Antifibrotic effect of placental multipotent mesenchymal stromal cells in liver fibrosis

Abstract

A large number of chronic liver diseases, such as chronic viral hepatitis, alcohol intoxication, primary sclerosing cholangitis, autoimmune hepatitis and others, lead to the development of liver fibrosis. In the pathogenesis of the development of liver fibrosis against the background of the action of an etiological factor, the leading importance is assigned to the activation of perisinusoidal liver cells and their subsequent differentiation into myofibroblast-like cells, which are central regulators of fibrogenesis. In this study, allogeneic transplantation of multipotent mesenchymal stromal cells (MMSCs) isolated from the placenta in animals with liver fibrosis is performed. These cells are capable of synthesizing a number of factors that can potentially be considered antifibrogenic.

The purpose of the study was to evaluate the possibility of using placental multipotent mesenchymal stromal cells for the treatment of liver fibrosis.    

Methods. The experiments were performed on 40 male mice aged 8-10 weeks. Fibrosis was modeled by intraperitoneal injection of carbon tetrachloride in an amount of 2 µl/g of animal weight for 6 weeks, 2 times a week. Further, the animals in which liver fibrosis was induced were divided into the main group and the comparison group. The main group consisted of mice that received a single intravenous injection of MMSC in the amount of 1×10⁶ cells/ mouse in 0.2 ml. The comparison group consisted of mice that were not injected with cells after fibrosis modeling. 5 weeks after modeling liver fibrosis, the effectiveness of the therapy was evaluated. The culture of placental MMSCs was isolated from the chorion of the placenta according to the method of A.S. Teplyashin et al. 2004. The viability of the isolated cells was evaluated using 7-AAD. The METAVIR scale was used to assess the severity of fibrosis. Quantitative analysis of the collagen content in the liver was performed using the Picro Sirius Red Stain Kit. In the histological preparation of the liver, the number of MMP9, MMP13, TIMP1, α-SMA positive cells, CD45 positive cells was determined.  The content of growth factors HGF and TGF-β, Pro-Collagen I alpha 1 β, was evaluated in liver homogenate. Serum levels of proinflammatory cytokines were determined: interleukin-1β, tumor necrosis factor-α. The obtained data were processed by the variational statistical method using the Student's t-test. In the absence of a normal distribution, the statistical significance of the differences was determined using the nonparametric Mann-Whitney criterion (U). The differences were considered statistically significant at p<0.05.

Results. 5 weeks after modeling liver fibrosis on the background of MMSC transplantation, a decrease in fibrosis activity on the METAVIR scale by 50% was noted. MMSC transplantation was accompanied by a decrease in the content of CD 45+ cells in the liver. This led to a decrease in the level of proinflammatory cytokines in the blood serum. The introduction of MMSC was also accompanied by a decrease in the area of connective tissue by 33.1%. The area of the histological preparation stained with α-SMA, as well as the number of α-SMA positive cells after the introduction of MMSC decreased by 28.3% and 31.0%, respectively.  The introduction of MMSCs led to a decrease in the number of MMP9 and an increase in MMP13 producing cells, a decrease in the number of TIMP1 positive cells. A decrease in the level of TGF-β and an increase in HGF in the liver were also revealed. When analyzing the biochemical parameters of serum, it was found that the introduction of MMSC led to a decrease in the activity of transaminases, restoration of protein-synthetic liver function, and a decrease in the level of total bilirubin.

Conclusion. The results obtained indicate a high therapeutic potential of placental MMSCs. The use of MMSCs isolated from the chorion of the placenta can be considered as a promising non-surgical method for the treatment of liver fibrosis.