To investigate the relationship of aberrant SMAD family member 7(SMAD7) signaling pathway and ferroptosis in myelodysplastic syndromes (MDS) and evaluate the effect of asiaticoside (AC)-modulating SMAD7 up-regulation to suppress ferroptosis in MDS cell lines.

Publicly available MDS-related datasets from the Gene Expression Omnibus (GEO) database were analyzed to identify differentially expressed genes (DEGs) between MDS patients and healthy controls. These DEGs were cross-referenced with ferroptosis-associated genes from the Ferroptosis Database (FerrDb) to identify potential ferroptosis-related targets in MDS. Bone marrow mononuclear cells (BMMNCs) were isolated from 18 MDS patients freshly diagnosed in the First Medical Center of Chinese PLA General Hospitaland and from 16healthy donors during October 2022 and November 2024. RT-qPCR was employed to detect the expression of SMAD7 and ferroptosis-related genes. Immunomagnetic bead sorting was applied to purify CD33⁺ cells, and then qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels. Human normal bone marrow cells (HS-5) and MDS cell lines (MUTZ-1, SKM-1) were treated with gradient concentrations of AC (SMAD7 activator) and ferrostatin-1 (Fer-1,ferroptosis inhibitor), and SMAD7 overexpression plasmids were transfected into MDS cells. qPCR and Western blotting were utilized to measure the expression of SMAD7 and ferroptosis-related biomarkers at mRNA and protein levels, and the contents of glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD), and reactive oxygen species (ROS) were detected. Flow cytometry of CD11b⁺ was performed to measure cellular differentiation.

① Bioinformatics analysis revealed significant down-regulation of SMAD7 in MDS patients, correlating with ferroptosis activation. Compared to the healthy controls, MDS patients exhibited decreased SMAD7 expression (P<0. 05), reduced levels of negative regulators of ferroptosis,glutathione peroxidase 4 (GPX4) and ferritin heavy chain (FTH1)(P<0. 05), and elevated expression of its positive regulator transferrin receptor protein 1 (TFRC)(P<0. 05). Consistent with this, when compared with the normal human bone marrow stromal cell line HS-5, the MDS cell lines MUTZ-1 and SKM-1 exhibited declined expression of SMAD7, GPX4, and FTH1, alongside elevated expression of TFRC (P<0. 05). ② Treatment with gradient concentrations of the ferroptosis inhibitors ferrostatin-1 (Fer-1), the expression levels of GPX4and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner (P<0. 05),while TFRC was markedly downregulated (P<0. 05). Additionally, GSH content and SOD activity were enhanced, whereas ROS levels and MDA content were significantly reduced (P<0. 05). These results suggest that Fer-1 effectively suppresses ferroptosis in MDS cells. ③ SMAD7 overexpression led to up-regulation of GPX4 and FTH1 ih MDS cell lines, while downregulation of TFRC, improved anti-oxidative ability and reduced ferroptosis, with enhanced CD11b⁺ expression and myeloid differentiation. ④ Following AC treatment, the expression levels of GPX4 and FTH1 in MDS cell lines were significantly upregulated in a concentration-dependent manner (P<0. 05), whereas the downregulation of TFRC did not reach statistical significance. Additionally, AC treatment effectively enhanced the antioxidant capacity of the cells, increased the proportion of CD11b⁺ cells (P<0. 05), and facilitated cellular differentiation.

AC activates SMAD7 in MDS cell lines, up-regulating GPX4 and FTH1 while suppressing TFRC expression. This mechanism alleviates oxidative damage and lipid peroxidation, thereby inhibiting ferroptosis in MDS cells.Concurrently, SMAD7 activation promotes cellular differentiation.

To investigate the protective effects of ferroptosis inhibitor ferrostatin-1 (Fer-1) on high-altitude hypoxic lung injury and explore novel preventive strategies for high-altitude hypoxia-induced lung injury.

①Eighteen SPF male Wistar rats (5~6 weeks old, 210~230 g) were randomly divided into 3 groups (n=6): normoxic control, hypoxic lung injury, and Fer-1 pretreatment groups. A hypobaric chamber was used to establish a rat model of high-altitude hypoxic lung injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to compare pulmonary protein profiles between normoxic and hypoxic groups, followed by bioinformatics analysis of pathways enriched with differentially expressed proteins (DEPs). Histopathological changes and lung injury scores were assessed with HE staining. ELISA was used to quantify the inflammatory cytokines, flow cytometry and immunofluorescence assay were employed to measure the production of reactive oxygen species (ROS), and spectrophotometry was utilized to determine the contents of Fe²⁺, glutathione (GSH), malondialdehyde (MDA), and superoxide dismutase (SOD) to evaluate oxidative stress and detect ferroptosis-related markers. ② Human bronchial epithelial cells (bronchial epithelium transformed with Ad12-SV40, BEAS-2B) and macrophages induced by tumor human peripheral blood monocytes-1 (THP-1) cells were placed in a low oxygen conditions for 48 h to establish a cellular model of hypoxic lung injury, on which Fer-1 was administered as a preventive group. Ferroptosis markers in BEAS-2B cells and inflammatory cytokine secretion in macrophages were analyzed.

① Proteomics identified 2962 proteins, with 357 DEPs (199 up-regulated, 158 down-regulated). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed ferroptosis as the most enriched pathway. Hypoxic lung injury resulted in elevated ROS, MDA, Fe²⁺, and inflammatory cytokines (P<0.05), reduced SOD, GSH, solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), and ferritin heavy chain 1 (FTH1), and increased acyl-coa synthetase long chain family member 4 (ACSL4) (P<0.05). Fer-1 pretreatment significantly mitigated oxidative stress (ROS, MDA, SOD, GSH; P<0.05), up-regulated SLC7A11 and FTH1, down-regulated ACSL4 (P<0.05), and reduced inflammation (P<0.05). ② In cellular models, Fer-1 increased SLC7A11, GPX4, FTH1, GSH, and SOD (P<0.05), declined ROS (P<0.05), and suppressed macrophage inflammatory cytokines (P<0.05).

Fer-1 alleviates high-altitude hypoxic lung injury by inhibiting ferroptosis in pulmonary epithelial cells and attenuating macrophage-driven inflammation, providing experimental evidence for novel therapeutic strategies.

To investigate the protective effects of dimethyl fumarate (DMF), an agonist of nuclear factor erythroid 2-related factor 2(NRF2), against acute lung injury (ALI) induced by high-altitude hypoxia, in order to found a basis for developing novel preventive strategies against hypoxic pulmonary injury.

① Twenty SPF-grade male Wistar rats (5 to 6 weeks old, 210 to 230 g) were randomly divided into (n=5): normoxic control (intraperitoneal injection of normal saline for 10 consecutive days followed by being placed in a normobaric normoxia chamber), hypoxic lung injury (intraperitoneal injection of normal saline for 10 consecutive days followed by being exposed to a hypobaric hypoxic chamber simulating 5000 m altitude for 48 h), DMF control (intraperitoneal injection of DMF for 10 consecutive days followed by being placed in a normobaric normoxic chamber), and DMF prophylaxis (DMF preconditioning for 10 d followed by hypobaric hypoxic modelling) groups. HE staining was used to observe the histopathological changes in lung tissues to pathologically score the lung injury. ELISA was employed to detect the inflammatory factors to assess inflammatory responses, immunofluorescence assay was utilized to measure the generation of reactive oxygen species (ROS), and spectrophotometry was performed to determine the glutathione (GSH) and malondialdehyde (MDA) levels, as well as superoxide dismutase (SOD) activity. Western blotting and real-time polymerase chain reaction (RT-qPCR) were applied to analyze ferroptosis-related pathway proteins at protein and mRNA levels. ② Hypoxic lung injury cellular model and normoxic control model were established by co-culturing human bronchial epithelial (BEAS-2B) cells and TNF-α-activated human peripheral blood monocyte (THP-1)-derived macrophages under hypoxic and normoxic conditions for 48 h. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to compare the protein profiles in BEAS-2B cells between normoxic control and hypoxic injury groups. Bioinformatics analysis was carried out to identify the key differentially expressed proteins (DEPs). A hypoxic injury prophylaxis group was established by BEAS-2B cells being pretreated with DMF for 24 h and then co-cultured with THP-1 cells. Then the ferroptosis markers, macrophage polarization phenotypes, and inflammatory cytokines were detected in BEAS-2B cells. ③ Additionally, solute carrier family 7 member 11(SLC7A11) gene was knocked down under DMF intervention to assess ferroptosis markers and macrophage-related inflammatory responses.

① Compared to the hypoxic injury group, the DMF prophylaxis group exhibited significantly decreased pulmonary ROS level and MDA content (P < 0.05), increased SOD activity and GSH level (P < 0.05), elevated expression levels of SLC7A11 and glutathione peroxidase 4(GPX4)(P < 0.05), along with markedly reduced lung injury score and inflammatory cytokine levels (P < 0.05). ② Proteomic analysis identified 5377 proteins and 613 DEPs (302 up-regulated, 311 down-regulated) in BEAS-2B cells, and among 9 ferroptosis-related DEPs, SLC7A11 exhibited the most significant alteration. In the cellular hypoxic ALI model, compared to the hypoxic injury group, DMF prophylaxis up-regulated SLC7A11 and GPX4 expression (P < 0.05), increased GSH level and SOD activity, decreased the M1 macrophage ratio and inflammatory factor levels (P < 0.05). ③ SLC7A11 knockdown reversed DMF's effects by increasing MDA level (P < 0.05), decreasing SOD activity and GSH level (P < 0.05), suppressing GPX4 expression (P < 0.05), and elevating M1 macrophage proportion and enhancing inflammatory cytokine secretion (P < 0.05). Conversely, overexpression of SLC7A11 enhanced the protective effects of DMF. Compared with the hypoxia-DMF group, the hypoxia-DMF-overexpression group showed significantly increased GSH level, SOD activity, and GPX4 expression (P < 0.05), along with a marked decrease in MDA content (P < 0.05) in BEAS-2B cells. Meanwhile, SLC7A11 overexpression further alleviated the inflammatory response, as indicated by significantly reduced expression of IL-1β, IL-6 and TNF-α(P < 0.05).

DMF attenuates high-altitude hypoxic ALI by suppressing ferroptosis in pulmonary epithelial cells via up-regulating the NRF2/SLC7A11 axis, modulating macrophage polarization, and mitigating inflammatory response, and thus, exerts a protective effect against hypoxic pulmonary injury.