A Combination of Ultrasound Targeted Microbubble Destruction with Transplantation of Bone Marrow Mesenchymal Stem Cells Promotes Recovery of Acute Liver Injury

Abstract

Bone marrow mesenchymal stem cells (BMSCs) has been considered as a therapeutic strategy for acute liver injury (ALI). However, the insufficient homing of BMSCs in vivo limited their applications. Ultrasound targeted microbubble destruction (UTMD) has shown the potential in promoting the homing of stem cells into ischemic myocardium. We therefore explored whether UTMD could promote BMSCs' homing in ALI rat models and its corresponding therapeutic effect. Methods: Bone marrow mesenchymal stem cells (BMSCs) were obtained from the femurs and tibias of Sprague-Dawley (SD) rats. The characteristics of BMSCs including the proliferative viability, diversified differentiation ability and specific cell surface markers' expression were verified by flow cytometry. In order to find the appropriate ultrasound parameters, the normal rat liver was exposed to different intensity of ultrasound (1.0 W/cm2, 1.5 W/cm2, 2.0 W/cm2) with 300µl microbubbles, and the expression of tumor necrosis factor $a$ (TNF-a) and stromal cell-derived factor 1 (SDF-1) were detected at 24h post-treatment. The ALI models were established via a single intraperitoneal injection of D-galactose on 32 SD rats, which then were randomly assigned into four groups: Control, BMSCs, UTMD, and UTMD+BMSCs. After the BMSCs were stably transfected with a lentivirus for expressing green fluorescent protein (GFP), these cells were then injected through tail vein in a concentration of 2×106/ml. Forty-eight hours after the treatment, the protein expression levels of SDF-1, intercellular cell adhesion molecule1(ICAM-1), vascular cell adhesion molecule 1(VCAM-1 and monocyte chemotactic protein 1 (MCP-1) of exposed liver were analyzed, and the number of GFP-transfected BMSCs were observed as well. Serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) activities were tested after 48 h/72 h/168 h of treatment. The histology of liver was evaluated at 168h. Results: The isolated rat BMSCs demonstrated good proliferation, osteogenic and adipogenic differentiation and the expression of CD29+, CD90+, CD45-, CD11b/c-. After the application of UTMD, the expression level of SDF-1 and TNF-a on exposed liver was markedly higher in 2.0 W/cm2 group than in the 1.5 W/cm2 and 1.0 W/cm2 group ( $p$ $p$ <0.05), as well as the protein expression of SDF-1, ICAM-1, VCAM-1 and MCP-1 (p<0.01). The ALT, AST and ALP activities were much lower in UTMD+BMSCs group. The pathological features of liver injury were alleviated to a large extent in UTMD+BMSCs group. Conclusion: Through upregulation of adhesion molecular and cytokines, UTMD could enhance the homing of BMSCs in rat models of ALI and improve the function of acute injured liver.