Ultrasonic microbubbles promote mesenchymal stem cell homing to the fibrotic liver via upregulation of CXCR4 expression

Abstract

Objective: To investigate the mechanism of ultrasound microbubbles (UTMB) promoting stem cells homing to fibrotic liver.

Methods: Bone marrow derived mesenchymal stem cells (BMSCs) were divided into 5 groups with or without ultrasound microbubbles and continuously irradiated with ultrasound conditions of frequency 1 MHZ and output power 0.6 W/cm² for different times, and then injected into a mouse model of liver fibrosis through the tail vein with or without ultrasound microbubbles, with sound intensity. The effect of ultrasound microbubbles on MSC expression of CXC chemokine receptor 4 (CXCR4) and homing fibrotic liver was evaluated by flow cytometry (FCM), western blot (WB) and immunohistochemistry (IHC) analysis.

Results: The level of CXCR4 expression was significantly higher in the ultrasound microbubble group than in the non-intervention group (P < 0.05), and the number of MSC and the rate of CXCR4 receptor positivity in the ultrasound microbubble-treated liver tissues were significantly higher than in the non-intervention group (P < 0.01).

Conclusion: Ultrasonic microbubbles can promote the expression of CXCR4 on the surface of MSCs, thus improving the homing rate of MSCs in fibrotic liver.

Keywords: CXCR4; Homing; Liver fibrosis; Mesenchymal stem cell; Preface; Ultrasound microbubble.

Fig. 1

The identification of microbubble (SonoVue®) and Mouse model. a all microbubbles are less than 5 μm (n = 40, ×100, scale bar = 25 μm). b most microbubble size distribution Between in 2.10–4. 70 μm, the average diameter of microbubbles is about 2.52 ± 0.43 μm. c The mice livers with H&E staining had visible fibrosis, destruction of hepatic lobules, a large amount of hepatocyte apoptosis, and infiltration of inflammatory cells (n = 10, HE ×100, scale bar = 100 μm). d Fluorescence microscopy of HE staining of mouse liver fibrosis

Fig. 2

Characteristics of mice MSCs (n = 3). a BMSCs transfected with lentiviral vectors were cultured until day 6, The adherent cells reached approximately 80% confluence (×100, scale bar = 100 μm). b Normol BMSCs were cultured until day 6, The adherent cells approximately 100% confluence (×100, scale bar = 100 μm). c The normal BMSCs have better proliferative capacity(*, P < 0.05), Otherwise, The BMSCs transfected with lentiviral vectors can eventually multiply in sufficient numbers. d MSCs were labeled with GFP (×100, scale bar = 100 μm). e FCM revealed that the GFP expression level in BMSCs was approximately 97.21 ± 0.19%. f FCM analysis indicated that the positive rates of MSCs-specific antigens CD29 and CD44 were 97.2 and 97.8%. g The positive rates of CD34 and CD45 were 2.06% and 2.74%

Fig. 3

Cell surface expression of CXCR4 using FCM (n = 3). a–e Representative examples of the membrane expression levels of CXCR4 on MSCs in the different groups. The number of CXCR4-positive cells in the M + UTMB60s group (c) was significantly higher compared with the M group(a) and the M + U60s group (b). There was no significant difference in the number of CXCR4-positive cells between the M + UTMB60s group (c), the M + UTMB90s group (d) and the M + UTMB180s group (e). f Quantification of CXCR4 expression by FCM assay in the experimental MSCs. All values are expressed as the mean ± SD. **P < 0.01; n = 3

Fig. 4

Expression of CXCR4 by IHC (n = 10). The surface of CXCR4 immunohistochemistry positive cells is brown (× 200, scale bar = 25 μm). a–e IHC of CXCR4 in the M group, M + U60s group, M + UTMD 60 s group, M + UTMD 90 s group, M + UTMD 180 s group. The number of CXCR4-positive cells was relatively smaller in the M group (a) and the M + U60s group (b), P > 0.05. The percentage of cells expressing surface CXCR4 in the M + UTMB60s group (c) higher than the M + U60s group (b) and the M group (a), P < 0.05. There was no significant difference in the number of CXCR4-positive cells between the M + UTMB60s group (c), the M + UTMB90s group (d) and the M + UTMB180s group (e), P > 0.05. f Quantification of CXCR4 expression was performed by Image-Pro Plus 6.0 software. All values are expressed as the mean ± SD. **P < 0.01; n = 10

Fig. 5

Expression of CXCR4 by WB (n = 6). a WB of CXCR4 in the M group, M + U60s group, M + UTMD 60 s group, M + UTMD 90 s group, M + UTMD 180 s group. b Quantification of CXCR4 expression was performed by Image J software. All values are expressed as the mean ± SD. **P < 0.01; n = 6

Fig. 6

Trypan blue staining for cell viability. The results showed that ultrasound treatment for 60 s and UTMD treatment for 60 s had no significant effect on cell viability. With the further increase of UTMD treatment time from 60 to 180 s, cell viability decreases from 86.33 ± 1.89% to 38.00 ± 1.63% after 48 h of treatment. All values are expressed as the mean ± SD. **P < 0.01; n = 3

Fig. 7

Representative photograph of GFP staining of MSCs in each group (n = 10). GFP-labeled MSCs were concentrated in the portal vein region under fluorescence microscopy images (×100, scale bar = 100 μm). a There are almost no GFP positive cells in the liver tissue and portal area of the group I. b There are a small number of GFP positive cells in the portal area of the group II. c There are GFP positive cells in the liver tissue and portal area of the group III. d There are a large number of GFP positive cells in the liver tissue and portal area of the group IV. e Quantitative analysis revealed that transplanted MSC in the group II (5.7 ± 1.26) was significantly increased compared to the group I (1.7 ± 1.09) (P < 0.01) and transplanted MSC in the group IV (31.9 ± 4.58) was significantly increased compared with other groups (P < 0.01). All values are expressed as the mean ± SD. **P < 0.01; n = 10

Fig. 8

Representative photograph of IHC staining of MSCs in each group (n = 10). Anti GFP antibody (ab183734) -labeled MSCs were concentrated in the portal vein region under microscopy images (× 100, scale bar = 100 μm). a a few of IHC positive cells (brown) in the liver tissue and portal area of the group I. b There are a small number of IHC positive cells in the portal area of the group II. c There are IHC positive cells in the liver tissue and portal area of the group III. d There are a large number of IHC positive cells in the liver tissue and portal area of the group IV. e Quantitative analysis revealed that transplanted MSC in the group II (9.8 ± 2.53) was significantly increased compared to the group I (2.4 ± 0.84) (P < 0.01) and transplanted MSC in the group IV (37.1 ± 5.11) was significantly increased compared with other groups (P < 0.01). All values are expressed as the mean ± SD. **P < 0.01; n = 10

Fig. 9

Expression of SDF-1 by WB (n = 3). a WB of SDF-1 in the Control group, US 60 s group, US + microbubble 60 s group, b Quantification of SDF-1 expression was performed by Image J software. All values are expressed as the mean ± SD. **P < 0.01; n = 3