Human umbilical cord mesenchymal stromal cell-derived exosomes protect against MCD-induced NASH in a mouse model

Abstract

Background and aims: Human umbilical cord mesenchymal stem cells (hUC-MSCs) are increasingly being studied in clinical trials of end-stage liver disease because of their good tissue repair and anti-inflammatory effects. hUC-MSC exosomes are vesicles with spherical structures secreted by cells that produce them. The diameter of exosomes is much smaller than that of hUC-MSCs, suggesting that exosomes might be a novel and safer therapeutic product of mesenchymal stem cells. As exosomes have been suggested to have biochemical functions similar to those of hUC-MSCs, this study investigated the efficiency of hUC-MSC-derived exosomes in protecting against nonalcoholic steatohepatitis using an MCD-induced mouse model.

Methods: Human umbilical cord mesenchymal stem cell-derived exosomes were extracted and purified. The effect of these exosomes on disease progression in an MCD-induced nonalcoholic steatohepatitis mouse model was investigated.

Results: The results showed that UC-MSC exosomes intravenously transplanted into mice with MCD-induced NASH improved MCD-induced body weight loss and liver damage in a mouse model. Additionally, the inflammatory cytokines in liver tissue were reduced, which may be caused by exosome-induced macrophage anti-inflammatory phenotypes both in vitro and in vivo. In addition, UC-MSC exosomes reversed PPARα level in ox-LDL-treated hepatocytes in vitro and in NASH mouse liver, which had been downregulated.

Conclusions: UC-MSC exosomes alleviate MCD-induced NASH in mice by regulating the anti-inflammatory phenotype of macrophages and by reversing PPARα protein expression in liver cells, which holds great potential in NASH therapy.

Keywords: MCD mouse model; Nonalcoholic steatohepatitis; PPARα; hUC-MSC exosomes.

Fig. 1

A. MSC surface marker expression analysis by flow cytometry. B. (a) Fifth-generation hUC-MSCs under a light microscope; (b and c) osteogenic and adipogenic differentiation of hUC-MSCs; (d) cartilage formation differentiation of hUC-MSCs. C. The vesicle diameter, number, purity, and morphology were tested by electrophoresis (red box, left panel) and Brownian motion video analysis laser scattering microscopy (right panel). D. Western blot detection of the expression of TS101, CD63, CD9, and GAPDH in hUC-MSC-derived exosomes. The negative control was the product extracted from the control medium (CM) according to exosome extraction steps. Full-length blots are presented in Additional file 1: Fig. S1. E. Transmission electron micrographs of hUC-MSC exosomes. Scale bar = 200 nm

Fig. 2

A. Weight changes of mice fed regular chow or MCD during 6 weeks period. In each group, the number of mice was n = 6. **p < 0.01. B. Photographs of liver sizes of mice from different experimental groups (upper panel). The weights of each group of mice were recorded and counted (n = 6, below panel). **p < 0.01. ns: not significant. C. Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of each mouse were recorded and counted (n = 6 in each group); *p < 0.05; D. Representative images of HE-stained liver sections of the indicated groups (microscope 40×). a mice with regular chow; b mice with MCD; c mice with MCD and intervention with control medium (CM) extract twice a week; d mice with MCD and intervention with hUC-MSC exosomes(Ex) twice a week. Scale bar = 50 μm. E. Pathology scores for balloon-like degeneration, steatosis, and lobular inflammation of groups of liver sections (n = 6). *p < 0.05, **p < 0.01

Fig. 3

A Plasma levels of TNF-α, IL-6, and IL-1β in groups of mice, n = 6. Protein levels were evaluated using the sandwich ELISA method. *p < 0.05. B. Western blotting analysis of phosphorylated NF-κB (p-P65) and total NF-κB (P65) in liver tissues in groups of mice (left panel). Three liver tissues were randomly selected from each group to compare NF-κB activation levels. GAPDH protein levels were used as a reference standard. The phosphorylated NF-κB protein was been quantified using ImageJ software and represented by histogram (fold change of phosphorylated NF-κB /GAPDH, right panel). Full-length blots are presented in Additional file 1: Fig. S2. C. Immunohistochemical assay targeting F4/80 was used to detect macrophages in groups of mouse livers. (a) Regular chow; (b) MCD group; (c) MCD with control medium (CM) extract intervention group; (d) MCD with hUC-MSC exosomes (Ex) group (left panel). Scale bar = 50 μm. Three fields were randomly selected to determine the number of macrophages in the livers of mice in each group, and the differences among groups were compared (right panel). *p < 0.05. D. Quantitative PCR results showing the endogenous mRNA levels of CD206, Arginase-1, and IL-10 in mouse livers in each group (n = 3). GAPDH mRNA served as reference standard. The fold change is shown by the histogram. **p < 0.01

Fig. 4

A. PKH-26 labeling of hUC-MSC exosomes taken up by RAW264.7 cells. Scale bar = 20 μm. B. Oil red O staining of foamy RAW264.7 cells (left panel). (a) Without any treatment; (b) Ox-LDL treatment for 48 h; (c) Ox-LDL treatment for 48 h after control medium (CM) extract treatment for 24 h; d. Ox-LDL treatment for 48 h after UC-MSC treatment for 24 h. Three fields were randomly selected to calculate the number of red lipid droplets and the number of cells. The fold change is shown by the histogram (right panel). C. Quantitative PCR results showing the endogenous mRNA levels of TNF-α, IL-6 and IL-1β in RAW264.7 cells. CM represents control medium extract treatment, and Ex represents UC-MSC exosome treatment. The independent experiment has been repeated for three times. *p < 0.05; **p < 0.01. D. Detection of the M2 polarization markers CD206, Arginase-1 and IL-10 in Ox-LDL-treated RAW264.7 cells by quantitative PCR. CM represents control medium extract treatment, and Ex represents UC-MSC exosome treatment. GAPDH mRNA served as reference standard. *p < 0.05; **p < 0.01

Fig. 5

A. Immunohistochemical assays were used to detect distribution and location of PPARα in groups of mouse livers. (a) Regular chow; (b) MCD group; (c) MCD with Control Medium (CM) extract intervention group; (d) MCD with hUC-MSC exosomes(Ex) group. The random view to zoom in on the panel shown is on the right. Scale bar = 20 μm. B. Western blotting analysis of PPARα in liver tissues in groups of mice. Three samples were selected in each group (upper panel). Protein levels were quantified using ImageJ software and are represented by a histogram (fold change of PPARα/GAPDH, panel below). Full-length blots are presented in Additional file 1: Fig. S3. C. PKH-26 labeling of MSC-exo taken up by HepRG cells. Scale bar = 20 μm. D. PKH-26 labeling of MSC-exo taken up by Huh1-6 cells. Scale bar = 20 μm. E. Western blotting analysis of PPARα in the human liver cell line HepRG under the indicated treatment. Protein levels were quantified using ImageJ software and represented by a histogram (fold change of PPARα/Histone H3). **p < 0.01. Full-length blots are presented in Additional file 1: Fig. S4. F. Western blotting analysis of PPARα in the mouse hepatoma carcinoma cell line Huh1–6 under the indicated treatment (fold change of PPARα/Histone H3). Protein levels were quantified using ImageJ software and represented by a histogram. **p < 0.01. Full-length blots are presented in Additional file 1: Fig. S5