

Keywords: c-Jun N-terminal kinase; estradiol; ferroptosis; histones H3; reactive oxygen species; sepsis.

Figure 1

Sepsis increases ferroptosis and extracellular histone H3 content, but reduces E2 concentration. (A) Determination of GSH/GSSG in serum sample of healthy subjects and sepsis patients; (B) determination of iron content in serum sample of healthy subjects and sepsis patients; (C) determination of histone H3 content in serum sample of healthy subjects and sepsis patients by ELISA; (D) determination of E2 level in serum sample of healthy subjects and sepsis patients by ELISA. All results are expressed as means ± standard deviation, and were analyzed by a student t‐test. ***p < .001. n = 22. E2, estradiol; ELISA, enzyme‐linked immunosorbent assay; GSH/GSSG, reduced and oxidized glutathione.

Figure 2

Extracellular histone H3 induces ferroptosis of HUVECs induced by LPS. (A) determination of histone H3 content in LPS‐exposed cells by ELISA; (B) detection of HUVEC viability after LPS or histone H3 treatment by CCK8; (C) determination of ROS level in HUVECs after LPS or histone H3 treatment; (D) determination of iron content in HUVECs after LPS or histone H3 treatment; (E) detection of Gpx4 and ACSL4 expression in HUVECs after LPS or histone H3 treatment by western blot analysis. All results are expressed as means ± standard deviation, and were analyzed by one‐way ANOVA. Three repetitions were implemented at least. *p < .05; **p < .01; ***p < .001. ELISA, enzyme‐linked immunosorbent assay; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharides.

Figure 3

E2 antagonizes the effect of extracellular histone H3 on LPS‐induced HUVEC ferroptosis. (A) Detection of HUVEC viability after LPS, histone H3 or E2 treatment by CCK8; (B) determination of ROS level in HUVECs after LPS, histone H3 or E2 treatment; (C) determination of iron content in HUVECs after LPS, histone H3 or E2 treatment; (D) detection of Gpx4 and ACSL4 expression in HUVECs after LPS, histone H3 or E2 treatment by western blot analysis. All results are expressed as means ± standard deviation, and were analyzed by one‐way analysis of variance. Three repetitions were implemented at least. *p < .05; **p < .01; ***p < .001. E2, estradiol; HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharides; ROS, reactive oxygen species.

Figure 4

Extracellular histone H3 promotes the ferroptosis of HUVECs by activating JNK pathway. (A) Determination of p‐JNK/JNK expression after LPS or histone H3 treatment by western blot analysis; (B) determination of iron content in HUVECs after LPS, histone H3 or SP600125 treatment; (C) detection of Gpx4 and ACSL4 expression in HUVECs after LPS, histone H3 or SP600125 treatment by western blot analysis. All results are expressed as means ± standard deviation, and were analyzed by one‐way analysis of variance. Three repetitions were implemented at least. *p < .05; **p < .01; ***p < .001. HUVECs, human umbilical vein endothelial cells; LPS, lipopolysaccharides; ROS, reactive oxygen species.

Figure 5

E2 reverses the promoting effect of extracellular histone H3 on sepsis. (A) Determination of serum E2 level in mice after CLP, histone, or EB treatment; (B) determination of histone H3 level in mice after CLP, histone, or EB treatment; (C) survival rate of mice after CLP, histone, or EB treatment; (D) pathological changes of lung tissues in mice after CLP, histone, or EB treatment; (E) determination of GSH/GSSG in lung tissues in mice after CLP, histone, or EB treatment; (F) determination of iron content in tissue homogenate in mice after CLP, histone, or EB treatment; (G) detection of Gpx4 and ACSL4 expression in lung tissues in mice after CLP, histone, or EB treatment by western blot analysis. All results are expressed as means ± standard deviation, and were analyzed by one‐way analysis of variance. *p < .05; **p < .01; ***p < .001. n = 10. CLP, cecal ligation and puncture; E2, estradiol; EB, estradiol benzoate;  GSH/GSSG, reduced and oxidized glutathione.