Exosomal miR-499a-5p from human umbilical wire mesenchymal stem cells attenuates liver fibrosis through focusing on ETS1/GPX4-mediated ferroptosis in hepatic stellate cells | Journal of Nanobiotechnology

MSCs mitigate liver fibrosis induced by CCl₄ and TAA in mice

To confirm the multidirectional differentiation potential of the bought MSCs and characterize their phenotypes, a number of in vitro experiments had been carried out. Oil crimson O, alizarin crimson and alcian blue staining confirmed adipogenic, osteogenic and chondrogenic differentiation, respectively, indicating profitable lineage-specific differentiation (Fig. S1A-C). Circulate cytometry additional validated the MSC phenotype by confirming constructive markers (CD73, CD90, CD105) and detrimental markers (CD34, CD45, HLA-DR) (Fig. S1D-E). These outcomes uniformly confirmed the MSC traits of the cells.

Two well-characterized mouse fashions of liver fibrosis had been generated utilizing CCl₄ and TAA injections. The timeline for the CCl₄-induced mouse mannequin and MSC administration is proven in Fig. 1A. The CCl₄ group exhibited a lower in physique weight in comparison with Controls, whereas the MSC group confirmed a noticeable improve relative to the CCl₄ group (Fig. 1B). To confirm MSC homing to the liver, immunofluorescence staining for CD73 and CD90 was carried out on liver tissues. The liver tissues of MSC-treated mice exhibited considerably elevated ranges of CD73 and CD90 in comparison with the Management and CCl₄ teams, indicating profitable MSC homing to the liver (Fig. 1C). In vivo fluorescence imaging revealed the presence of DiR-labeled MSCs within the livers of each Management mice and people with CCl₄-induced liver fibrosis. Ex vivo fluorescence imaging revealed important MSC accumulation within the lungs, with detectable distribution within the liver and spleen, however no important DiR alerts within the coronary heart, kidneys, or intestines. The liver of fibrotic mice exhibited stronger fluorescence alerts, indicating that MSCs have an enhanced propensity emigrate towards broken tissues below pathological situations (Fig. 1D). Gross liver examination revealed shrinkage and a granular floor within the CCl₄ group, in distinction to the Management, whereas the MSC group displayed decreased granularity and a smoother floor (Fig. 1E). H&E staining revealed that MSC therapy markedly improved CCl₄-induced hepatocyte structural disarray and perivascular inflammatory cell infiltration. A discount in collagen fibers and fibrosis was noticed following MSC therapy, as proven by Masson and Sirius crimson staining (Fig. 1F, Fig. S1F). Immunohistochemical evaluation revealed elevated ranges of α-SMA and Col1a1 within the CCl₄ group, which decreased following MSC therapy (Fig. 1G, Fig. S1G). Blood biochemical evaluation revealed substantial reductions within the ranges of ALT, AST, and ALP, alongside a rise in ALB, indicating improved liver operate following MSC therapy (Fig. 1H). MSC therapy downregulated the expression of α-SMA, Tgfb1, Desmin, and Vimentin in mice uncovered to CCl₄ (Fig. 1I-J).

MSC therapy mitigates CCl₄-induced liver fibrosis. (A) Schematic of the CCl₄ modeling timeline. (B) Physique weight adjustments in Management, CCl₄, and CCl₄ + MSCs teams. ***P < 0.001, CCl₄ vs. Management group, ****P < 0.0001, CCl₄ vs. Management group; # P < 0.05, CCl₄ + MSCs vs. CCl₄ group. (C) Immunofluorescence staining of CD73 and CD90 in liver tissue. Scale bar = 20 μm. (D) In vivo imaging of DiR-labeled MSCs 24 h post-injection in mice and ex vivo fluorescence imaging of liver, coronary heart, lungs, spleen, kidneys, and intestines. (E) Consultant liver morphology within the three teams. (F) Liver sections had been stained with H&E, Masson, and Sirius Purple within the three teams. Scale bar = 50 μm. (G) Immunohistochemical staining of α-SMA and Col1a1 in liver sections. Scale bar = 50 μm. (H) Liver operate exams for serum ALT, AST, ALP, and ALB ranges within the mouse teams. (I) qRT-PCR evaluation of α-SMA and Tgfb1 mRNA expression in liver tissues. (J) Western blot evaluation of α-SMA, Desmin, and Vimentin protein expression in liver tissues. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

The timeline for the TAA-induced mouse mannequin and MSC therapy schedule is illustrated in Fig. S2A. Gross liver examination, histopathology, immunohistochemistry, biochemical assays, ELISA, qRT-PCR, and Western blot outcomes demonstrated the alleviation of liver fibrosis by MSCs (Fig. S2B-H, Fig. S1H-I). In abstract, MSCs mitigate liver fibrosis induced by CCl₄ and TAA in mice.

MSCs inhibit ferroptosis in liver fibrotic tissues

Complete gene expression profiling of liver tissue samples from Management, CCl₄-induced liver fibrosis, and MSCs-treated teams was performed utilizing RNA sequencing to deepen the understanding of the therapeutic mechanisms of MSCs. Initially, a comparability between the CCl₄ -induced fibrosis group and the conventional Management recognized 1,846 considerably upregulated and 1,767 considerably downregulated genes. After MSC therapy, 2,019 genes had been upregulated and a couple of,056 genes had been downregulated in comparison with the CCl₄-induced fibrosis group. Notably, 1,162 downregulated and 1,233 upregulated genes induced by CCl₄ had been reversed by MSC therapy, suggesting these DEGs could play essential roles in MSCs’ therapeutic results (Fig. 2A). Subsequently, genes with important expression variations throughout the three teams had been visualized utilizing heatmaps (Fig. 2B). Additional GO evaluation confirmed that these 2,395 DEGs had been notably enriched in processes related to oxidative stress response, and iron ion binding (Fig. 2C). KEGG pathway evaluation recommended that these DEGs had been concerned in metabolic pathways, glutathione metabolism, and ferroptosis (Fig. 2D).

Ferroptosis in liver fibrotic tissues is inhibited by MSCs. (A) Venn diagram figuring out DEGs related to MSC therapy. (B) Heatmap of RNA sequencing outcomes from Management, CCl₄, and CCl₄ + MSC teams. (C) GO evaluation of DEGs modulated by MSCs. (D) KEGG evaluation of DEGs modulated by MSCs. (E) Western blot of Cox2 and Sod1 expression in liver tissues. (F) Serum iron ranges measurement. (G) SOD ranges measurement in liver tissues. (H) MDA ranges measurement in liver tissues. (I) Immunofluorescence staining of BODIPY in liver tissue. Scale bar = 20 μm. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

Protein assays for oxidative stress and ferroptosis markers had been performed in mouse liver tissues. Outcomes indicated a big upregulation of Cox2 and a notable downregulation of Sod1 protein expression within the liver fibrosis mannequin group. These adjustments had been considerably reversed by MSC therapy (Fig. 2E). Serum iron ranges, together with SOD and MDA in liver tissues, had been additionally measured. Fibrotic mice exhibited elevated serum iron and MDA ranges, with decreased SOD ranges in comparison with Controls. MSC therapy decreased serum iron and MDA ranges whereas rising SOD ranges (Fig. 2F-H). BODIPY fluorescence staining revealed the next lipid peroxidation degree in fibrotic tissues in comparison with the Management group, demonstrated by elevated fluorescence depth, whereas MSC therapy considerably decreased lipid peroxidation (Fig. 2I). Collectively, these outcomes counsel that MSCs could exert their results by modulating ferroptosis in fibrotic tissue.

MSCs inhibit HSCs activation by inducing ferroptosis

Though earlier research have proven that MSCs shield towards acute liver harm by inhibiting hepatocyte ferroptosis, their impact on HSC ferroptosis stays unclear [18]. Contemplating the central function of HSCs in liver fibrosis and the rising recognition of ferroptosis in cell destiny regulation, it’s important to find out whether or not MSCs can affect ferroptosis in HSCs. Therefore, we investigated whether or not MSCs might inhibit HSC activation by inducing ferroptosis.

Immunofluorescence evaluation confirmed that within the CCl₄-treated group, each α-SMA and Cox2 expression elevated with out colocalization, suggesting no ferroptosis in HSCs. Nevertheless, after MSC therapy, Cox2 expression decreased, and colocalization with α-SMA elevated, indicating that MSCs promoted ferroptosis in HSCs, contributing to the discount of liver fibrosis (Fig. 3A). Following 48 h of therapy with MSCs-CM conditioned medium, Fer-1, and Eras, the viability of mHSCs and LX-2 cells was evaluated through the CCK-8 assay. Outcomes demonstrated that MSCs-CM therapy decreased cell viability, with an extra decline within the MSCs-CM + Eras group. In distinction, cell viability was partially restored within the MSCs-CM + Fer-1 group (Fig. 3B-C). HSCs had been co-cultured with MSCs and handled with Fer-1 (Fig. 3D). MSCs considerably decreased the mRNA expression of fibrosis markers α-SMA and TGFB1 in HSCs whereas rising the mRNA ranges of ferroptosis markers Acsl4 and COX2. Notably, even with the ferroptosis inhibitor Fer-1, MSCs nonetheless partially regulated these markers (Fig. 3E-F). On the protein degree, MSCs decreased Vimentin, Desmin, α-SMA, TGFB, Sod1, and SOD2 expression in HSCs whereas enhancing COX2 expression. These results had been partially inhibited by Fer-1 (Fig. 3G-H, Fig. S3A-B). Utilizing DCFH-DA and JC-1 fluorescent probes, MSCs considerably elevated ROS technology and decreased mitochondrial membrane potential in HSCs. In distinction, the Fer-1 group confirmed no important improve in ROS ranges, and mitochondrial membrane potential remained unchanged. Within the Fer-1 + MSCs group, though ROS ranges had been greater than within the Fer-1 group, and mitochondrial membrane potential decreased, the impact was much less pronounced than within the MSCs group (Fig. 3I-J). Fe²⁺ and MDA ranges had been considerably elevated within the MSC-treated group in comparison with the Management, with Fer-1 partially inhibiting this improve (Fig. 3Okay-L). MSC-treated HSCs exhibited ferroptosis-related adjustments, together with mitochondrial shrinkage, decreased or absent cristae, and elevated electron density (Fig. 3M-N). These findings counsel that MSCs could induce ferroptosis by modulating particular molecular expressions in HSCs, thereby inhibiting their activation.

HSC activation is inhibited by MSCs by way of the promotion of ferroptosis. (A) Immunofluorescence staining of α-SMA (inexperienced) and Cox2 (crimson) in liver tissues. The white triangular area signifies colocalization. Scale bar = 50 μm. (B) and (C) CCK-8 assays of mHSCs and LX-2 cells viability. (D) Schematic of mHSCs and LX-2 cells handled with MSCs and Fer-1 for 48 h. (E) qRT-PCR evaluation of α-SMA, Tgfb1, and Acsl4 mRNA ranges in mHSCs. (F) qRT-PCR evaluation of α-SMA, TGFB1, and COX2 mRNA ranges in LX-2 cells. (G) Western blot of Vimentin, Desmin, α-SMA, Cox2, and Sod1 protein ranges in mHSCs. (H) Western blot of Desmin, α-SMA, TGFB1, COX2, and SOD2 protein ranges in LX-2 cells. (I) ROS ranges in mHSCs and LX-2 cells. Scale bar = 50 μm. (J) Mitochondrial membrane potential in LX-2 cells utilizing JC-1 probes. Scale bar = 50 μm. (Okay) and (L) Fe²⁺ and MDA ranges in mHSCs and LX-2 cells. (M) and (N) TEM of mitochondrial adjustments in mHSCs and LX-2 cells. Scale bar = 0.5 μm. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

MSCs induce ferroptosis in HSCs by downregulating GPX4

A complete of 83 potential ferroptosis-related genes regulated by MSCs had been recognized by intersecting 564 ferroptosis-related genes with the two,395 MSC-regulated genes from our earlier RNA-seq evaluation (Fig. 4A). Important gene expression variations between aHSCs and qHSCs had been recognized by way of evaluation of the GSE229291 dataset (Fig. 4B-C). A comparability of the genes upregulated and downregulated in aHSCs with the 83 ferroptosis-related genes regulated by MSCs means that MSCs could regulate 30 upregulated and 22 downregulated ferroptosis-related genes in aHSCs (Fig. 4D, Tab. S6). Notably, the GPX4 gene, considerably upregulated in aHSCs throughout liver fibrosis, emerged as a gene of specific curiosity. The evaluation of the GSE14323 dataset demonstrated that GPX4 expression ranges had been markedly elevated in cirrhosis sufferers relative to wholesome people (P = 0.004), underscoring the crucial function of GPX4 in liver fibrosis (Fig. 4E).

MSCs suppress HSC activation by selling ferroptosis by way of the downregulation of GPX4 in HSCs. (A) Ferroptosis-related genes regulated by MSCs. (B) and (C) Volcano plot and heatmap of DEGs between aHSCs and qHSCs. (D) Intersection of 83 ferroptosis genes with up- or down-regulated genes in aHSCs. (E) Violin plot displaying GPX4 expression in management and cirrhosis sufferers. (F) Violin plot of Gpx4 expression throughout three pattern teams (RNA-seq). (G) Immunohistochemistry of Gpx4 expression in Management, CCl₄, and CCl₄ + MSCs teams. Scale bar = 50 μm. (H) Immunohistochemistry of Gpx4 expression in Management, TAA, and TAA + MSCs teams. Scale bar = 50 μm. (I) qRT-PCR and (J) Western Blot evaluation of Gpx4 expression in Management, CCl₄, and CCl₄ + MSCs teams. (Okay) qRT-PCR and (L) Western Blot evaluation of Gpx4 expression in mHSCs and LX-2 cells. (M) and (N) Fe²⁺ and MDA ranges in Management, si-NC, and si-GPX4 teams in mHSCs and LX-2 cells. (O) Western Blot photographs for Gpx4, Cox2, Sod1, Vimentin, and α-SMA in mHSCs. (P) Western Blot photographs for GPX4, COX2, ACSL4, and α-SMA in LX-2 cells. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

The upregulation of Gpx4 expression in CCl₄ and TAA-induced fibrotic mouse fashions was validated utilizing RNA-seq and immunohistochemistry in subsequent experiments. Gpx4 expression was considerably downregulated by MSC therapy (Fig. 4F-H, Fig. S3C-D). This downregulation was additional confirmed by detecting Gpx4 mRNA and protein ranges in liver tissue samples (Fig. 4I-J). In vitro experiments additionally confirmed that MSCs downregulated GPX4 expression in mHSCs and LX-2 cells, even with the ferroptosis inhibitor Fer-1 current, demonstrating a powerful regulatory impact (Fig. 4Okay-L). To additional discover GPX4’s function in ferroptosis regulation, siRNA was employed to particularly knockdown GPX4. Knockdown of GPX4 in mHSCs and LX-2 cells considerably elevated Fe²⁺ and MDA ranges, indicating elevated oxidative stress (Fig. 4M-N). In mHSCs, Acsl4 and Cox2 mRNA ranges had been upregulated, whereas α-SMA and Tgfb1 had been downregulated (Fig. S4A). Equally, in LX-2 cells, COX2 mRNA ranges elevated, whereas α-SMA and TGFB1 ranges decreased (Fig. S4B). Gpx4 knockdown elevated Cox2 protein ranges in mHSCs and concurrently decreased α-SMA, Vimentin, and Sod1 proteins (Fig. 4O, Fig. S4C). In LX-2 cells, α-SMA protein expression decreased, whereas COX2 and ACSL4 proteins elevated (Fig. 4P, Fig. S4D). These findings counsel that GPX4 downregulation influences the expression of ferroptosis- and fibrosis-related genes in HSCs, probably affecting oxidative stress and fibrosis development.

MSCs promote HSCs ferroptosis through the exosome pathway

In earlier analysis, the results of MSCs on ferroptosis and HSC activation had been investigated utilizing a transwell co-culture system, which eradicated direct cell-to-cell contact. As MSCs primarily exert their results through exosomes (Exos), this research commenced with exosome extraction and characterization. TEM revealed that Exos displayed a attribute cup-shaped bilayer membrane construction (Fig. 5A), and NanoFCM evaluation indicated a mean particle measurement of 85.2 nm for the extracted Exos (Fig. 5B). Particular markers CD9, and TSG101 had been recognized in purified exosome samples through Western blot evaluation. The endoplasmic reticulum marker Calnexin was detected in MSCs however not within the Exos. These outcomes counsel that the exosome samples had been extremely purified, successfully avoiding contamination from intracellular elements (Fig. 5C). The uptake of PKH26-labeled Exos by mHSCs and LX-2 cells was visually confirmed following co-incubation (Fig. 5D).

Ferroptosis in HSCs is induced by MSC-Exos, which alleviates liver fibrosis. (A) Exos morphology was decided by TEM. Magnification = 60,000x. Scale bar = 100 nm. (B) The particle measurement of Exos was measured by NanoFCM. (C) Exosome-specific markers CD9 and TSG101, together with the endoplasmic reticulum marker Calnexin, had been assessed by Western blot. (D) The uptake of PKH26-labeled Exos by mHSCs and LX-2 cells was noticed utilizing confocal microscopy. The white triangular area signifies exosomes. Scale bar = 10 μm. (E) and (F) qRT-PCR evaluation of GPX4, α-SMA, and COX2 expression in response to Exos therapy, together with the blocking impact of GW4869 pre-treatment on MSCs in mHSCs and LX-2 cells. (G) Liver sections from CCl₄ and CCl₄ + Exos teams had been stained with H&E, Masson, and Sirius Purple. Scale bar = 50 μm. (H) Liver sections from CCl₄ and CCl₄ + Exos teams had been stained for α-SMA immunohistochemistry. Scale bar = 50 μm. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

Additional practical research demonstrated that Exos exhibited results just like these of MSCs. Pre-treatment with GW4869 on MSCs inhibited MSC-mediated expression of GPX4, α-SMA, and COX2, confirming the important function of Exos in inducing HSC ferroptosis and inactivation (Fig. 5E-F). In vivo experiments additional validated the therapeutic results of Exos, displaying decreased inflammatory cell infiltration and collagen deposition (Fig. 5G, Fig. S4E). Immunohistochemistry outcomes confirmed that Exos therapy considerably decreased the elevated α-SMA expression attributable to CCl₄(Fig. 5H, Fig. S4F). In abstract, these findings present direct proof that HSCs endure ferroptosis and inactivation pushed by exosomes secreted by MSCs.

miR-499a-5p in Exos promotes ferroptosis in HSCs by downregulating GPX4

On condition that MSC-Exos comprise a wide range of hsa-miRNAs, mHSCs had been chosen as an alternative of LX-2 cells to make sure that the miRNA sequences recognized had been predominantly these transferred by MSCs, slightly than endogenous hsa-miRNAs, which could have been underrepresented. Consequently, miRNA sequencing was performed below co-culture situations (Fig. 6A), resulting in the identification of two considerably upregulated hsa-miRNAs: hsa-miR-499a-5p and hsa-miR-127-5p (Fig. 6B-C). The goal genes of miR-499a-5p and miR-127-5p had been predicted utilizing miRDB and TargetScan, adopted by practical enrichment analyses to establish miRNAs probably influencing GPX4 expression through transcriptional regulation. Notably, GPX4 was not among the many predicted goal genes of both miRNA. GO evaluation confirmed that concentrate on genes of miR-499a-5p had been considerably enriched in processes comparable to transcription regulatory area DNA binding and nucleic acid binding (Fig. 6D), immediately linked to transcriptional regulation. KEGG pathway evaluation additional indicated that miR-499a-5p goal genes are concerned in RNA degradation, cGMP-PKG signaling, and mTOR pathways, all intently related to transcriptional regulation and mobile metabolism (Fig. 6E). Conversely, miR-127-5p goal genes are primarily concerned in post-transcriptional regulatory mechanisms, together with the ubiquitin ligase advanced, mobile protein modification, and ubiquitin-mediated proteolysis (Fig. S5A-B). This implies that miR-127-5p possible influences protein stability and degradation by way of post-transcriptional mechanisms, slightly than immediately regulating transcription. Due to this fact, given the hidden function of miR-499a-5p in transcriptional regulation and the relevance of its goal genes to the GPX4 expression course of, we selected miR-499a-5p for additional research.

Ferroptosis in HSCs is promoted by miR-499a-5p by way of the downregulation of GPX4. (A) A schematic diagram illustrating miRNA sequencing preparation. (B) and (C) Heatmap and volcano plot of miRNA sequencing outcomes. (D) GO enrichment evaluation for the goal genes of miR-499a-5p. (E) KEGG enrichment evaluation for the goal genes of miR-499a-5p. (F) and (G) Effectivity of miR-499a-5p mimic transfection in mHSCs and LX-2 cells. (H) qRT-PCR evaluation of α-SMA, Acsl4, and Cox2 in mHSCs transfected with the miR-499a-5p mimic. (I) qRT-PCR evaluation of α-SMA, TGFB1, ACSL4, and COX2 in LX-2 cells transfected with the miR-499a-5p mimic. (J) and (Okay) qRT-PCR evaluation of GPX4 in mHSCs and LX-2 cells transfected with the miR-499a-5p mimic. (L) and (M) Fe²⁺ and MDA ranges in mHSCs and LX-2 cells following miR-499a-5p mimic transfection. (N) and (O) TEM observations of mitochondrial adjustments in mHSCs and LX-2 cells. Scale bar = 0.5 μm. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

To additional validate the switch and practical relevance of miR-499a-5p, we investigated its enrichment in MSC-Exos and its impact on HSCs. We first confirmed the enrichment of miR-499a-5p in MSC-Exos (Tab. S7). Subsequent evaluation revealed that co-culture with MSCs or therapy with MSC-Exos considerably upregulated miR-499a-5p expression in mHSCs and LX-2 cells. Nevertheless, pre-treatment of MSCs with GW4869 resulted in a marked downregulation of miR-499a-5p expression (Fig. S5C-D). MSC therapy elevated the expression of miR-499a-5p in mouse liver tissue (Fig. S5E). These findings revealed that MSCs could ship miR-499a-5p to fibrotic liver tissue by way of MSC-Exos. To analyze additional the biology of miR-499a-5p, miR-499a-5p mimic was transfected into mHSCs and LX-2 cells (Fig. 6F-G). Following miR-499a-5p upregulation, α-SMA expression decreased, whereas Acsl4 and Cox2 expression elevated in mHSCs (Fig. 6H). Equally, in LX-2 cells, α-SMA and TGFB1 expression decreased, and ACSL4 and COX2 expression elevated (Fig. 6I), aligning with earlier findings. GPX4 expression was considerably downregulated in each mHSCs and LX-2 cells following miR-499a-5p mimic transfection (Fig. 6J-Okay). Put up-transfection will increase in Fe²⁺ and MDA ranges had been noticed (Fig. 6L-M), together with mitochondrial shrinkage and cristae disappearance, indicating ferroptosis (Fig. 6N-O). These knowledge counsel that miR-499a-5p could downregulate GPX4 expression, inducing ferroptosis in HSCs and inhibiting their activation.

miR-499a-5p targets ETS1 to downregulate GPX4 in Exos-treated HSCs

miR-499a-5p is hypothesized to control GPX4 expression on the transcriptional degree by focusing on transcription elements, primarily based on earlier GO and KEGG analyses. To refine the potential goal genes of miR-499a-5p, miRDB, TargetScan, and miRTarBase databases had been utilized, figuring out 11 candidate genes (Fig. 7A). GTRD and human TFBD databases had been used to foretell transcription elements for human GPX4, whereas GTRD and animal TFBD databases had been employed for mouse Gpx4. By intersecting these predictions with the 11 candidate genes, a standard gene, ETS1, was recognized in each human and murine datasets (Fig. 7B). No intersecting genes had been recognized of miR-127-5p, as anticipated (Fig. S5F-G). A Twin-luciferase reporter assay in LX-2 cells was performed to confirm the direct interplay between miR-499a-5p and ETS1. Transfection with miR-499a-5p mimic considerably decreased luciferase exercise within the wild-type ETS1 mRNA 3’-UTR group, with no important change within the mutant group, indicating particular binding (Fig. 7C, Fig. S5H).

miR-499a-5p targets ETS1 to downregulate GPX4 in Exos-treated HSCs. (A) Venn diagram displaying the intersection of miR-499a-5p goal genes predicted by miRDB, TargetScan, and miRTarBase databases. (B) Venn diagram displaying the intersection of 11 goal genes with GPX4 transcription elements predicted by GTRD, human TFBD, and animal TFBD databases. (C) Twin-luciferase reporter assay of LX-2 cells co-transfected with wild-type or mutant ETS1 mRNA 3’-UTR luciferase plasmids and miR-499a-5p mimic. (D) and (E) mRNA expression ranges of ETS1 and GPX4 in mHSCs and LX-2 cells after Exos intervention. (F) and (G) Protein expression ranges of ETS1 and GPX4 in mHSCs and LX-2 cells after Exos intervention. (H) Protein expression ranges of Gpx4, Ets1, and Acsl4 in mHSCs after transfection with miR-499a-5p mimic. (I) Protein expression ranges of ETS1, GPX4, SOD1, and ACSL4 in LX-2 cells after transfection with miR-499a-5p mimic. (J) ETS1 motifs had been recognized utilizing the JASPAR database. (Okay) Schematic illustration of potential ETS1 binding websites within the GPX4 promoter. (L) ChIP-qPCR evaluation confirmed ETS1 binding at BS2, BS3, and BS4 websites on the GPX4 promoter. (M) The optimum binding mannequin of the GPX4 promoter DNA sequence with the ETS1 protein is proven in blue. (N) ETS1 binds to the GPX4 base sequence CCAGGAAACT (BS2). (O) Twin-luciferase reporter assay was performed to guage the transcriptional exercise of the GPX4 promoter by ETS1. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

Constructing on our prior analysis, the interplay between miR-499a-5p and ETS1, together with the potential mechanism involving ETS1 and GPX4, was additional investigated. It was initially noticed that Exos intervention considerably decreased the mRNA and protein expression of ETS1 and GPX4 in each mHSCs and LX-2 cells (Fig. 7D-G, Fig. S6A). Additional experiments demonstrated that transfection with a miR-499a-5p mimic considerably inhibited ETS1 mRNA expression, confirming miR-499a-5p’s detrimental regulatory function on ETS1 (Fig. S6B). Moreover, after transfection with the miR-499a-5p mimic, Gpx4 and Ets1 protein expression decreased and Acsl4 protein expression elevated in mHSCs (Fig. 7H, Fig. S6C), whereas in LX-2 cells, GPX4, ETS1 and SOD1 protein expression decreased and ACSL4 protein expression elevated (Fig. 7I, Fig. S6D).

In mouse liver tissues, CCl₄ injection upregulated Ets1 expression, whereas MSC and MSC-Exos therapy decreased Ets1 mRNA and protein expression (Fig. S6E-F). The regulatory function of ETS1 on GPX4 as a transcription issue was explored by figuring out two ETS1 motifs utilizing the JASPAR database (Fig. 7J). Inside the 2000 bp upstream area of the GPX4 transcription begin website, encompassing the promoter space, 4 potential binding websites (BS) have been predicted (Fig. 7Okay). ChIP-qPCR evaluation confirmed that BS2, BS3, and BS4 are potential binding websites for ETS1 (Fig. 7L, Fig. S5I). The optimum binding mode between ETS1 and the GPX4 promoter sequence was recognized by way of molecular docking evaluation (Fig. 7M). ETS1 interacts with the GPX4 base sequence CCAGGAAACT (BS2) (Fig. 7N). Twin-luciferase reporter assays demonstrated that ETS1 markedly will increase the transcriptional regulation of the GPX4 promoter (Fig. 7O, Fig. S6G).

ETS1 knockdown experiments had been carried out in HSCs to discover the operate of ETS1. In mHSCs, Ets1 knockdown resulted in decreased mRNA expression of Gpx4 and Tgfb1, and elevated expression of Acsl4 and Cox2 (Fig. S6H). In LX-2 cells, ETS1 knockdown decreased the mRNA expression of GPX4, α-SMA, and TGFB1, whereas rising ACSL4 expression, supporting ETS1 as a constructive regulator of GPX4 (Fig. S6I). Immunofluorescence evaluation of mouse liver tissues revealed that Gpx4 is primarily localized in fibrotic areas, suggesting its potential function in fibrosis. Ets1 was notably detected within the nuclei of cells inside these fibrotic areas, implying its operate as a transcriptional regulator. MSC and MSC-Exos therapy considerably decreased Ets1 and Gpx4 expression ranges (Fig. S7A). Collectively, these knowledge counsel that miR-499a-5p regulates ferroptosis in HSCs by focusing on ETS1, resulting in downregulation of GPX4 and fibrosis-related genes.

ETS1 overexpression rescues miR-499a-5p-induced ferroptosis

To discover the function of ETS1 in miR-499a-5p-mediated ferroptosis in HSCs, EV and ETS1-overexpressing (OE ETS1) mHSCs and LX-2 cells had been established for practical rescue experiments. qRT-PCR and Western blot analyses had been carried out after transfecting the cells with miR-499a-5p mimic to evaluate ETS1, GPX4, ACSL4, COX2, and α-SMA expression ranges throughout completely different teams. Within the OE ETS1 group, ETS1, GPX4, and α-SMA mRNA and protein ranges had been considerably greater than within the EV group, indicating that ETS1 overexpression promotes GPX4 expression and prompts HSCs. Additional evaluation confirmed that within the OE ETS1 + miR-499a-5p mimic group, ETS1, GPX4, and α-SMA expression ranges had been considerably greater in comparison with the EV + miR-499a-5p mimic group, whereas ACSL4 and COX2 ranges had been markedly decreased (Fig. 8A-D). Moreover, Fe²⁺ and MDA ranges decreased within the OE ETS1 + miR-499a-5p mimic group in comparison with the EV + miR-499a-5p mimic group (Fig. 8E-F). These findings counsel that ETS1 overexpression can partially counteract miR-499a-5p-mediated ferroptosis, cut back Fe²⁺ and MDA ranges, and restore HSCs exercise.

ETS1 overexpression rescues cells from miR-499a-5p-induced ferroptosis. (A) mRNA ranges of Ets1, Gpx4, α-SMA, and Acsl4 in mHSCs after Ets1 overexpression and miR-499a-5p mimic transfection. (B) mRNA ranges of ETS1, GPX4, α-SMA, and COX2 in LX-2 cells after ETS1 overexpression and miR-499a-5p mimic transfection. (C) and (D) Protein ranges of ETS1, GPX4, α-SMA, and ACSL4 in mHSCs and LX-2 cells after ETS1 overexpression and miR-499a-5p mimic transfection. (E) and (F) Fe²⁺ and MDA ranges in mHSCs and LX-2 cells after ETS1 overexpression and miR-499a-5p mimic transfection. The information are offered because the imply ± SEM from not less than three unbiased experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 point out important variations between the indicated teams

To discover the function of ETS1 in liver fibrosis, we analyzed single-cell sequencing knowledge from the Human Protein Atlas (HPA) database. The evaluation confirmed that ETS1 was extremely expressed in fibroblasts from human liver tissue (Fig. S7B), suggesting its involvement in liver fibrosis, as activated HSCs purchase fibroblast-like traits through the fibrotic response. These findings align with our experimental outcomes, the place MSC and MSC-Exos therapies alleviated liver fibrosis by probably downregulating ETS1. This highlights ETS1 as a crucial regulator of ferroptosis in HSCs and a possible therapeutic goal for liver fibrosis.