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. 2023 Mar 28;5(7):100746. doi: 10.1016/j.jhepr.2023.100746

Fig. 3.

Fig. 3

MSC-ex activate AMPK-mediated PPARα/CPT-1A and the SREBP-1C/FASn signalling pathway in hepatocytes and liver tissues.

(A and B) mRNA-seq analyses were performed on L02 cells that were untreated (normal group) or subjected to OPA stimulation (2.0 mM, ratio of 2:1) combined with MSC-ex (800 μg/ml) (MSC-ex group) or PBS treatment (PBS group) for 24 h, n = 3 biological replicates per group. Histogram (A) and Venn diagram (B) analyses of differentially expressed genes. (C) Gene ontology analyses of differentially activated biological processes. (D) The top 10 enriched pathways as determined by the KEGG database. (E) Heat map of differentially expressed genes in the AMPK signalling pathway. (F and G) Evaluation of protein expression by immunoblotting (F) and quantification of the results (G). n = 3 biological replicates per group; ∗p <0.05, ∗∗∗p <0.001 vs. normal group; #p <0.05, ##p <0.01, ###p <0.001 vs. PBS group. (H and I) mRNA-seq analyses were performed on liver tissues in mice placed on a HFD for 10 weeks followed by 10 mg/kg MSC-ex or PBS treatment for 4 weeks. n = 3 biological replicates per group. The top 10 enriched pathways as determined by the KEGG database (H) and heatmap (I) of differentially expressed genes in the AMPK signalling pathway. (J and K) Immunoblotting analyses of AMPK signalling proteins in mice placed on a HFD for 10 weeks followed by 10 mg/kg MSC-ex or 10 mg/kg HFL1-ex or deMSC-ex and PBS treatment for 4 weeks (J) and quantification of the results (K). ∗p <0.05, ∗∗p <0.01, ∗∗∗p <0.001 vs. normal chow diet group; #p <0.05, ##p <0.01, ###p <0.001 vs. HFD group. (L) Immunofluorescence images of staining for p-AMPK, PPARα, CPT-1A, SREBP-1C, FASn, and p-IRS1 (green) in liver sections of mice. Nuclei were labelled with DAPI (blue). Scale bars, 100 μm. Data are represented as the mean ± SEM. Statistical analyses by a one-way ANOVA (G and K). AMPK, AMP-activated protein kinase; CPT-1A, carnitine palmitoyltransferase 1A; deMSC-ex, MSC-ex-free conditional medium supernatant; FASn, fatty acid synthase; HFD, high-fat diet; HFL1-ex, foetal HFL1-derived exosomes; HFL1, human lung fibroblast 1; IRS1, insulin receptor substrate 1; KEGG, Kyoto Encyclopedia of Genes and Genomes; mRNA-seq, mRNA sequencing; MSC-ex, MSC-derived exosomes; MSC, mesenchymal stem cell; NAFLD, non-alcoholic fatty liver disease; OPA, oleate and palmitate; p-AMPK, phosphorylated AMPK; p-IRS1, phosphorylated IRS1; PPARɑ, peroxisome proliferator-activated receptor alpha; SREBP-1C, sterol regulatory element-binding protein-1C; TCA, tricarboxylic acid cycle; TNF, tumour necrosis factor.