Sodium Houttuyniae attenuates ferroptosis by regulating TRAF6-c-Myc signaling pathways in lipopolysaccharide-induced acute lung injury (ALI)

Abstract

The impact of Sodium Houttuyniae (SH) on lipopolysaccharide (LPS)-induced ALI has been investigated extensively. However, it remains ambiguous whether ferroptosis participates in this process. This study aimed to find out the impacts and probable mechanisms of SH on LPS-induced ferroptosis. A rat ALI model and type II alveolar epithelial (ATII) cell injury model were treated with LPS. Enzyme-linked immunosorbent assay (ELISA), hematoxylin-eosin (HE) staining, and Giemsa staining were executed to ascertain the effects of SH on LPS-induced ALI. Moreover, Transmission electron microscopy, Cell Counting Kit-8 (CCK8), ferrous iron colorimetric assay kit, Immunohistochemistry, Immunofluorescence, Reactive oxygen species assay kit, western blotting (Wb), and qRT-PCR examined the impacts of SH on LPS-induced ferroptosis and ferroptosis-related pathways. Theresults found that by using SH treatment, there was a remarkable attenuation of ALI by suppressing LPS-induced ferroptosis. Ferroptosis was demonstrated by a decline in the levels of glutathione peroxidase 4 (GPX4), FTH1, and glutathione (GSH) and a surge in the accumulation of malondialdehyde (MDA), reactive oxygen species (ROS), NOX1, NCOA4, and Fe²⁺, and disruption of mitochondrial structure, which were reversed by SH treatment. SH suppressed ferroptosis by regulating TRAF6-c-Myc in ALI rats and rat ATII cells. The results suggested that SH treatment attenuated LPS-induced ALI by repressing ferroptosis, and the mode of action can be linked to regulating the TRAF6-c-Myc signaling pathway in vivo and in vitro.

Keywords: Acute lung injury; Ferroptosis; Sodium Houttuyniae; TRAF6-c-Myc pathway.

Fig. 1

SH significantly ameliorated LPS-induced ALI in vivo. (A) The W/D ratio of lung tissues. (B) Representative bright field images of HE staining (scale bar = 50 μm). (C) Lung injury score. (D) Representative bright field images of Giemsa staining (scale bar = 50 μm), Neutrophils (blue), and eosinophils (purple). (E-H) ELISA detected the IL-18, IL-1β, and IL-6 concentrations in the BALF (n = 4 for every group). Data are shown as the mean ± SD, ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

SH significantly attenuated LPS-induced ferroptosis by inhibiting the TRAF6-c-Myc signaling pathway in vivo. (A) The level of Fe²⁺ was evaluated by a ferrous iron colorimetric assay kit. (B-C) The levels of GSH and MDA was detected by Elisa kits. (D-E) Western blotting was used to detect the expression of GPX4, FTH1, NOX1, NCOA4, TRAF6, and c-Myc protein. (F) The mRNA expression of GPX4, FTH1, NOX1, NCOA4, TRAF6, and c-Myc was detected by qRT-PCR. (G) Representative immunohistochemical images of the expression of the TRAF6 in the lung tissue (scar bar = 50 μm). (H) The statistics of immunohistochemical staining. (I) Detection of c-Myc protein expression level by immunofluorescence staining (scar bar = 50 μm, n = 4 for every group). Data are shown as the mean ± SD, ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

SH significantly attenuated LPS-induced ferroptosis in vitro. (A) Observation of mitochondrial structure by transmission electron microscopy in rat ATII cells (scale bar is 500 nm). (B) CCK8 was used for the detection of cell proliferation of rat ATII cells. (C) The cellular iron levels were evaluated by the ferrous iron colorimetric assay kit. (D) Representative ROS fluorescence picture of rat ATII cells. (E) ROS fluorescence intensity. (F-G) Concentrations of GSH and MDA. (H-L) Western blotting was used to detect the expression of FTH1, GPX4, NCOA4, and NOX1 protein. Data are shown as the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

SH significantly attenuated LPS-induced ferroptosis by inhibiting the TRAF6-c-Myc signaling pathway in vitro. (A) CCK8 was used for the detection of cell proliferation of rat ATII cells. (B) The cellular iron levels were detected by the ferrous iron colorimetric assay kit. (C-D) Concentrations of GSH and MDA. (E) Representative ROS fluorescence picture of rat ATII cells. (F) Fluorescence intensity of ROS. Data are shown as the mean ± SD, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

SH significantly improve liver injury in LPS induced ALI rats in vitro. The rats were induced by the instillation of an intratracheal LPS to develop a rat model of ALI and ascertain the protective impact of SH towards LPS-induced ALI in rats. The activities of ALP (B), AST (C), and LDH (A) were measured. Data are shown as the mean ± SD, ^#p < 0.05; ^##p < 0.01; ^###p < 0.001; ^####p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

Mechanisms underlying the effects of SH on LPS-induced lung injury by inhibiting ferroptosis