Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Inhibit the TGF-β1 Pathway in Hepatic Fibrosis Rats Through a Paracrine Regulation Process

Abstract

The aim is to verify the therapeutic effect and possible mechanism of human umbilical cord Wharton's jelly-derived transplantation of mesenchymal stem cells (UMSCs) on CCl4-induced hepatic fibrosis rats through in vivo studies and to explore the regulatory mechanism of UMSCs on fibrosis of hepatic stellate cells (HSCs) through in vitro experiments. In vivo experiment: Rats were randomly divided into blank control group and hepatic fibrosis group. During the entire trial, the blank control group received subcutaneous injection of normal saline, while in the hepatic fibrosis group received injections of 50% CCl4-olive oil subcutaneously for 10 weeks to establish the rat model of liver fibrosis. Hepatic fibrosis rats were then randomly and evenly divided into umbilical cord mesenchymal stem cell (UMSC) group, bone marrow mesenchymal stem cell (BMSC) group, UMSC-culture medium (CM) group, and control group. Rats in each group were infused with the following substances through the caudal vein as follows: 1 mL UMSCs (2 × 106/mL) in UMSC group, 1 mL BMSCs (2 × 106/mL) in BMSC group, 1 mL UMSCs-CM in CM group, and 1 mL saline in control group. Rats of each group were closely observed (weight, hair condition, activity, appetite, diarrhea, etc.), venous blood samples were collected, the number of white blood cells and lymphocytes were measured, and liver function indicators (ALT, AST, TBIL, ALB) were determined. Three weeks later, rat liver specimens were taken, HE stained, pathological changes were examined and quantified. In vitro experiments: HSCs were seeded in 6-well plates at 1.0 × 105/mL, with a serum-free medium for 24 hours. Then, 2 mL of UMSCs-CM was added in the study group, while an equal amount of complete medium was added to the control group. RT-PCR was used to detect TGF-β1, Collagen-I, TIMP-2 mRNA expression in HSCs, and western blot was used to detect TGF-β1 protein expression in HSCs. In vivo experiment: Compared with the control group, after the transplantation, the activity status (weight, spirit, appetite, movement, hair, diarrhea, etc.) of rats in the UMSC group, BMSC group, and CM group were improved. The liver function indexes of these groups, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were significantly decreased (p < 0.05), while albumin (ALB) levels were mildly but not significantly increased (p > 0.05). The Knodell score (reflecting the degree of liver inflammation) and Chevallier score (reflecting the degree of liver fibrosis) of liver specimens in pathological examination were also significantly reduced, and the difference in the quantitative scores of those indexes was statistically significant (p < 0.05). There was no statistically significant difference in the number of venous white blood cells and lymphocytes, liver function indexes (ALT, AST, TBIL, ALB), Knodell score, and Chevallier score of liver samples among the UMSC group, BMSC group, and CM group. In vitro experiments: After treatment with UMSCs-CM, the expression of TGF-β1, Collagen-I, and TIMP-2 mRNA in HSCs was significantly down-regulated compared with that of the control group (treated with complete medium), and it gradually decreased with the extension of the treatment time. Compared with the control group, the expression of TGF-β1 protein in the HSCs of the experimental group was down-regulated, and this effect was time-dependent, specifically, the control group (2.49 ± 0.43) > the experimental group at 48 hours (1.98 ± 0.26) > the experimental group at 72 hours (1.62 ± 0.20) (F = 7.796, p < 0.05). In rats with liver fibrosis, transplantation of UMSCs can improve liver function and reduce the inflammatory activity and fibrosis of the liver, possibly through the paracrine mechanism. UMSCs inhibit HSCs fibrosis through a paracrine mechanism, which is time-dependent, possibly by targeting TGF-β1 and its downstream gene products.