Human umbilical cord mesenchymal stem cells ameliorate liver fibrosis by inhibiting hepatocyte ferroptosis and macrophage polarization via the miR-455-3p/PLAU axis

Abstract

Background: Peripheral infusion of human umbilical cord mesenchymal stem cells (hUC-MSCs) may therapeutically treat liver fibrosis. hUC-MSCs can influence liver fibrosis progression and immune microenvironment remodeling via microRNAs (miRNAs).

Methods: Bioinformatics showed the miR-455-3p/PLAU axis might regulate ferroptosis in liver fibrosis and affect the immune microenvironment. hUC-MSCs were transplanted into CCL4 induced liver-fibrotic male C57BL/6 mice. Liver tissues were stained (Hematoxylin and eosin (H&E), Masson, Sirius Red) to assess fibrosis; Alpha-smooth muscle actin (α-SMA) expression was detected by immunohistochemistry (IHC). Macrophage polarization was measured by immunofluorescence (IF), quantitative real-time polymerase chain reaction (qRT-PCR), and flow cytometry. Dual-luciferase and RNA pull-down assays verified miR-455-3p/PLAU regulation. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) evaluated liver function. Reactive oxygen species (ROS), glutathione peroxidase 4 (GPX4), iron content, and ferrous iron (Fe²⁺) assessed ferroptosis. Enzyme-linked immunosorbent assay (ELISA) detected cytokines in supernatant.

Results: In fibrotic vs. normal liver tissues, miR-455-3p was down-regulated, PLAU up-regulated, and ferroptosis increased. hUC-MSCs transplantation improves CCL4 induced liver fibrosis by inhibiting ferroptosis of hepatocytes through the miR-455-3p/PLAU axis. It also alleviated macrophage M1 polarization and liver damage.

Conclusion: hUC-MSCs inhibit hepatocyte ferroptosis via the miR-455-3p/PLAU axis, alleviate macrophage M1 polarization, and slow liver fibrosis. These findings offer insights for future liver fibrosis research and treatment.

Keywords: Ferroptosis; Human umbilical cord mesenchymal stem cells; Liver fibrosis; Macrophage polarization; MiR-455-3p/PLAU.

Fig. 1

Differential mRNAs and miRNAs between liver fibrosis and normal liver. a Volcano plots of differential genes from GSE33857 dataset; b Volcano plots of differential genes from GSE77271 datasets; c Venn diagram of jointly differential miRNAs from two miRNA datasets (GSE33857,GSE77271); d Volcano plots of differential genes from GSE54238 dataset; e Volcano plots of differential genes from GSE197112 dataset; f Venn diagram of jointly differential mRNAs from two mRNA datasets (GSE54238, GSE197112); g–h Heatmap of differential genes from the mRNA datasets (GSE54238, GSE197112); i Intersection of mRNAs predicted by co-differentiated miRNAs and differential mRNAs taken from the aforementioned differential mRNAs, obtaining the four regulatory axes

Fig. 2

Prediction of miR-455-3p/PLAU downstream pathway and its impact on the tumor immune microenvironment. a–c Results of KEGG enrichment analysis and GO (including Biological process, Cellular component, and Molecular function) enrichment analysis of differential genes from GSE54238 dataset; d–f Results of KEGG enrichment analysis and GO (including Biological process, Cellular component, and Molecular function) enrichment analysis of differential genes from GSE197112 dataset; g Results of GSEA enrichment analysis of GSE54238 dataset; h immune infiltration analysis of GSE54238 dataset

Fig. 3

hUC-MSCs alleviate CCL4-induced liver fibrosis and ferroptosis. a Representative images of liver of H&E, Masson, Sirius red, and α-SMA immunohistochemistry staining of liver sections of three groups (Control group, CCL4 group and CCL4 + hUC-MSC group, n = 5); b The effect of hUC-MSCs transplantation on liver function was assessed by measuring ALT, AST, TBIL and albumin, data are presented as mean ± SD (n = 5); c qRT-PCR to detect the expression levels of miR-455-3p in liver of the three groups, data are presented as mean ± SD (n = 5); d qRT-PCR to detect the expression levels of PLAU in liver of the three groups, data are presented as mean ± SD (n = 5); e The expression level of GPX4 and ROS in liver of the three groups, data are presented as mean ± SD (n = 5). ns = not significant, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001

Fig. 4

hUC-MSCs inhibit macrophage M1 polarization. a Immunofluorescence staining for F4/80, CD86 and CD206 from liver sections of three groups (Control group, CCL4 group and CCL4 + hUC-MSC group); b Statistical results of immunofluorescence staining for F4/80, CD86 and CD206, data are presented as mean ± SD (n = 5); c Flow cytometry analysis of F4/80⁺ and CD86⁺ macrophages in the liver tissue of three groups; d Quantitative analysis of F4/80⁺/CD86⁺ in the liver tissue of three groups, data are presented as mean ± SD (n = 5). ns = not significant, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001

Fig. 5

MiR-455-3p directly targets PLAU in primary hepatocytes. a qRT-PCR to detect the expression levels of miR-455-3p in primary hepatocytes extracted from the livers of three groups (Control group, CCL4 group and CCL4 + hUC-MSC group, data are presented as mean ± SD (n = 5)); b qRT-PCR to detect the expression levels of PLAU in primary hepatocytes extracted from the livers of three groups, data are presented as mean ± SD (n = 5); c TargetScan to predict that PLAU is a potential target of miR-455-3p; d miR-455-3p mimic reduced luciferase ability of PLAU wild-type mRNA 3'-UTR, while it did not change PLAU mutant 3'-UTR in primary hepatocytes extracted from the livers of the control group and CCL4 group, data are presented as mean ± SD (n = 5); e Western blot to detect PLAU protein expression levels in control, miR-455-3p mimic, pc-PLAU, and miR-455-3p mimic + pc-PLAU groups, data are presented as mean ± SD (n = 5); f Western blot and qRT-PCR were used to detect the expression level of PLAU in the RNA-pulldown assay, data are presented as mean ± SD (n = 5). ns = not significant, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001

Fig. 6

MiR-455-3p/PLAU axis regulates the ferroptosis process in hepatocytes. a–b Western blot assay was performed to detect the level of PLAU, GPX4, SLC7A11, ACSL4 in primary hepatocytes of control and miR-455-3p mimic group; c–d Western blot assay was performed to detect the level of PLAU, GPX4, SLC7A11, ACSL4 in primary hepatocytes of control and pc-PLAU group treated with Fre-1 or not; e The molecular docking diagram of PLAU and ITCH; f–g Western blot analysis of GPX4 protein in PLAU-overexpressing hepatocytes treated with cycloheximide (CHX; 25 µg mL⁻¹) for 0, 8, 16, and 24 h; h Co‐IP and western blot analysis showing the interaction of PLAU and GPX4 in hepatocytes; i Co‐IP and western blot analysis showing the interaction of PLAU and ITCH in hepatocytes; j At 6 h post‐treatment with MG132 (10 µm), cell lysates were obtained and immunoprecipitated with anti‐GPX4 antibody. Western blot analysis was performed with antibody to GPX4 to detect endogenous GPX4 ubiquitination from PLAU-overexpressing hepatocytes relative to the control group. k–n Levels of total iron, ferrous iron, MDA, and lipid ROS were measured in hepatocyte of control and miR-455-3p mimic group, data are presented as mean ± SD (n = 5). o–r Levels of total iron, ferrous iron, MDA, and lipid ROS were measured in hepatocyte of control and pc-PLAU group, data are presented as mean ± SD (n = 5). ns = not significant, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001

Fig. 7

hUC-MSCs inhibited M1 polarization and pro-inflammatory cytokine release in BMDMs. a Immunofluorescence staining for CD86 from BMDMs co-cultured with hUC-MSCs or not; b Statistical results of immunofluorescence staining for CD86, data are presented as mean ± SD (n = 5); c–d qRT-PCR detected the expression of CD86, NOS2, ARG1 and MRC1, in BMDM co-cultured with hUC-MSCs or not, data are presented as mean ± SD (n = 5); e–f The levels of IL-1β, TNF-a, IL-10, TGF-β, in the supernatant of BMDMs co-cultured with hUC-MSCs or not, data are presented as mean ± SD (n = 5). ns = not significant, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001